System biology of multiple organ dysfunction: formation of a ceramide-enriched macro-domain in SIRS/sepsis

Generation of bioactive lipids such as ceramide (Cer) and the formation of Cer-enriched macrodomains are regarded as mediators of SIRS and the development of multiple organ failure. Therefore, we addressed the question of whether there is a difference in the plasma activity of the secreted isoform of the Cer-forming enzyme sphingomyelinase (SMPD1) in patients with various degrees of SIRS/sepsis of different origin as well as in a murine loss of function model. We found plasma activity in critically ill patients (median 262.3 pmol/ml*hour) was significantly higher than age-matched controls (123.6). In patients with fatal outcome, activity increased (+77.4) in comparison with survivors (-252.1). A severity-dependent increase was also analyzed in patients with multiple organ dysfunction syndrome (MODS) following elective cardiac surgery. Beyond immunological detection of increased pSMPD1 in septic patients, we found an increase in Cer-enriched macrodomains in endothelial cells after stimulation with patients' plasma, endotoxin, or TNF. 
 
We also found formation of Cer-enriched macrodomains by immunostaining using specific antibodies directed against Cer, CD14, and Fas. In a loss of function model, we identified 315 transcripts differentially regulated in circulating white blood cells, liver, and lung by use of microarray technology as well as in the cytokine pattern/organ function parameters following polymicrobial cavity infection. Furthermore, host responses in knockout mice were more pronounced with respect to bacterial load in lung, liver, and blood, plasma cytokine levels, thrombocytopenia as well as delayed migration of neutrophils into hepatic tissue. 
 
In conclusion, the results provide demonstration of a biofunctional relevant activity of SMPD1 resulting in altered signal transduction in SIRS, which may contribute to the development of MODS.

Introduction Hydroxyethyl starch (HES) is widely used for volume therapy in intensive care. In critically ill and sepsis patients, HES use was dose-dependently associated with increased renal failure, tissue storage with organ failure, and increased long-term mortality. There are other safety concerns with regard to coagulopathy, pruritus, and mortality. However, third-generation HES 130/0.4 is considered to have an improved risk profi le. Therefore, we wanted to assess whether published studies on HES 130/0.4 resuscitation are suffi ciently well designed to draw conclusions about the safety of this compound. Methods We derived clinically relevant outcome parameters to analyze safety outcomes from the literature and provided exemplary power calculations. Randomized controlled trials (RCT) on fl uid resuscitation with HES 130/0.4 were systematically searched and analyzed for clinical condition, sample size, study duration, cumulative dose, control fl uids, endpoints, and colloidcrystalloid volume ratios in studies with a goal-directed fl uid regimen. Due to the heterogeneity of included studies, all analyses were descriptive (SPSS 17.0). Results A total of 56 RCTs were included. Only two studies included severe sepsis patients, 80% were from the elective surgical setting and one study from the emergency surgical setting. In general, studies were underpowered (median sample size 25 patients in HES 130/0.4 groups, range 10 to 90 patients); of short duration (median study period 12 hours, range 0.5 to 144 hours) and with low cumulative HES doses (median 2,465 ml, range 328 to 6,229 ml). Sepsis studies (n = 2) included 18 patients (median, range 10 to 26 patients), study period was 96 hours (median, range 72 to 120 hours) and total fl uid volumes was 3,000 ml in one study. Sixty percent of control fl uids were synthetic colloids (other starches, gelatins, or dextran) that carry a similar risk profi le. Primary endpoints with power calculation (in 87% of studies) were mostly unspecifi c or clinically irrelevant. Only one sepsis study provided a primary endpoint, which was extravascular lung water. This did not diff er in comparison with the albumin 20% control group. Conclusions There is no reliable evidence from published clinical data that third-generation HES 130/0.4 is safe in septic patients or in the emergency or elective surgical setting.

Introduction
The triad interaction of neonatal-maternal-bacterial determinants plays a crucial role in the increased incidence of bacterial sepsis during the neonatal period. This study was undertaken to determine whether neonatal-maternal predisposing factors and bacterial pathogens aff ect the risk of early or late onset sepsis. Methods Three hundred neonates in the NICU of two hospitals in Tehran were studied. Blood cultures from neonates with suspected sepsis were performed on BHI broth followed by identifi cation of isolates and testing for their susceptibility to antimicrobial agents. Collectively, neonatal and maternal risk factors such as birth weight, gestation age, PROM, Apgar score, and others were studied in the cultures of proven cases of neonatal sepsis. In univariate binary logistic regression models, the impact of neonatal and maternal factors on sepsis risk was estimated in terms of odds ratio (OR) with 95% confi dence interval (CI). Results The present study revealed the impact of bacterial pathogens and neonatal and maternal predisposing factors on sepsis as follows. Bacterial pathogens: 14/300 (4.7%) of neonates developed septicemia. Among infected neonates, 64.3% and 35.7% were considered with early-onset and late-onset sepsis, respectively. The most isolated Gram-negative organism was Stenotrophomonas maltophilia (42.8%) followed by Klebsiella pneumonia (28.6%), Escherichia coli (21.4%) and Serratia liquefaciens (7.2%). Neonatal factors: the mean age of neonates ± SD with early-onset sepsis (1.56 ± 0.88) was lower than that of those with late-onset sepsis (10.40 ± 5.50) and this diff erence was statistically signifi cant (P <0.05). Low birth weight (LBW) <2,500 g increased the risk of sepsis to more than twofold (OR = 2.9, 95% CI = 1.17 to 9.86; P <0.01). Gestation age (GA) <29 weeks was signifi cantly associated with sepsis (P <0.01). The septicemia, in turn, increased the risk of death up to more than fi vefold (OR = 5.5; 95% CI = 1.98 to 15.3; P <0.01). More than one-half of septic neonates had positive result for CRP whereas only 1.9% of neonates with sepsis were CRP-negative, and this diff erence was statistically signifi cant (P <0.001). Maternal factors: PROM aff ected the sepsis risk to more than threefold (OR = 3.8; 95% CI = 1.37 to 10.56; P <0.05). Conclusions The present study reveals that specifi c neonatal and maternal factors are associated with increased risk of sepsis. Among the studied factors, prematurity of neonates explained as GA and LBW are the most important contributors to morbidity in neonate who suff ered from sepsis. Furthermore, PROM as a maternal risk factor predisposes a child to neonatal sepsis.
Introduction Mortality from sepsis is greater in the elderly than in the young although incidence only increases slightly. Pulmonary infections caused by Staphylococcus aureus that progress into sepsis are a major cause of death in elderly patients. Bacterial pneumonia is a common precipitating cause of sepsis. Gram-positive bacteria are increasing as causative agents of pneumonia in the elderly. Objective To investigate age-dependent changes in the intrapulmonary response to staphylococcal challenge. Methods Cell wall components lipoteichoic acid (LTA, 200 ng) and peptidoglycan (PGN, 660 ng) from S. aureus were instilled intratracheally to young (3 to 4 months) and old (>18 months) C57Bl6 mice (n >5/group). Controls received saline alone. After 6 hours, mice were euthanized by exsanguination, and blood saved for analysis. One-sided lavage was performed, the nonlavaged lung tissue collected and total RNA isolated. Results Using this relatively mild challenge of LTA and PGN, we observed signifi cant and age-dependent diff erences in the infl ammatory response. Macrophage migration inhibitory factor (MIF) protein was signifi cantly and agedependently increased in BAL and plasma. A trend toward lower levels of total cells and neutrophils in the lung was noted in the old following stimulation, although the variation of the response was large. The dynamics of MIF at a transcriptional level in the lung was age-dependently altered, with a marked downregulation in the young mice after stimulation, whereas levels of MIF mRNA remained unchanged in the old mice. Transcriptional changes were also noted for the anti-infl ammatory cytokine IL-10 and other mediators involved in lymphocyte, macrophage and neutrophil recruitment. Interestingly, explanted lung cells from young and old mice showed a similar expressional pattern, with atypical expression levels in cells originating from old mice lungs. Conclusions The fi ndings support a hyperinfl ammatory response in the older individual at the measured time point. Interestingly, the diff erences were sustained in vitro in cells explanted from young and old mice. Together the data suggest an altered infl ammatory response to infectious challenge of the aged lung. Explanted cells from old animals may be a valuable tool in determining age-dependent diff erences in infl ammatory response and identifying novel targets for intervention.
Introduction Sepsis is a complex immunological response to infection characterized by a sinusoidal pattern that represents early hyperinfl ammatory Critical Care 2010, Volume 14 Suppl 2 http://ccforum.com/supplements/14/S2 S3 signals [1] followed by severe and protracted immunosuppression, suggesting that a multimarker approach has the greatest clinical utility in early detection within a clinical environment focused on SIRS diff erentiation. Preclinical research using an equine endotoxemia model identifi ed a panel of gene expression biomarkers that defi ne the aberrant immune activity during early sepsis. Thus, the primary objective was to apply these gene expression biomarkers to distinguish patients with sepsis from those who had undergone major open surgery and had clinical outcomes consistent with systemic infl ammation due to physical trauma and wound healing. Methods This was a multicenter, prospective clinical trial conducted across four tertiary critical care settings in Australia. Sepsis patients were recruited if they met the 1992 Consensus Statement [2] and had clinical evidence of systemic infection based on microbiology diagnoses (n = 27). Participants in the post-surgical (PS) group were recruited preoperatively and blood samples collected within 24 hours following surgery (n = 36). Healthy controls (HC) included hospital staff with no known concurrent illnesses (n = 19). Each participant had minimally 5 ml PAXgene blood collected for RNA isolation and gene expression analyses. Aff ymetrix Exon array and multiplex tandem (MT)-PCR studies were conducted to evaluate gene expression using a set of molecular markers that had been identifi ed a priori. A LogitBoost algorithm was used to create a machine-learning diagnostic rule in which to predict sepsis outcomes. Results Based on preliminary exon array analyses comparing HC and sepsis groups, a panel of 42 gene expression markers was identifi ed that linked to key innate immunity, cell cycle, endothelial, coagulation, and apoptotic pathways. When sepsis and PS groups were combined, the test had an ROC area >95%. Using subsets of these biomarkers in the MT-PCR assay, the ROC AUC for sepsis prediction was between 85 and 90%. Conclusions This novel molecular biomarker test has a clinically relevant sensitivity and specifi city profi le, and has the capacity for early detection of sepsis via the monitoring of critical care patients.
Introduction Nitric oxide (NO) plays a key role in innate immune system controlling microbial infection; however, during septic shock its exacerbate formation is associated with several deleterious complications. Cholecystokinin (CCK) was fi rst described as a gastrointestinal hormone, but immune cells express their receptor, suggesting a possible involvement of this hormone in modulation of infl ammatory response. Our aim was to evaluate the role of CCK on NO production during endotoxemia in rats as well as lipopolysaccharide (LPS)-stimulated macrophages. Methods Male Wistar rats received an intravenous injection of CCK (0.4 and 40 μg/kg) 10 minutes before LPS (1.5 mg/kg) administration. The mean arterial pressure was monitored during 6 hours after endotoxin injection. Blood was collected for plasma nitrate level and vasopressin measurement at 2, 4 and 6 hours after LPS. Thioglicollate-elicited macrophages were obtained by peritoneal lavage and cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum and antibiotics. Macrophage culture was treated with CCK (10 -14 , 10 -12 , 10 -10 , 10 -8 , 10 -6 M) 30 minutes before LPS stimulation (1 μg/ml) and supernatant nitrite concentration was determined at 6, 24 and 48 hours. The iNOS expression was evaluated by quantitative real-time PCR and the amount of gene transcription was measured using the delta-delta method. The presence of iNOS was analyzed by indirect immunofl uorescence at 12 and 24 hours after LPS incubation.

Results
The LPS-induced hypotension was reverted by the pretreatment with CCK only at the lower dose. Moreover, CCK increased vasopressin levels at 2 and 4 hours after LPS administration and reduced nitrate levels during 2 and 6 hours. LPS-stimulated macrophages increased rapidly nitrite levels in supernatant and also iNOS expression. The pretreatment with CCK at all tested concentrations signifi cantly reduced nitrite levels at 6, 24 and 48 hours after LPS stimulation when compared with the LPS group (P <0.05). The iNOS/GAPDH expression ratio were also lower in CCK-treated cells at 6 and 24 hours (P <0.001). The qualitative analysis of iNOS protein was assessed at 12 and 24 hours after LPS stimulus by immunocytochemistry. In CCK-treated macrophages, a reduction of fl uorescence emission in comparison with the LPS group was observed. In control groups (without LPS), fl uorescence was not observed, suggesting the absence of iNOS protein in non-infl ammatory conditions. Conclusions These data suggest that CCK restores hypotension and reduces NO formation during endotoxemia in rats. Furthermore, CCK regulates negatively iNOS expression and also NO synthesis in LPS-activated peritoneal macrophage culture.

P11
Sepsis induces platelet mitochondrial uncoupling and a gradual increase in respiratory capacity that is negatively associated with clinical outcome F Sjövall 1,2 , S Morota 1 , MJ Hansson 1,3 , H Friberg 4 , E Gnaiger 5 , E Elmér 1,6 Introduction Mitochondrial dysfunction has been suggested as a contributing factor in the pathogenesis of multiple organ dysfunction syndrome (MODS) and sepsis is the leading cause of MODS. Also, restoration of mitochondrial function, known as mitochondrial biogenesis, has been implicated as a key factor for the recovery of organ function in patients with sepsis. Here we investigated platelet mitochondrial respiratory function in patients with sepsis during the fi rst week after disease onset. Methods Platelets were isolated from blood samples taken from 18 patients with severe sepsis or septic shock within 48 hours of their admission to the intensive care unit. Subsequent samples were taken on days 3 to 4 and days 6 to 7. Eighteen healthy blood donors served as controls. Platelet mitochondrial function was determined by high-resolution respirometry. Endogenous respiration of intact platelets suspended in their own plasma or PBS glucose was determined and, in order to investigate the activity of individual complexes of the respiratory system, platelets were permeabilized with digitonin and stimulated with complex-specifi c substrates and inhibitors. Results There was a signifi cant increase in maximal respiratory capacity of platelets from days 1 to 2 to days 6 to 7 as well as compared with controls in both intact platelets and permeabilized platelets oxidizing complex I and/or II linked substrates. Platelets suspended in their own septic plasma exhibited increased leak respiration compared with platelets suspended in PBS glucose and to controls. No inhibition of respiration was detected in septic patients compared with controls. Mortality at 90 days was 33% (6/18). Nonsurvivors had a signifi cantly more elevated respiratory capacity at days 6 to 7 as compared with survivors. No correlation between respiratory capacity and severity of disease as measured by APACHE II, SAPS II, SOFA or noradrenaline dose were found. Platelet content of mitochondria-specifi c cytochrome c increased signifi cantly, but no change in mitochondrial DNA was detected over the time interval studied.

Conclusions
The results indicate the presence of a soluble plasma factor in the initial stage of sepsis inducing uncoupling of platelet mitochondria but not inhibition of oxidative phosphorylation. Further, the mitochondrial uncoupling was paralleled by a gradual and substantial increase in respiratory capacity that may refl ect mitochondrial biogenesis as a response to severe sepsis or septic shock. The enhanced respiratory capacity developing over the fi rst week seems to refl ect the severity of the condition and may be used as a prognostic marker of mortality.

Introduction
The endotoxin activity assay (EAA) is a rapid whole-blood chemiluminescent test for endotoxin that has proven clinical utility in the detection and risk stratifi cation of clinically ill patients with suspicion of sepsis. Methods The EAA was studied in a cohort of 153 septic patients admitted to the ICU. At the same time, IL-6 (chemiluminescent enzyme immunoassay), C-reactive protein (CRP), procalcitonin (PCT, chemiluminescent enzyme immunoassay) and plasminogen activator inhibitor-1 (PAI-1, latex photometric immunoassay) were measured within 24 hours after ICU admission. The patients were divided into the following three groups: L group: EAA <0.4, M group: 0.4 ≤EAA <0.6, H group: 0.6 ≤EAA. Nonrepeatedmeasures ANOVA was used to compare over three groups or conditions. Statistical signifi cance was assumed for values of P <0.05. Normally distributed data are presented as mean ± SD, and abnormally distributed data are presented as median values. Results Of the 153 patients, the L group contained 61 patients, M group 41 patients, and H group 51 patients, respectively. On the day of ICU admission, the rate of EAA ≥0.4 was 60.1% (MEDIC study: 57.2%). APACHE score in the L group was 21.0 ± 7.9, M group 24.8 ± 8.4, H group 26.4 ± 8.9, and SOFA score in the L group was 8.2 ± 4.3, M group 8.9 ± 4.1, H group 9.5 ± 4.3, respectively. There was no statistically signifi cant diff erence among the groups. The median value of PCT in the L group was 1.1 ng/ml, M group 5.9 ng/ml, H group 8.5 ng/ml, respectively. PCT values of the M and H groups were signifi cantly higher than those of the L group. Median IL-6 level of the H group was signifi cantly higher than that of the L group (H group: 2,635 pg/ ml, L group: 177 pg/ml). Conclusions EAA has no signifi cant correlation with other sepsis-related markers, but may be associated with body insults (infl ammation or infection).

P13
A fast and accurate diagnostic test for severe sepsis using model-based insulin sensitivity and clinical data JD Parente 1 , D Lee 2 , J Lin 3 , JG Chase 1 , GM Shaw 4 Introduction Severe sepsis occurs frequently in the ICU and is a leading cause of admission, mortality, and cost. Management guidelines defi ne treatment objectives within the fi rst 6 hours of clinical syndrome presentation. However, blood culture test confi rmation may return in up to 48 hours, with only 30 to 50% of presentations having positive blood cultures. Early treatment compliance has demonstrated a decrease in sepsis mortality. Thus, there remains a serious need for an early and accurate diagnostic test for severe sepsis. Insulin sensitivity (SI) is known to decrease with worsening condition and infl ammatory response, and could thus be used to aid clinical treatment decisions. Some glucose control protocols are able to accurately identify SI in real time, without high rates of hypoglycemia [1]. This research explores the diagnostic test properties of a real-time test for severe sepsis. Methods A diagnostic biomarker for severe sepsis was developed from retrospective SI and concurrent temperature, heart rate, respiratory rate, blood pressure, and SIRS score from 36 adult patients with sepsis. Patients were identifi ed as having severe sepsis based on a clinically validated sepsis score (ss). Kernel density estimates were used for the development of joint probability density profi les for ss ≥2 and ss <2 data hours (213 and 5,858, respectively, of 6,071 total hours) and for classifi cation. From the receiver operator characteristic (ROC) curve, the optimal probability cutoff values for classifi cation were determined, as well as AUC, positive and negative likelihood ratios (LHR), predictive values, and diagnostic odds ratios (DOR) for in-sample and out-of-sample estimates, respectively.

Results
A biomarker including concurrent insulin sensitivity and clinical data for real-time diagnosis of severe sepsis (ss ≥2) achieves 69 to 94% sensitivity, 75 to 94% specifi city, 0.78 to 0.99 AUC, 3 to 17 LHR+, 0.06 to 0.4 LHR-, 9 to 38% PPV, 99 to 100% NPV, and 7 to 260 DOR for optimal probability cutoff values of 0.32 and 0.27 for in-sample and out-of-sample data, respectively. The overall result lies between these minimum and maximum error bounds. See Figure 1. Conclusions The clinical biomarker shows good to high accuracy and may provide useful information as an early real-time diagnostic test for severe sepsis.  Results According to our results, PCT levels in all patients with bacteremia caused by Gram-negative bacteria (75/75) were >2 ng/ml. In more details in 41 patients with Gram-negative bacteremia (54.7%) the PCT levels were 2 to 10 ng/ml and in 34 patients (45.3%) were >10 ng/ml while in patients with CNS bacteremia the PCT levels were >2 ng/ml only in 14% (6/56). In addition, in all patients with bacteremia caused by S. aureus the PCT levels were >2 ng/ ml and by Streptococcus spp., C. perfrigens, and C. acnes the PCT levels were 2 to 10 ng/ml. Conclusions PCT levels were markedly higher in patients with bacteremia associated with Gram-negative bacteria than in those with Gram-positive bacteremia, especially caused by CNS. Future research is needed to confi rm our results. stimulates the immune and endocrine systems, inducing acute phase of sickness and stress responses. Neonatal LPS exposure has been shown to alter many aspects of adult physiology, including neuroendocrine, neurochemical, and febrile responses. The aim of this study was to evaluate the eff ects of neonatal immune challenge on adults during septic shocklike condition assessing mean arterial pressure and heart rate, plasma vasopressin (AVP) concentration, body temperature (Tb), and macrophage nitric oxide (NO) synthesis. Male Wistar rats were exposed to LPS (100 μg/ kg i.p.; nLPS) or saline administration (nSal) 14 days after birth (P14). On day 50 after birth, endotoxemic shock was induced by intraperitoneal injection of 10 mg/kg LPS, on rats previously implanted with polyethylene catheters in the femoral artery and loggers for Tb measurements. A diff erent set of animals was used to assess the eff ect of neonatal LPS exposure on NO synthesis by peritoneal macrophage in vitro, with (1 μg/ml) or without LPS, added to the culture. In nSal rats, LPS injection induced a transitory increase in AVP plasma concentration, a decrease in mean arterial pressure with a concomitant increase in heart rate, which were statistically signifi cant from 1 hour (P <0.01) up to 6 hours (P <0.001) after treatment. LPS-induced hypothermia (P <0.05) was observed for 2 hours after LPS administration, and was followed by an increased Tb (P <0.01). We also observed a signifi cant increase in nitrate plasma concentration as well as in macrophage culture medium after LPS stimulation. In nLPS rats we observed an attenuation to the development of hypotension, no signifi cant change in heart rate (P <0.05), an increased hypothermia, and a decreased febrile response, and further increased (P <0.01) AVP plasma levels were observed, in response to LPS administration. Interestingly, nitrite released in the culture medium was attenuated in nLPS animals. Neonatal exposure to LPS induces attenuation in hypotension during septic shock-like conditions and this response may involve an increased AVP release.

P16
Neonatal LPS exposure reduces stress fever in adult rats: modulation by glucocorticoids and PGE 2 LGS Immune challenges during the neonatal period may permanently program immune responses later in life, including endotoxin fever. We tested the hypo thesis that neonatal endotoxin exposure aff ects stress fever in adult rats. In control rats (treated with saline as neonates; nSal) body temperature peaked ~1.5°C during open-fi eld stress, whereas in rats exposed to endotoxin (lipopolysaccharide, LPS) as neonates (nLPS) stress fever was signifi cantly attenuated. Following stress, plasma corticosterone levels signifi cantly increased from 74.29 ± 7.05 ng/ml to 226.29 ± 9.87 ng/ml in nSal rats, and from 83.43 ± 10.31 ng/ml to 324.7 ± 36.87 ng/ml in nLPS rats. Animals treated with LPS as neonates and adrenalectomized 1 week before experimentation no longer displayed the attenuated febrile response to stress. This attenuated stress fever caused by an increased corticosterone secretion is likely to be linked to an inhibitory eff ect of glucocorticoids on cyclooxygenase activity/ PGE 2 production in the preoptic/anteroventral third ventricular region (AV3V) since stress failed to cause a signifi cant increase in PGE 2 in nLPS rats, and this eff ect was reverted by adrenalectomy. Altogether, the present results indicate that endogenous glucocorticoids are key modulators of the attenuated stress fever in adults rats treated with LPS as neonates, and they act downregulating PGE 2 production. Moreover, our fi ndings also support the notion that neonatal immune stimulus aff ects programming of stress responses during adulthood, despite the fact that infl ammation and stress are two distinct processes mediated largely by diff erent neurobiological mechanisms.
Introduction Alcohol consumption is a signifi cant risk factor for mortality in patients with sepsis. Alcohol is the most widely abused substance worldwide and numerous studies have revealed that it has widespread eff ects on the immune system and leaves abusers at increased risk of a variety of infections. An increased predisposition to infection among patients with alcohol use problems may also mediate an association with sepsis.
Objective The present study was carried out to investigate the mechanisms by which acute ethanol exposure alters the course of sepsis and the eff ect of TLR4 signaling. Methods Two diff erent strains of mice, C3H/HeJ (TLR4-mutants) and C3H/ HeOuJ (wildtype), were treated with a dosage of 6 g/kg ethanol, which yields a blood-ethanol concentration of ~0.4%, similar to the blood-ethanol levels that occur in ethanol-dependent humans. Viable, indigenous Escherichia coli, log-phase, grown in LB broth was administered intraperitoneally. The dosage of E. coli was 2 × 10 8 per mouse, which serves as a model for loss of intestinal integrity and release of bacteria in large numbers. Blood samples were obtained retro-orbitally while the animal was under halothane anesthesia. After euthanasia, peritoneal lavage was performed and samples of this fl uid were used to quantify bacteria by making serial dilutions in LB agar, and for cell-counting, for cytospin and cytokine and chemokine study. Spleen was also harvested from all the mice for carrying out bacterial quantifi cation, RNA analysis, and fl ow-cytometry analysis.
Results Ethanol administration decreases resistance to E. coli and causes a decrease in the ability to clear bacteria both from the peritoneal cavity as well as the spleen. At early time points, ethanol also suppresses the production of proinfl ammatory cytokines (for example, IL-1, IL-17, IFNγ, TNFα, and so forth) and chemokines (for example, Eotaxin, RANTES, MIP-1, MIG, LIX, and so forth). Most (80 to 90%) of the cells in the peritoneal cavity were found to be macrophages (full of bacteria) and hardly any neutrophils could be found. See Figure 1.
Conclusions Ethanol decreases clearance of bacteria in the peritoneal cavity and increases mortality. Ethanol also decreases production of most proinfl ammatory cytokines and chemokines. A large number of macrophages in the peritoneal fl uid indicates decreased attraction of neutrophils to the peritoneal cavity, decreased clearance of bacteria by macrophages and neutrophils in the peritoneal cavity, and, hence, increased mortality. TLR4 is dispensable for survival in E. coli sepsis but it also contributes to lethality in wildtype mice. Although TLRs have been implicated as an important element of host defense against infections, evidence indicates that these receptors may also play a crucial role in the pathophysiology of sepsis.
Introduction Sepsis hyperglycemia is poorly understood. It is not known whether there is a role in sepsis hyperglycemia for glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), crucial for normal glucose metabolism. We developed an in vitro model of sepsis employing monocytes (crucial cells in mediating sepsis) and hepatocytes (crucial cells in carbohydrate homeostasis) to clarify the role of the incretin system in sepsis.
Objective To establish an in vitro model of sepsis employing monocytic (U937) and hepatocytic (HUH7) cell lines by co-incubation with lipopolysaccharide (LPS) and to determine whether receptor expression for GIP, GLP-1, and insulin (INS) was altered.
Methods U937 (monocyte cell line) and HUH7 (hepatocyte cell line) cells were cultured with diff erent concentrations of LPS for 24 hours. Real-time RT-PCR quantitation of gene expression was used to compare the rates for relative expression. Results U937 and HUH7 cells expressed mRNA GIPR (including GIPR protein expression in HUH7 cells), and INSR, but only HUH7 expressed GLP-1R. There was an inverse relationship between the LPS dose and mRNA expression for GIPR (P <0.05). For example at 5 μg/ml LPS, the expression of GIPR was reduced to 86% and INSR 72% of control in U937: while in HUH7 cells at 1 μg/ml LPS, the GIPR expression was decreased to 63%, GLP-1R 95% and INSR 89% compared with control (P <0.001). A direct signifi cant relationship between LPS and infl ammatory cytokines IL-1 (P <0.05) and IL-6 (P <0.05) in both cell lines validated our model.

Conclusions
We not only show for the fi rst time GIPR mRNA expression on U937 cells and expression of GIPR and GLP-1R on hepatocyte cell line, but also their downregulation with LPS. The LPS-mediated alteration in incretin receptor expression on these cell lines may be relevant to changes in cytokine secretion and carbohydrate metabolism in sepsis.

P19
The new sepsis marker, sCD14-ST, induction mechanism in the rabbit sepsis models K Shirakawa, K Naitou, J Hirose, M Nakamura, T Takeuchi is a fragment of CD14 and is markedly increased in sepsis patients. We developed a new immunoassay to detect sCD14-ST and evaluated the effi cacy of this marker for diagnosis of sepsis. For developing the strategies of sCD14-ST as a sepsis diagnostic marker, the induction mechanism must be known. Methods To determine the kinetics of sCD14-ST in the rabbit endotoxin shock model and the cecal ligation and puncture (CLP) model, we prepared the rabbit sCD14-ST immunoassay. Induction by infl ammatory inducers and inhibition of sCD14-ST production were assessed using rabbit abdominal cavity granulocytes. Fragmentation of CD14 by N-aspartic protease was analyzed by western blot analysis and immunoassay. Results sCD14-ST was induced in the CLP model. However, sCD14-ST was not induced in the endotoxin shock model. These results suggested that sCD14-ST was not induced after stimulation by physiologic activating agent but induced by bacterial infection. sCD14-ST was not induced after stimulation of rabbit granulocytes by LPS, IFNγ, FMLP, and PMA. In contrast, it was induced by adding Escherichia coli, indicating that sCD14-ST is produced by phagocytosis rather than infl ammation. The phagocytosis inhibitors cytochalasin D and wortomanin inhibited the production of sCD14-ST in vitro. Additionally, N-asparagin protease inhibitor inhibited the production of sCD14-ST from granulocytes. Additionally sCD14-ST was detected from recombinant CD14 digested supernatant by cathepsin D enzyme.
Conclusions These data suggested that induction mechanism of sCD14-ST is dependent on the phagocytosis and cathepsin D is one of the enzymes for fragmentation of CD14. This mechanism is strong evidence for explanation of the production of sCD14-ST in sepsis patients. Introduction There is evidence that early delivery of antibiotics following the recognition of severe sepsis leads to decreased morbidity and indeed mortality. It is estimated that 36,800 people die annually in the UK as a result of severe sepsis, claiming more lives than bowel and breast cancer combined [1]. Patients admitted to ICUs with severe sepsis have a 39.8% risk of death [2], and for each hour delay in antibiotic administration, a 7.6% increase in mortality [3]. The Surviving Sepsis Campaign 2008 recommends that appropriate antimicrobial therapy be administered within 1 hour following recognition of severe sepsis [4].

Impact of delayed antimicrobial therapy in septic ITU patients
Methods We conducted a prospective audit of consecutive patients with severe sepsis admitted to an ITU between February and June 2010. The patients were identifi ed as those who fulfi lled two or more components of the systemic infl ammatory response syndrome (SIRS) criteria, and had evidence of organ dysfunction requiring critical care. Compliance to the Surviving Sepsis Campaign's antibiotic care bundle was audited. The relationship between time of antibiotic administration and mortality was also determined.
Results During the study period, 33 patients out of 187 admissions met the inclusion criteria. The population demographics are illustrated in Table 1. The mean time from fulfi lling SIRS criteria to delivery of antibiotics was 4.32 hours. Only eight (25%) of the patients received antibiotics within the hour, with the mortality rate for this group being 25%. Those patients who received antibiotics after 4 hours had a lower mortality rate than the group that received antibiotics after 12 hours (67% vs. 80%). See Figure 1.
Conclusions Our results support published evidence that a delay in antibiotic delivery greater than 1 hour is associated with increased mortality in patients treated in the ITU. As a result of this study we have developed a standardized sepsis protocol to integrate into the AE triage pro forma, as well as a pathway to help instigate treatment earlier to those patients identifi ed as septic on the wards. Recruitment period has not concluded. More data analysis will be presented later.
Introduction Acute lung injury (ALI) caused by smoke inhalation with or without bacterial pneumonia remains a signifi cant cause of morbidity and mortality among burn patients. Bacterial pneumonia in an ALI patient is particularly worrisome because it often leads to sepsis. Although much of the literature surrounding ALI and pneumonia-induced sepsis has rightfully focused on pulmonary and endothelial changes, a major consequence of ALI and an area of continued research and drug development is coagulopathy. The objective, therefore, of our study was to determine whether coagulopathy diff ers between types of ALI and whether the dysregulation can be estimated using a disease progression model. Methods Nineteen sheep with acute lung injury were incorporated into this pneumonia-sepsis model. Pneumonia was induced by inoculating the airway with ~2.5 × 10 11 colony-forming units (CFUs) methicillin-resistant Staphylococcus aureus (MRSA), while smoke injury was created through inhalation of cotton smoke. The injury groups studied were as follows; MRSA and smoke inhalation (M+S), MRSA untreated (M), MRSA treated (M+T), and smoke inhalation only (S). Data were modeled over 24 hours. First, all the sheep were modeled together to determine a rank-order of the injury groups. After rank-ordering the groups, the groups became model inputs and in conjunction with other clinical and laboratory variables were used to estimate the output parameter, prothrombin time (PT). In order to minimize overparameterization of a small patient population, the model was allowed to estimate PT using only two parameters.

Results
The number of sheep in each group was as follows; seven M+S, three M, three M+T, and six S. The rank-order of injury from least to greatest severity was M+T, S, M, M+S. The two highest-ranking parameters in estimating PT were calcium and injury. When using calcium and injury alone, the model estimate agreement with measured PT was r 2 = 0.70 and r 2 = 0.36, respectively. Allowing the model to combine the inputs did not improve the model estimate (r 2 = 0.70) compared with when calcium was used alone. Conclusions The progression model allowed all individual sheep to be characterized as to the severity of resulting coagulopathy and identifi ed some important co-factors. Acute lung injury can lead to systemic coagulopathy even without MRSA infection, but the extent and severity is greater with infection.

P22
Overexpression of PD- Introduction Septic syndromes are a culmination of multiple partially under stood dynamic processes. However, it is now established that, after a transient exacerbated proinfl ammatory response, a counter-regulatory phase develops, rapidly inducing immune alterations that are thought to play a major role in patients' mortality and susceptibility to nosocomial infections. Programmed Death-1 (PD-1) receptor and its ligands PD-L1 and PD-L2 constitute a newly described pathway that negatively controls immune responses. Recently, improved bacterial clearance and decreased mortality were observed in PD-1 knockout mice [1]. The objective of the present study was to investigate PD-1-related molecule expressions in septic shock patients.
Methods PD-1-related molecule expressions were measured by fl ow cytometry on circulating leukocytes from 64 septic shock patients and 49 healthy individuals. Severity scores (SAPS II, SOFA), clinical events (28-day mortality, occurrence of nosocomial infections) and the usual biomarkers of sepsis-induced immunosuppression (monocyte HLA-DR expression, lymphocyte phenotyping including Treg, plasmatic IL-10 concentration) were assessed. Ex vivo functional assays such as lymphocyte proliferation ([ 3 H] thymidine incorporation) in response to phytohemagglutinin, and cytokine release (TNFα and IL-10 assessed by Bio-Plex technique) after overnight LPS incubation, were performed in the presence of blocking antibodies against PD-1-related molecules.
Results Patients presented with typical features of sepsis-induced immunosuppression (decreased mHLA-DR expression, increased Treg percentage, decreased LPS-induced TNFα release). At days 1 to 2 and days 3 to 5 after the onset of shock, patients displayed increased PD-1 and PD-L1 expressions on CD4 + T lymphocytes and enhanced PD-1, PD-L1 and PD-L2 expressions on monocytes. See Figure 1 overleaf. Nonsurvivors presented with increased mono cyte PD-L1 expression while enhanced monocyte PD-1 or PD-L2 expressions were associated with the occurrence of secondary nosocomial infections. In addition, decreased mitogen-induced lymphocyte proliferation was negatively correlated with increased lymphocyte PD-1 and PD-L1 expressions whereas monocyte PD-1-related molecule expressions were highly correlated with increased circulating IL-10 concentration. No benefi cial eff ects of anti-PD-1-related molecule antibodies were observed. Conclusions We describe here for the fi rst time the overexpression of PD-1-related molecules on circulating leukocytes in septic shock patients. Importantly, these increased expressions were signifi cantly associated with the occurrence of immune dysfunctions, secondary nosocomial infection, and death after septic shock. Taken together, our results suggest that PD-1related molecules may constitute an additional regulatory system involved Introduction The severe form of sepsis is associated with multiple organ dysfunction syndrome (MODS), but the precise mechanisms by which MODS develops remain unclear. Neutrophils are essential cellular components of the innate immune system that have conserved roles in bacterial containment. Paradoxically, however, neutrophils also mediate tissue injury in varied human diseases, including sepsis.

Objective
In the present study, we investigated the role of chemokine receptor CCR2 in driving neutrophil infi ltration and eliciting tissue damage in remote organs during sepsis.

Methods and results
We demonstrated that neutrophils, which are normally unresponsive to CCR2 chemokines, acquired substantial chemotaxis to CCL2 and CCL7 when exposed to LTA (4.33-fold and 3.02-fold increase, respectively) or LPS (4.67-fold and 3.29-fold increase, respectively). Moreover, consistent with the functional response, we found that TLR2 and TLR4 signaling through the MyD88/NF-κB pathway mediates the upregulation of CCR2 and chemotactic responsiveness to CCR2 ligands on neutrophils.
In vivo, intravenous injection of TLR ligands or induction of cecal ligation and puncture (CLP)-induced sepsis triggered chemotaxis of circulating neutrophils to CCR2 chemokines, which was completely abolished in MyD88defi cient mice. Notably, CCR2-defi cient (CCR2 -/-) or WT mice treated with CCR2 antagonist (RS504393, 2 mg/kg) showed a signifi cant increased survival rate after CLP when compared with WT mice. Defi ciency or pharmacology blockade of CCR2 attenuated neutrophil infi ltration (by myeloperoxidase activity) into the lungs, heart, and kidneys, which was associated with reduction of serum biochemical markers of organ injury/dysfunction. Importantly, neutrophils from septic patients (n = 19, prospected in survivors (S) and nonsurvivors (NS)) showed an increase of median of fl uorescence intensity (MFI) of CCR2 by fl ow cytometry (S = 5.76 ± 2.30 vs. NS = 9.12 ± 1.72, MFI), which was related to the chemotactic response to CCL2 (S = 6.35 ± 0.68 vs. NS = 10.56 ± 2.38, neutrophils/fi eld). Furthermore, there was a positive correlation between SOFA scores with the neutrophil response to CCL2 (r 2 = 0.62, P <0.01). Introduction The prognostic capability of TNFα and IL-6 is limited in septic shock. Previous studies were performed prior to publication of current therapeutic guidelines recommending aggressive early resuscitation. The objective of the present study was to evaluate the impact of early fl uid resuscitation on serial TNFα and IL-6 levels and its association with mortality in severe sepsis. Methods This is a substudy of a previously completed prospective, observational multicenter investigation of patients with severe sepsis. Inclusion criteria were age >17, infection with ≥2 SIRS, hypotension despite fl uid challenge, treatment with a standardized quantitative resuscitation protocol, and identifi cation within 3 hours of treatment initiation. Blood samples were obtained at enrollment, 6 hours, and 24 hours. Therapeutic amounts of intravenous crystalloid fl uid was defi ned by ≥5 l and <5 l over 24 hours (initial 2 l fl uid challenge over 4 hours followed by 150 ml/hour for 20 hours). Data analysis compared absolute levels of TNFα and IL-6 at each time point between survivors and nonsurvivors. The magnitude and direction of serial cytokine levels was quantifi ed by the percentage diff erence of each marker for each patient between 0 and 6 hours and 0 and 24 hours. Statistical analysis was performed using the Wilcoxon-rank-sum test or the Student t test.
Results Forty patients were enrolled; 11 died. Vasopressors were required in 60% of all patients. Absolute values of IL-6 (pg/ml) were higher in nonsurvivors than survivors at enrollment (5,479 vs. 710); 6 hours (4,180 vs. 405), and 24 hours (5,710 vs. 377) (P <0.05). There was no diff erence in TNFα values between the two groups (P = NS at 0, 6, 24 hours). Nonsurvivors had a larger percentage (diff erence) in both TNFα and IL-6 than survivors at 24 hours. See Figure 1. Treatment with ≥5 l intravenous fl uid over 24 hours was associated with a 32% decline in IL-6 compared with a 64% increase in IL-6 with <5 l fl uid therapy. See Figure 2.
Conclusions In the context of a quantitative protocol for the treatment of severe sepsis, high-volume fl uid resuscitation is associated with a decline in the percentage diff erence of IL-6. Trends in the percentage diff erence of both TNFα and IL-6 diff erentiate survivors from nonsurvivors. Further investigation is needed into the impact fl uid resuscitation has on decreasing the infl ammatory insult and the use of serial cytokine measurements as a measure of therapeutic eff ectiveness. Introduction Prognostic and severity-of-illness scoring systems are valuable tools for predicting mortality and choosing the site of care for patients with community-acquired pneumonia (CAP) [1]. Legionnaires' disease (LD) is a pneumonia caused by Legionella spp. and carries a higher mortality rate (5 to 30%) than CAP of most other etiologies. The aim of our study was to evaluate fi ve scoring systems commonly used in CAP for predicting mortality in patients with Legionella pneumophila serogroup 1 infection admitted during a large LD outbreak [2,3]. Methods Patients with microbiologically verifi ed LD (n = 103) and CAP patients with epidemiological association to the outbreak with no other bacteriological etiology identifi ed (n = 32) were included. A clinical protocol was initiated during an early phase of the outbreak, and clinical and biochemical data were collected from patients on admission to the regional hospital. The fi ve evaluated scoring systems were: pneumonia severity index (PSI), CURB-65 (confusion, uremia, respiratory rate ≥30, low blood pressure, age ≥65) and CRB-65 score, the modifi ed American Thoracic Society (ATS) score, and the IDSA/ATS guidelines. The endpoint was defi ned as 28-day mortality.   PPV, positive predictive value; NPV, negative predictive value.

Results
The overall mortality rate was 12% (16/135), and 19% (25/135) were admitted to the ICU. The discriminatory power was highest for PSI, CURB-65 and CRB-65 with area under the receiver operator characteristic curve (AUC) of 0.79, 0.78, and 0.75, respectively. The AUC of the modifi ed ATS score and IDSA/ATS guidelines were 0.61 and 0.69, respectively. Table 1 shows that a PSI class IV or V, and a CURB-65 and CRB-65 score ≥2 yielded the highest sensitivity for prediction of mortality, but the specifi city and positive predictive value was low. Conclusions The PSI, the CURB-65 and CRB-65 scores proved sensitive in predicting mortality in patients with Legionella pneumonia admitted during an LD outbreak, but the low specifi cities and positive predictive values necessitate thorough clinical judgment in patients with a high severity score. The modifi ed ATS score and IDSA/ATS guidelines, which are decision recommendations for ICU admission, were not sensitive in predicting mortality from LD in this study.  [1]. Olive oil is composed of diff erent polyunsaturated fatty acids such as omega 3 and 6, but the monounsaturated fatty acid omega 9, also known as oleic acid (OA), that is the main component of olive oil, is highly consumed in the Mediterranean diet.
Objective We aim to investigate the role of OA in an experimental model of sepsis.
Methods Swiss mice were given daily doses (orally) of OA, at 282 μg/animal, for 15 days. Control animals received saline. On the 16th day, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Immediately after the procedure, all mice received volemic reposition and after 6 hours animals were given imipenem. Twenty-four hours after surgery, mice were euthanized and the peritoneal cavity was rinsed with sterile saline. Total leukocyte counts were performed in a Neubauer chamber and diff erential leukocyte were stained with May-Grunwald Giemsa. The supernatant and plasma were collected for cytokine quantifi cation. In another set of experiments, the survival rate was determined daily for 7 days in separate groups of 10 animals for each condition. Results Mice fed with OA were resistant to sepsis, with a 64% survival rate at 168 hours compared with saline-treated mice (33%). OA supplementation in CLP-subject animals led to a signifi cant decrease in the total leukocyte counts (10.69 × 10 6 ± 1.71), mainly neutrophils, compared with mice that received saline (20.30 × 10 6 ± 2.69). However, in mice that consumed OA the levels of TNFα, IL-10 and IL-6 were not signifi cantly diff erent from mice fed with saline submitted to CLP. Interestingly, preliminary data showed that mice fed with OA had a lower level of bacteria in the peritoneal lavage leukocyte compared with mice submitted to CLP. See Figure 1.
Conclusions Our data suggest that treatment with OA reduces mortality in an experimental model of sepsis and attenuates infl ammation. One mechanism involved may be due to an increased bacterial clearance in mice fed with OA. More data are required to clarify this mechanism of increased survival.
Introduction Sepsis is a leading cause of death in critically ill patients and is characterized by a marked increase of the host proinfl ammatory cytokine release that is precipitated by infectious agents. The sepsis response to therapy has not appreciably improved. The procalcitonin (PCT) concentration is increased in the serum samples of septic patients and correlates with severity of the illness. LPS is a pivotal bacterial product involved in pathogenesis of sepsis and septic shock. Preventing the beginning of infl ammatory systemic cascade by means of LPS modulating agents might have a valuable eff ect in the control of such deadly illness. The aim of the present study was to evaluate the potential eff ect of procalcitonin on in vitro LPS-induced release of cytokines from human PBMC. Methods S. typhimurium LPS was preincubated with human PCT and then was added to freshly isolated human PBMC cultures in RPMI 1640. In such cultures the fi nal concentrations of LPS and PCT were 10 ng/ml and either 5,000 or 500 ng/ml respectively. A panel of cytokines was evaluated on culture supernatants by Biochip microarray (Randox) and PCT was tested by ELFA. Data analysis was carried out by a nonparametric method (Mann-Whitney U test, Graph Pad Prism version 4.03) to establish statistical diff erences between groups. Results Both of the PCT concentrations used signifi cantly (P <0.05 vs. LPSstimulated PCT-free controls) reduced TNFα (after 4-hour incubation with LPS) and MCP-1 (24 hours following LPS challenge). The lower concentration of PCT was also able to signifi cantly (P<0.05 vs. LPS-stimulated PCT-free controls) decrease the TNFα and IL-2 levels in the 24-hour samples.
Conclusions PCT, besides the well-known role as marker of sepsis, could be a potentially useful molecule to control systemic infl ammatory mediators in both sepsis and septic shock. Introduction Sepsis is a major cause of morbidity and mortality in intensive care clinics and the incidence is continuously increasing. Estimated mortality rates are over 30% for patients with severe sepsis and over 50% for patients with septic shock. Endotoxins, or lipopolysaccharides (LPSs), are a major component of the cell membrane of Gram-negative bacteria. LPS is well known to induce a strong response from the immune system leading to release of infl ammatory mediators and occasionally sepsis, or even septic shock. The aim of our study is to investigate the clinical eff ects of adding treatment with the Alteco® LPS Adsorber (Alteco Medical AB, Lund, Sweden) to standard therapy for patients with septic shock. Methods Our study included 12 patients with septic shock and endotoxemia randomized 1:1 to standard therapy plus LPS adsorption (adsorber group (AdG), n = 6) or standard therapy alone (reference group (RefG), n = 6). Randomization of patients will be performed using sealed envelopes.
This included fi ve women and seven men; mean age 47.3 ± 24.8 years. All patients needed inotropic support and mechanical lung ventilation. The mean APACHE II score at start of treatment was 26.6 ± 2.3. Both groups in the study received standard therapy (Surviving Sepsis Campaign International Guidelines 2008) for patients in septic shock. For patients in the study group, treatment with the Alteco® LPS Adsorber was added to standard therapy. The adsorber treatment was initiated immediately after inclusion in the study; that is, as soon as possible after onset of septic shock. The duration of Alteco® LPS Adsorber treatment was 120 minutes repeated twice within 24 hours. Hemofi ltration/hemoperfusion in the study group was not carried out during the perfusion. Samples for endotoxin and PCT analysis shall be taken from the arterial line. Hemodynamic parameters are registered according to routines at the clinic (PICCO technology).
Results Hemodynamic data, laboratory results, and blood gas analysis are summarized in Table 1. LPS was signifi cantly lower in all patients in the study group after treatment. No complications related to the use of the Alteco® LPS Adsorber were seen and all patients in this group were discharged from the ICU after 45 to 68 days, respectively. Conclusions We have received a statistically signifi cant improvement in hemodynamics, oxygenation, and reduced markers of endotoxemia in group therapy with Alteco® LPS Adsorber compared with traditional therapy. These eff ects were attributed with the removal of endotoxin from the systemic circulation. Only in one case using hemofi ltration for acute renal failure in the study group (in the reference group in all patients), 28-day mortality was 16.7% and 66.7% respectively. Negative eff ects were negligible.
Conclusions Critical sepsis is characterized by an excessive release of extracellular glucose, lactate, and glycerol, with the latter refl ecting probably increased lipolysis. These mirror the well-known sepsis-related blood metabolic alterations. Thus, chemical monitoring with subcutaneous MD is accurate in severely ill septic patients.
Introduction New endogenous antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffi n cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides.
Methods PMNs were treated with diff erent concentrations of CgAderived peptides in the presence of several drugs. Calcium mobilization was observed using fl ow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy were performed to analyze the intracellular localization of the peptides. The calmodulin-binding and iPLA2-activating properties of the peptides were shown by surface plasmon resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMN treatment with CgA-derived peptides was performed using HPLC and nano-LC MS-MS. Results Using fl ow cytometry we fi rst observed that after 15 seconds, in the presence of extracellular calcium, chromofungin (CHR) or catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in the absence of extracellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes of exposure. Treatment with 2-aminoethoxydiphenyl borate, a store-operated channel blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding Figure 1 (abstract P32). CHR and CAT induce store-operated channel (SOC) activation.
Introduction Septic shock is characterized by cardiac collapse and decreased peripheral resistance due to systemic resistance vessel dilatation, generally induced by large nitric oxide (NO). G-protein-coupled receptor (GPCR) kinases (GRKs), specifi c kinases interacting with GPCR proteins, induce receptor phosphorylation and thereby signal desensitization in the continuing agonist presence. Then, an increased expression of GRKs could augment adrenergic receptor desensitization and in turn reduce cardiovascular responses. Thus, we hypothesized that the hyporesponsiveness observed in sepsis could result from signal adrenergic receptor desensitization mediated by GRK2 via a NO-dependent mechanism. Methods C57Bl/6 mice were submitted to cecal ligation and puncture (CLP) surgery and sham-operated animals as controls. The cardiovascular responsive ness activity was evaluated in aorta rings or in cardiac ventricles. Aorta rings were contracted with phenylephrine (Phe; 1 μM), whereas ventricles were contracted with isoproterenol (Iso; 1 μM). The tissues responsiveness was evaluated 6, 12, and 24 hours after CLP surgery in the presence or absence of NO synthesis inhibitor (1400W; 100 μM; 30 min). GRK2 expression was analyzed on heart and aorta 6, 12, and 24 hours after CLP from sham, septic, and 1400W (1 mg/kg)-treated septic mice by immunofl uorescence analysis. The procedures have been approved by the Animal Use Ethics Committees of UFSC (PP003).

Results
The vascular responsiveness to vasoconstrictor Phe was signifi cantly reduced in aorta rings from septic mice evaluated 6 hours (55%), 12 hours (57%), and 24 hours (78%) after CLP. However, the 1400W incubation prevented this vascular hyporesponsiveness 6 and 12 hours after CLP. The cardiac responsive ness to Iso was signifi cantly reduced in ventricles from septic mice evaluated 12 hours (73%) and 24 hours (88%) after CLP. Conversely, the 1400W incubation prevented this cardiac hyporesponsiveness 12 hours after CLP. Moreover, high expression of GRK was detected in aorta 6 hours (65%), 12 hours (70%), and 24 hours (88%), and heart of septic mice 12 hours (52%) and 24 hours (63%) after CLP. The 1400W treatment reduced the GRK high expression on the aorta (75%) and heart (79%) of septic mice. Conclusions Our fi ndings identify that NO seems to activate GRK, which may induce adrenergic receptors' desensitization to agonists, contributing to severe cardiovascular hyporesponsiveness observed during septic shock. Therefore, the results suggest that GRK could be a new potential target to sepsis pharmacotherapy.  Introduction Older patients comprise an increasing proportion of ICU admissions. Advanced age and multiple co-morbidities compromise their immunity and hence they may be more prone to succumbing to severe infection and have poorer outcome.
Objective To assess the impact of severe sepsis on mortality in the old and very old subgroups of patients admitted to a medical ICU. Methods All patients admitted to a medical ICU of a tertiary care institute with severe sepsis or septic shock were prospectively included. Patients were divided into young (age below 60 years), old (age between 60 and 80 years), and very old (age above 80 years) groups. Data regarding baseline patient characteristics, admission APACHE II score, and ICU course including need for organ support and ICU length of stay were noted. Qualitative data were analyzed using the chi-squared test or Fisher exact test as appropriate and quantitative data were analyzed using Student's t test. Inter-group and intragroup comparison for quantitative data was done by one-way ANOVA. The primary outcome measure was ICU mortality.

Results
Of 387 patients who were admitted with signs of SIRS or sepsis during the study period of 20 months, 132 patients who fulfi lled the criteria for severe sepsis/septic shock were included in the analysis. The most common suspected site of infection was the lungs (60 patients, 45.5%), followed by the urinary tract (28 patients, 21.2%) and the abdomen (22 patients, 16.7%). ICU mortality in younger patients was 45.6% as compared with 60.7% in old patients and 78.9% in very old patients (P = 0.035). The odds ratio (OR) and relative risk (RR) for dying in the old age group was 1.32 (95% CI = 0.655 to 2.659) and 1.125, respectively, and OR and RR for dying was 3.313 (95% CI = 1.035 to 10.6) and 1.487 in the very old age group. There was an increased need for organ support in the old and very old population as compared with the younger population. See Table 1.

Conclusions
The risk of dying from severe sepsis is considerably higher in the old and very old subgroup of patients. Hence, early aggressive care to recognize and manage severe sepsis is required to improve outcome. Introduction Risk assessments of patients should be based on objective variables, such as biological markers that can be measured routinely. Such a prediction remains a diffi cult challenge. The acute response to stress causes the release of catecholamines from the chromaffi n cells of the adrenal medulla accompanied by numerous proteins, peptides such as chromogranin A (CGA) and its natural fragments. To date, no study has evaluated the prognostic value of CGA in critically ill ICU patients.

P37
Methods We conducted a prospective study of ICU patients, admitted for a life-threatening condition with at least two organ failures, by measuring plasma procalcitonin (PCT), C-reactive protein (CRP), the Simplifi ed Acute Physiological Score II (SAPS II), and CGA on the 24th hour after admission. Continuous data are reported as the median (interquartile range), and group diff erences were evaluated with the Mann-Whitney U test or the Kruskal-Wallis test. A Cox proportional hazards regression model was used to evaluate the eff ect of the logarithmically transformed CGA concentration on the endpoint and to calculate hazard ratios (HRs) with 95% CIs. All statistical analyses were performed with the SPSS statistical package (SPSS for Windows version 11.5).

Introduction
The genus of Chlamydiae comprises obligate intracellular pathogens that occasionally can disseminate and even cause septic infections. Chlamydophila pneumoniae causes acute and chronic respiratory tract infections from sinusitis to severe pneumonia, and phagocytes can transmit the bacteria from the lungs to the vasculature. We have previously shown in IL-10 knockout mice that IL-10 limits the severity of infl ammation but prolongs the clearance of the C. pneumoniae pneumonia [1].
Objective Although IL-10 could contribute to the resolution of C. pneumonia infection by regulating the T-helper cell balance (Th1/Th2), we were interested in the direct eff ects of IL-10 and the IL-10-regulated genes in modulating C. pneumoniae growth in macrophages and in respiratory epithelial cells.
Methods We investigated the eff ect of IL-10 and the expression of an IL-10-responsive anti-infl ammatory factor on mRNA and protein level in C. pneumoniae infected human monocyte/macrophage (MonoMac6 and Thp-1) and in lung epithelial adenocarcinoma (A549) and in HL (human lung) cell lines. We also applied a luciferase promoter assay to study the regulation of the anti-infl ammatory gene expression during the C. pneumoniae infection.

Results
In agreement with the previous studies, C. pneumoniae proliferated in epithelial cells, while in monocyte/macrophages the infection was often nonproductive and aberrant forms of bacteria were observed. The IL-10 responsive anti-infl ammatory factor was diff erentially regulated at transcriptional level in A549 and MonoMac6 cells in response to C. pneumoniae infection, which could potentially aff ect the outcome of infection. The luciferase promoter assay showed that the transcription was mediated via the E-box regulatory element of the gene. Conclusions Our results imply that the anti-infl ammatory response to intracellular C. pneumoniae infection varies in diff erent cell types and ongoing studies are needed to clarify the role of IL-10 response in limiting Chlamydia growth in these cells.
Introduction In a smaller sample of patients with severe sepsis, resuscitation with the synthetic colloids hydroxyethyl starch (HES) as well as gelatin (GEL) increased the occurrence of acute kidney injury (AKI) in comparison with crystalloids (CRYS). We now performed the analysis in a large-scale sample with over 1,100 septic patients on our ICU. Methods A prospective controlled before-and-after study in patients with severe sepsis on a mixed ICU. AKI was defi ned by RIFLE criteria [1]. Statistical analysis was performed using SPSS 18.0. ; P = 0.000). Univariate analysis for assessing the risk factors for ICU mortality among bacteremic patients was done in which age (P = 0.061), sex (P = 0.253), type of admission (P = 0.203), type of organism, severity of illness (P = 0.234), and site of infection (P = 0.250) were analyzed, but only previous antibiotic use was statistically associated with higher mortality (P = 0.011). Bacteremic patients who were already on antibiotics had a signifi cantly higher mortality (54.2% vs. 8.3%) (OR 12.9, 95% CI: 1.6 to 100). Mortality was higher in patients with Pseudomonas bacteremia (72.7%) although it was not statistically signifi cant (P = 0.08). See Tables 1 and 2.

Results
Conclusions Blood cultures may be positive in only a minority of patients with suspected infection admitted to the ICU as most of these patients may already be taking antibiotics. Nevertheless, the prognosis of those patients with positive blood culture is worse, especially if culture is positive in spite of the patient being on antibiotics.
Introduction Severe septic syndrome remains one of the most frequent causes of death in ICUs. One of the main players in this pathology is the endothelium integrity. Our laboratory has demonstrated in preliminary clinical studies among the various biomarkers of endothelial dysfunction that blood levels of endocan (ESM-1), a pulmonary vascular endothelial cell-specifi c molecule participating in the control of endothelial-leukocyte interactions, are associated with the severity and evolution of septic states.
On the other hand, we showed in vivo that antiprotease therapy is associated with a decrease of the leukocyte rolling and fi rm leukocyte adhesion to endothelium in sepsis. This decrease in the leukocyte-endothelial cell contacts was associated with an increase of blood endocan levels, suggesting a linkage between leukocyte proteases, endocan, and infl ammation during sepsis.
Methods In order to characterize this linkage, we set up a mouse model of nonlethal shock induced by endotoxin (LPS), in mice genetically defi cient in cathepsin G (CG -/-) or double defi cient in cathepsin G and elastase (CGEL -/-). The neutrophil and endothelial activation biomarkers included myeloperoxidase, sE-selectin, and endocan ELISAs in mouse serum. In vitro tests of endocan proteolysis were also performed.
Results During the nonlethal endotoxemia, clinical scores as well as E-selectin levels were maximal at 24 hours and progressively returned to the baseline. Circulating myeloperoxidase also increased early at 24 hours but remained elevated until 72 hours. By contrast, circulating endocan decreased early at day 1, remained undetectable at days 2 and 3, and then normalized at day 5. A strong inverse correlation was observed between endocan and myeloperoxidase levels. Similar fi ndings were observed CG -/-. However, . In the animal model after MAOA inhibition, the survival rate was signifi cantly higher (risk reduction 40%, P <0.05) and less bacterial burden was found in the blood, lung, and liver (1 log, P <0.05). Furthermore, less organ damage shown by LDH, ASAT and ALAT was observed (P <0.05). These results were associated with less ROS production in granulocytes (P <0.05).
Conclusions MAOA is strongly upregulated during severe sepsis on RNA as well as on protein level. In septic mice, higher survival rates were observed by blocking MAOA. Inhibition of MAOA might have potential in sepsis and so provide a novel method for therapeutic intervention.
foci, thereby favoring increased bacteremia and ultimately leading to mortality. We have previously shown that the selective gastrin-releasing peptide receptor antagonist RC-3095 can reduce organ dysfunction in experimental sepsis. Thus the aim of the present study is to report a novel link between GRPR and TLR-4 signaling and its relationship with infl ammatory parameters in in vitro and in vivo experimental models as well as in sepsis patients.
Methods For the in vitro experiment, RAW 264.7 macrophages were stimulated with LPS and treated with RC-3095 for RT-PCR analyses of TLR-4 mRNA, immunoblotting of pERK1/2, pJNK, pAkt, and EMSA of NF-κB and activator protein 1 (AP-1). In the in vivo studies, male Wistar rats were divided and submitted, into sham surgery, cecal ligation and puncture (CLP) surgery, and CLP plus RC-3095. Six hours after, all rats were anesthetized and sacrifi ced by cardiac puncture. Blood was collected for bacterial count and cytokine analyses; bronchoalveolar lavage fl uid for cell count, levels of TLR-4 and cytokines; peritoneal lavages for bacterial count; and lung tissue for levels of TLR-4 and RT-PCR analyses of TLR-4 mRNA. In a human study 12 patients, admitted to an adult medical ICU with a clinical diagnosis of septic shock, received a continuous infusion with RC-3095 over a period of 12 hours and concentrations of IL-6 and IL-10 in plasma were determined. Results are expressed as means ± SD. Diff erences between groups were determined by ANOVA, followed by Tukey's post hoc test. Diff erences between two groups were determined by t test.
Results RC-3095 inhibited expression of TLR-4 and reduced phosphorylation of extracellular signal-regulated kinase (ERK-1/2), c-Jun NH2-terminal kinase (JNK), and Akt, leading to decreased activation of NF-κB and AP-1 in macrophages. In a rat model of sepsis, RC-3095 treatment decreased lung TLR-4 content, reduced the migration of infl ammatory cells to the lung, reduced systemic cytokine levels, and attenuated bacterial dissemination. Continuous infusion with RC-3095 for 12 hours decreased IL-6 plasma levels in septic patients, but did not signifi cantly aff ect IL-10 plasma levels.
Conclusions These fi ndings demonstrate the benefi cial action of GRPR antagonists in controlling the infl ammatory response in sepsis through a mechanism involving inhibition of TLR-4 signaling.

Introduction
Having the ability to predict organ failure could impact treatment decisions and potentially lessen the consequences of acute lung injury (ALI) and sepsis. While Acute Physiology and Chronic Health Evaluation (APACHE II) and the Pneumonia Severity Index are useful in predicting the risk of morbidity and mortality, they do not predict the risk of developing organ failure. Currently no accepted practice allows for the prediction of the development of organ failure. The objective, therefore, of our study was to predict the development of organ failure at 24 hours using only the data available from the fi rst 4 hours post inoculation. Methods This pneumonia-sepsis model included 19 sheep with ALI. Inoculation of ~2.5 × 10 11 colony-forming units methicillin-resistant Staphylococcus aureus (MRSA) induced pneumonia, while smoke injury was created through inhalation of cotton smoke. Four diff erent groups were studied and are as follows: MRSA and smoke inhalation (M+S, n = 7), MRSA untreated (M, n = 3), MRSA treated (M+T, n = 3), and smoke inhalation only (S, n = 6). In order to use the injury group as a model input, all the sheep were modeled independent of group and a rank order of severity was determined. Additional inputs included a number of clinical and laboratory parameters.
Only the fi rst 4 hours of data were allowed to be used as an input. The model outputs were prothrombin time (PT) and mean arterial pressure (MAP) over the entire 24-hour time frame. To minimize overparameterization, only two inputs per output were used for prediction.

Results
The rank order of injury group from least to greatest severity was M+T, S, M, M+S. PT was best predicted by calcium and injury. The agreement between predicted and measured PT using only calcium as the input was r 2 = 0.24. Adding the second input, in this case injury group, improved the model's predictive ability (r 2 = 0.48). MAP was best predicted by lactate with an agreement between predicted and measured of r 2 = 0.64. Unlike PT, the model was not able to better predict MAP by adding a second input (r 2 = 0.64). Conclusions Our model was able to provide an accurate prediction of MAP using only the fi rst 4 hours of data, while PT was less accurately predicted. However, this early study suggests that continued refi nement of the progression model could provide a viable tool to predict organ failure in sepsis.
MRSA pneumonia-sepsis model in sheep mimics the vascular state in human sepsis. In the present study, we sought to combine sheep and clinical data from humans to determine whether common parameters existed across sepsis with regard to coagulopathy. Secondly, we wanted to model and provide estimates of MRSA bacterial load in both species. Methods Nineteen sheep with acute lung injury and 14 human patients were incorporated into this sepsis model. In sheep, pneumonia was induced by inoculating the airway with ~2.5 × 10 11 colony-forming units (CFU) MRSA. Thirteen of the sheep had smoke injury induced through inhalation of cotton smoke. All human patients were retrospectively studied and were bacteremic with MRSA from varying primary infection sites. Initial bacterial load in humans was modeled using clinical and microbiologic data available at the start of sepsis, while the initial load in sheep was the inoculating amount of bacteria. Load continues throughout the study period and is modifi ed by vital signs and antibiotic coverage. The bacterial load as well as clinical and laboratory parameters are inputs, with the output parameter being prothrombin time (PT). In order to minimize overparameterization of the population, the model was allowed to estimate PT using only three parameters. Data were modeled for 24 to 48 hours. Results Bacterial load was estimated to range from between 10 8 and 10 11 CFU, with the high end of the range being similar to the inoculum used to induce pneumonia in the sheep. The highest-ranking parameters in estimating PT were calcium, potassium, and bacterial load. When using calcium alone, the model estimate agreement with measured PT was r 2 = 0.25. Combining calcium and potassium improved agreement (r 2 = 0.34), while using all three parameters further improved the estimate (r 2 = 0.37).
Conclusions Through progression modeling we were able to provide prediction of coagulopathy and bacterial load across two diff erent species of animals infected with the same organism. Understanding the mechanisms of bacterial immune modulation will teach us more about pathogenesis of infection and could lead to new strategies in anti-infl ammatory therapy during sepsis. Various approaches are already used to identify bacterial immune modulating proteins. However, these are ineffi cient and time consuming. Immune modulating proteins need to be secreted in order to act on their targets outside the bacterial cell. In the present study, phage display technology was used to specifi cally identify secreted immune modulating proteins with high effi cacy. Phage display technology is a technique to express a protein fused to a coat protein of a fi lamentous phage. The most widely used coat protein is pIII encoded in the phage genome by gIII. This gene contains a signal sequence that is essential for production of stable phage particles. When a bacterial genome is randomly fragmented and these fragments are inserted into a phage vector containing gIII lacking the signal sequence, intact phage particles are formed only when the inserted bacterial genomic fragment contains a signal sequence. This allows for selective expression of a bacterial secretome since secreted proteins also contain a signal sequence. The resulting secretome phage library can be used to select displayed proteins that specifi cally bind to various components of the immune system. This powerful technique has several advantages: it can be used for diff erent Gram-positive and Gram-negative bacterial genomes. There is no need for extensive culturing of bacteria so it can be used for diffi cult or slow-growing bacteria. Expressed proteins are not hampered by solubility problems. There is a direct relation between expressed protein and coding gene that allows for rapid identifi cation of selected proteins. As a proof of principle, a Staphylococcus aureus library was constructed. The goal is to evaluate the proportion of previously described secreted immune modulators that can be recovered and to identify new immune modulators. In order to express most of the 300 secreted proteins encoded by this microorganism, the library reached a diversity of 108 clones. The secretome phage library was screened for interaction with leukocytes and for modulation of the coagulation and complement pathways, which are highly activated in sepsis process. Introduction Healthcare-associated infection (HAI) control is one of the most important challenges in quality and safety. Defi ned as the intensive use of the information and communication technologies, e-health can be a major part of this matter. Aiming for implementation of an infection control strategy based on the e-health concept in a 280-bed, paper-free, general hospital, the Infection Control Committee (ICC), along with the Quality and Antibiotics Committees and the IT team, has been working on the implementation of new tools for widespread use.

Building up an infection control strategy based on the e-health concept
Methods The authors used the electronic medical record, a special program for infection surveillance and data-mining, and some additional resources, such as messages and alerts sent by email or by SMS, as a global approach for improving infection control. On the electronic medical record (Soarian®; Siemens Medical Solutions), interventions are made at diff erent levels. On patient admission, through the fulfi llment by the physician of a questionnaire on detection of increased risk for infection/colonization, several protocols regarding isolation and screening testing are automatically activated and the ICC is informed via email, thus minimizing the spread of epidemiologically important microorganisms. During hospitalization, new templates for prescribing microbiological tests are in progress to improve the availability of clinical information to the laboratory and to the Vigiguard® (Biomérieux) program. New context-sensitive templates for antimicrobial prescription are being implemented to improve the quality of such therapeutics. Alerts to the pharmacy are sent when there is some inaccuracy in terms of the chosen antimicrobial or the duration of therapy, thus reducing the emergence of new drug resistances and minimizing costs. New fi elds were created to individualize infection control issues in the patient's history, and to generate an automatic note on epidemiologically important issues at the transfer or discharge of the patient, thus complying with the recommendations for information transfer. Finally, surveillance of several infections/colonizations will be obtained in real time from Vigiguard®, a tool with a data-mining engine.
Conclusions The authors hope that the application of the e-health concept to the infection control policy in a paper-free hospital will improve quality of and reduce the risk of HAI.