key: cord-0007041-2tdn1vr9
authors: nan
title: Abstracts 4th International Congress “Sepsis and Multiorgan Dysfunction”: September 9–12, 2009 Weimar
date: 2009
journal: Infection
DOI: 10.1007/s15010-009-1004-1
sha: 2dffef28fe66a944fcc070772bfe759121bb02de
doc_id: 7041
cord_uid: 2tdn1vr9

nan

Understanding the pathogenesis of septic shock remains imperative as mortality from septic shock has been unimproved for decades. Septic circulatory failure is characterized by strong vascular hyporeactivity to vasopressors such as the alpha-1 receptor agonist norepinephrine.We recently described a systemic downregulation of alpha-1-adrenergic receptors which contributes to septic circulatory failure (Bucher et al. Crit Care Med 2003; 31:566-571) Objectives: Here we investigate the role of NF-κB in the pathogenesis of cecal ligation and puncture-(CLP)-induced cardiovascular failure with downregulation of alpha-1 adrenergic receptors. Methods: Sepsis was induced by cecal ligation and puncture (CLP) in wildtype-mice and mice with deficiencies for proinflammatory cytokines. Furthermore, mice were injected with TNF-α, IL-1β, IFN-γ, and IL-6. Animals were treated with pyrroldine dithiocarbamate (PDTC) blocking NF-κB activity. Hemodynamic parameters and expression of alpha-1 receptors were examined. Finally, hemodynamic dose-respsonse studies with norepinephrine were performed. Results: CLP treatment resulted in a hyperdynamic circulatory failure, a diminished vascular reactivity to norepinephrine and a significant downregulation of alpha-1-receptors. Proinflammatory cytokines also downregulated alpha-1-receptors and caused cardiovascular dysfunction. However, suppression of single proinflammatory cytokines in cytokine knock-out mice did not diminish CLP-induced downregulation of alpha1-receptors. In contrast, blocking of NF-κB via PDTC significantly attenuated liberation of multiple cytokines and CLP-induced downregulation of alpha-1-receptors. Thereby, PDTC prevented septic cardiovascular failure and improved survival of septic mice. Conclusions: Our findings define a critical role for NF-κB in the pathogenesis of septic shock as NF-κB ameliorates sepsis-induced downregulation of alpha-1 receptors, indicating that targeting NF-κB is a desired therapeutic strategy to treat septic vasoplegia.

Introduction: Abdominal sepsis is still a major clinical problem in surgical intensive care units. The development of multiple-organ failure due to sepsis continues to be one of the leading causes of mortality and morbidity. The occurrence of thrombozytopenia in critically ill patients has been observed for many years, and platelets play an important role in the induction of organ dysfunction. Platelets express many receptors and store a lot of influential cytokines and chemokines in their α-granules, which underly their role in inflammation. Colon ascendens stent peritonitis (CASP) is a clinical relevant animal model of polymicrobial sepsis in mice and can be used to follow the function of platelets in sepsis. To induce septic conditions in an in vitro model, cells can be stimulated with lipopolysaccharide, a component of the outer membrane of gram-negative bacteria. LPS is an important agent in the induction of systemic inflammation. It binds to the TLR4-MD2complex, which is also expressed on the surface of platelets Objectives: In recent years evidence has accumulated that platelets have many effects beyond their role in hemostasis and thrombosis. They are involved in pathways of inflammation and react themselves to exogenous noxa like LPS. The aim of our research is to examine the role of platelets in systemic inflammation and the part played by the chemokine receptor CCR4 which they express on their surface. In the first readout we looked at the development of liver dysfunction in murine sepsis. In the second approach we analyzed in vitro the changes of human CCR4-mRNA in platelets after stimulation with LPS. Methods: CASP was performed in CCR4-/-and C57Bl/6 wild type mice. 20 hours after CASP immunohistochemistry was used to assess the number of platelets in murine liver microcirculation and to determine whether the CCR4 receptor influences the recruitment of platelets during experimental sepsis. The processing of CCR4 mRNA after treatment with LPS was followed using qRT-PCR. Platelet activation was induced by LPS (E.coli O:127) directly injected in a human platelet concentrate with a final concentration of 1μg LPS per ml. After injection the concentrate was gently moved over 20 h to avoid formation of clots and unwanted activation and was stored at room tem-perature. After different time points a small amount of platelet rich plasma was taken out and RNA from 2x107 cells was isolated by using the TRIZOL-method. Results: In murine liver a significant increase of platelets is observed 20 h after CASP, in both CCR4-/-and C57Bl/6 wild type mice compared to the non-septic control group. The progression curves of real time PCR show that E. coli LPS induced the human platelet CCR4 RNA processing. The increase in spliced CCR4 RNA after LPS exposure was time dependent. Maximal accumulation of processed RNA occurred 20h after stimulation, where it was 3 times higher than without LPS. Conclusions: These findings indicate that sepsis leads to a recruitment of platelets in the microvascular system of the liver which is independent of the CCR4 receptor. Nevertheless the chemokine-receptor 4 is part of the platelet response during inflammation, because its mRNA is processed and increased by the spliceosome in platelets after LPS treatment. These findings lead to new experiments which should be done in future. If the receptor protein is really synthesized is currently checked by western blot. Also the RNA of its ligands MDC (macrophages-derived chemokines) and TARC (thymus-and activation regulated chemokine), which are stored in the α-granules of platelets will be concerned in following LPS-experiments.

Infection 2009; 37 (Suppl. III): 5

Muscle failure during sepsis can be explained by direct action of the pro-inflammatory cytokine IL-1 in stabilising the inhibitory Mg2+ binding site of RyR1 in skeletal muscle spective Se-supplementation trials indicated Se supplementation as a promising adjuvant therapeutic option in severe sepsis. Objectives: Aim of the project was to study the influence of Se-supplementation on the acute phase response (APR) in male and female mice after lipopolysaccharide (LPS) injection, and to ascertain whether there is a sex-specific difference of the effect. Methods: We have injected male and female mice on different Se status with LPS to induce an APR. Se-levels were determined by Total reflection X-ray fluorescence analysis (TXRF) and Selenoproteins were detected by WesternBlot or Enzyme Activity Assays. A multiplex biometric immunoassay was used to determine circulating cytokine-levels. Results: Serum Se showed a very pronounced decline to approx. 50% of control values during the acute phase response. Cytokine production was strongly affected by the Se-status. The Se poor animals displayed significantly enhanced concentrations of the circulating cytokines IL-6 (2-fold, p<0,05) and MCP-1 (2-fold, p<0,01) compared to the Se-supplemented mice. Se-supply increased the glutathione peroxidase activity (pGPx) and selenoprotein P (SePP) concentration in serum. Upon LPS injection, both pGPx activity and SePP concentrations declined in both male and female mice. The effect was especially pronounced in well-supplied mice, but the final levels were still above those insufficient concentrations observed in the mice on the Se-poor diet. At the same time, the cytokine-regulated selenoprotein S (SELS) increased tissuespecifically in liver and seems to profit from the deranged Se metabolism.

Conclusions: Se supplementation influences selenoprotein expression and the APR in a beneficial way. Cytokine production was strongly affected by the Se-status. The observed effects differ between males and females, and appear to represent an adaptive mechanism controlling the privileged biosynthesis of immune-relevant selenoproteins. These findings are in agreement with the clinical data from a respective study with human sepsis patients (SIC). We conclude that Sebased adjuvant supplementation efforts might improve selenoprotein expression and immune function and reduce mortality during sepsis.

Infection 2009; 37 (Suppl. III): 6

Interrelation of selenium and acute phase response in mice Introduction: Cardiac depression is a frequent manifestation of organ dysfunction during sepsis. Interplay and contributions of underlying mechanisms remain incompletely understood although various molecular events are discussed, including cardiosuppressive effects of proinflammatory mediators, derangement of calcium homeostasis and cardiomyocyte apoptosis. Objectives: In a murine model of polymicrobial peritonitis resulting in severe sepsis we determined myocardial gene expression alterations in a set of selected genes which are implicated in inflammatory (S100a9, C5ar1) and apoptotic pathways (Fas, Tnfrsf1a, Smpd1) or have been noticeable in preliminary cardiac microarray studies of our research group (Bcr, Myc, Cth) . Histological examination was performed to evaluate apoptotic cell death of cardiomyocytes. Methods: Female C57BL/6J mice received either intraperitoneal injection of human fecal suspension (PCI = peritoneal contamination and infection) or sterile sodium chloride solution. Hearts were harvested after 6 or 24h and genes were analyzed for transcriptional changes utilizing real-time quantitative PCR. After normalization using three unvaried housekeeping genes, expression values were calculated as log2 fold changes and subjected to statistical evaluation (MWU test, Levene's test; Bonferroni-Holm adjusted p-value < 0.05). Apoptosis was determined via TUNEL staining. Results: Murine heart tissue exhibited elevated gene expression levels of death receptors Fas and Tnfrsf1a in septic animals. However, histological sections revealed no significant apoptosis in cardiomyocytes following PCI. Furthermore extensive up-regulation of S100a9, encoding a small calcium binding protein, was observed in the sepsis groups. Increased transcription of proto-oncogenes Myc and Bcr could be confirmed, while mRNA of the hydrogen sulfide generating cystathionine-γ-lyase (Cth) was down-regulated. Conclusions: Low apoptotic rate of cardiomyocytes in this model is consistent with clinical studies and indicates that septic cardiomyopathy may be due to functional and molecular rather than structural alterations. Roles of proto-oncogenes and Cth in the course of cardiac dysfunction need to be further elucidated. As hydrogen sulfide is described to act anti-inflammatory and cardioprotective in myocardial ischemia/reperfusion, down-regulation of Cth may be a crucial event for the development or aggravation of septic cardiomyopathy. Introduction: During sepsis immune cells and inflammatory mediators are released from the gastrointestinal tract into the mesenteric lymph causing severe pulmonary dysfunction (Glatzle JOGS 2007) . Basic science and clinical studies indicate a protective effect of long chain fatty acids during sepsis although the involved pathways still have to be clarified. Objectives: We investigated the effect of an enteral immune modulating diet with long chain fatty acids on distinct leukocyte release of the gastrointestinal tract during sepsis and the ability of enteral fatty acids to inhibit the activity of gut derived leukocytes released in mesenteric lymph during sepsis. Methods: Lymph fistula rats were used to collect mesenteric lymph. The animals were intestinally infused with either a 5% glucose solution (control) or a solution of 5% glucose and 2% long chain fatty acids in form of olive oil. Mesenteric lymph was collected before and after LPS (E.coli 5mg/kg i.p.) injection and immune cells were analyzed with FACS. Furthermore lymph leukocytes (≈106) were isolated and in vitro stimulated with LPS (10ng, 100ng, 1µg, 10µg). TNFα concentration was determined in the supernatant of the in vitro stimulation by ELISA. Results: Interestingly the release of T-cells (approximately 70% of total cell content), macrophages and dendritic cells (approximately 7% of total cell content) from the gut during LPS-induced sepsis was not significantly different in rats treated either with glucose or long chain fatty acids. However, in vitro LPS-stimulation of mesenteric lymph leukocytes harvested from rats enterally treated with long chain fatty acids caused an up to fourfold lower release of TNFα compared to the control group (TNFα pg/ml, control vs. long chain fatty acids, basal: 11±6 vs. 6±3; 10 ng: 11±7 vs. 14±5; 100ng: 45±19 vs. 21±11; 1µg: 31±12 vs. 8±5*; 10µg: 33±8 vs. 11±5*; *p<0,05 vs. control). Conclusions: Abdominal sepsis leads to activation of leukocytes in the gut wall and subsequent release of inflammatory mediators such as TNFα. Here we report that an intestinal immune modulating diet with long chain fatty acids in form of olive oil is able to suppress TNFα release of gut derived immune cells. It is likely that the so called cholinergic anti-inflammatory pathway accounts for this effect, thereby inhibiting the release of pro-inflammatory cytokines into the circulation. Anti-inflammatory effects of drotrecogin alfa (activated) on organ tissue in murine sepsis

Protein-2 (MIP2) upon stimulation with pro-inflammatory cytokines [1] . Objectives: To investigate whether DAA provides effects on murine heart in sepsis. Methods: Sepsis was induced by cecal ligation and puncture (CLP) in male NMRI-mice (n=20, body weight 30±3g). Animals were randomly assigned to vehicle infusion (control), or CLP sepsis with DAA infusion (DAA; 24µg/kg/hr). A third group received only sham operation and vehicle infusion (Sham). 48 hrs prior to CLP all mice were given a permanent central i.v.-line and an arterial transmitter (PA-C10, St. Paul, MN, USA) to measure heart rate (HR) and mean arterial pressure (MAP). CLP was adjusted to survive 24hrs. After 12hrs of sepsis, heart tissue was snap frozen in liquid hydrogen and plasma samples were collected and frozen following manufacturers guidelines until measurement. RNA was extracted of frozen tissue (RNeasy, Qiagen, Hilden, Germany) and analysed by real-time-PCR with mouse specific primers to CD14 and MIP2 normalized to ß-Actin (all: biomers, Ulm, Germany) using an iCycler (Biorad, Hercules, USA). Determination of Interleukin (IL)-6, IL10, and monocyte chemoattractant protein-1 (MCP-1) was performed using a cytometric bead array (CBA, BD Biosciences, Heidelberg, Germany). Differences between groups were evaluated by 1-way ANOVA and Pair-wise fixed reallocation randomisation test© for PCR diagnostic. All data are given as mean±SD. P<0.05 was considered significant Results: There were no significant differences in HR between the groups (Sham 650±38/min; DAA 547±175/min; Control 530±143/min). MAP was significantly higher in sham group (p=0.031) and non-significantly higher in DAA group when compared to control (Sham 137±15mmHg; DAA 111±26mmHg; Control 97±4mmHg). Normalized expression ratio of CD14 was 28.7 fold higher in control group compared to sham group. DAA treatment resulted in an 18.1 fold expression ratio (p=0.001). MIP2 expression ratio was 25.4 fold higher in control and 11.0 fold higher in DAA group. IL-6 (DAA 4663±6229 pg/ml, p=0.027; Control 10906±8324 pg/ml; Sham 54±32 pg/ml, p<0.001), IL-10 (DAA 220±338 pg/ml, p=0.072; Control 2539±5251; Sham 7±11, p=0.049) and MCP-1 (DAA 2708±3401 pg/ml, p=0.031; Control 7010±7095; Sham 75±107, p=0.001) were significantly lower after treatment with DAA compared to control. Conclusions: Our data provide evidence of DAA-induced direct antiinflammatory effects on murine heart cells, suggesting that DAA plays an important role in the anti-inflammatory response to septic injury.

Infection 2009; 37 (Suppl. III): 9

Serum, brain, heart, and liver: Proteomic analysis of sepsis-induced protein alterations in different tissues Hinkelbein J, Kalenka A Department for Anesthesiology and Intensive Care Medicine, University Mannheim, Germany.

Introduction: During the course of sepsis, multiple organ dysfunction syndrome (MODS) is a common complication in patients. MODS is the major cause of mortality and morbidity in septic patients but the underlying molecular mechanisms remain widely unclear. Objectives: The present study provides an overview on alterations in protein expression of serum, brain, heart, and liver using proteomic techniques in a cecal ligature and puncture (CLP) model of rat sepsis [1] [2] [3] [4] . Methods: After approval by the local institutional review board, 62 male Wistar rats were investigated and assigned to three sham groups (n=16) and three sepsis groups (n=46). Rats of the sepsis groups and control groups were analyzed at 12, 24, and 48 hours after sepsis induction. Sepsis was induced by CLP and organs removed after decapitation. 2D-gel electrophoresis (2D-PAGE) and mass spectrometry (MS) as well as bioinformatic network pathway analysis (Pathway Studio ® , Ariadne Genomics, USA) were used to identify and interpret alterations in protein expression between septic and non-septic samples. Results: Mortality was 59 % in the CLP groups but no rat of the sham group died. In a total of 72 gels, 1,277±10 protein spots per gel were discriminated with the proteomic method. 118 spots were differentially regulated (serum: n=55; brain: n=38; heart: n=12; liver: n=13) and proteins identified with MS. Using Pathway Studio, oxidative stress, cell death, and NO-biosynthesis as important cell processes were found to be associated with protein alterations. Additionally, bioinformatic analysis showed relations to inflammation, endothelial cell dysfunction, and diabetes mellitus. Conclusions: The complete array of protein alterations indicates a strong association with inflammation and endothelial cell dysfunction. Oxidative stress and severely compromised cellular function following sepsis seem to play a major role for developing MODS. Lymphocyte apoptosis in severely septic rats receiving lipid emulsions Introduction: Severe sepsis remains a leading cause of death in intensive care units. The long-term parenteral administration of lipid emulsions enriched with polyunsaturated fatty acids (PUFA) has been shown to differentially modulate immune functions and apoptotic pathways in vivo and in vitro depending on their respective content of ω-6and ω-3-FA. However, the short-term effects of PUFA in the context of severe sepsis remain unclear. Objectives: The present study was designed to compare the immediate effects of a continuous perfusion with ω-6-enriched soybean oil (SO) or ω-3-enriched fish oil (FO) on pro-inflammatory cytokines, on lymphocyte apoptosis in the spleen and on the plasma profile of PUFA in a rodent model of severe sepsis induced by cecal ligation and incision (CLI). Methods: After governmental approval of this prospective randomized study, 34 male Sprague-Dawley rats (501±33 g) received tracheotomy and mechanical ventilation. I.v. anesthesia and additional lipid perfusion were administered throughout the entire observation time of 390 min to the following six groups: 1. Laparotomy (LAP, n=4); 2. LAP + 0.06 g/kg/h Intralipid-10% (LAP-SO, n=6); 3. LAP + 0.06 g/kg/ h Omegaven-10% (LAP-FO, n=6); 4. CLI (n=6); 5. CLI-SO (n=6); 6. CLI-FO (n=6). At the end of observation time, plasma samples were analysed for their content of free FA (chromatography) and for Interleukin-(IL-)1β and IL-6 (ELISA). Spleen lysates were analysed for cleaved caspase-3 expression (Western Blot) and splenic lymphocytes for characteristic apoptotic morphology (FACS). Statistical analysis was performed using Two-Way ANOVA with Dunn s post-hoc test. Differences were considered significant if p < 0.05. Results: Within 390 min after sepsis induction, IL-1β and IL-6 plasma levels were significantly induced in the CLI-FO-group compared to CLI and to CLI-SO. Moreover, both SO and FO administration induced splenic apoptosis and cleaved caspase-3 expression regardless of the surgical procedure. The CLI-sepsis led to a loss of total free FA and to a simultaneous relative increase in arachidonic acid. Conclusions: ω-6-enriched SO and ω-3-enriched FO possess characteristic immunomodulating properties in this short-term model of severe sepsis, hitherto only been described after long-term administration. The data suggest that the mode of action of lipid emulsions de- Objectives: To compare the influence of HES and GEL resuscitation on hemodynamic variables and microvascular oxygenation as well as renal function in a rat model of peritoneal sepsis and septic shock. Methods: Peritoneal sepsis was induced by cecal ligation and puncture in 20 Sprague-Dawley rats weighing between 350-400g. Intravenous fluid resuscitation with either 25mg/kg/h of HES (Voluven© 6% HES 130/0.4) or GEL (Gelafundin© 4%) was performed for two hours when mean arterial pressure (MAP) decreased to 80 mmHg. MAP, heart rate (HR), and cardiac output (CO) were continuously measured and recorded. Microvascular pO2 (µpO2) was measured in the buccal mucosa using oxygen dependent quenching of phosphorescence.

Besides conventional renal markers such as serum creatinine and blood urea nitrogen (BUN), neutrophil gelatinase associated lipocalin (NGAL) concentration was determined in kidney tissue homogenates using a rat specific ELISA kit (BioPorto© Diagnostics, Gentofte, Denmark). Results: Hemodynamics, µpO2 as well as markers of kidney injury did not differ significantly between groups before fluid resuscitation. GEL and HES were equally effective in restoring sepsis induced decreases in CO one and two hours after start of treatment (HES vs. GEL: 203±42 vs. 185±31 ml/min at 1h ; 207±28 vs. 218±29 ml/min at 2h). This was also true for HR and MAP. µpO2 was significantly higher after one hour in GEL treated animals (13.5±3.9 vs. 18.4±4.5 mmHg; p=0.017), probably due to stronger dilution effects of HES (Hemoglobine 7.2 ± 1.6 vs. 9.1 ± 1.8 mg/dl; p=0.031). Creatinine serum levels as well as BUN were equally elevated in GEL and HES treated animals (Creatinine: 1.1±0.6 vs. 1.4±0.4 mg/dl; Urea: 88.3±34.3 vs. 99.7±14.5 mg/dl; p=NS). NGAL as early biomarker behaved in a similar manner (24153±16466 vs. 14242±4750 U/ml; p=NS). Conclusions: In this animal model of peritoneal sepsis and septic shock, GEL showed comparable hemodynamic properties without significantly improved markers for renal integrity as compared to HES. µpO2 was significantly higher in GEL treated animals, eventually due to dilution effects by the higher osmolarity of HES. Introduction: Microcirculation, cellular energy state as well as hepatic transport determine hepatocellular excretory function which is often impaired during sepsis. However, the underlying mechanisms of a sepsis-related breakdown in excretory function including the fate of xenobiotics which are subject to biliary clearance remain poorly understood.

Objectives: To investigate the three determinants of excretory function in a rodent model of severe polymicrobial sepsis, using the fluorescent dye indocyanine green (ICG) as an example for solely biliary excreted xenobiotics. Methods: Applying in vivo imaging and fluophotometry, plasma clearance, hepatocellular uptake and biliary excretion of ICG as well as microvascular perfusion and hepatocellular energy state were studied at 15 hours after induction of a peritoneal cavity infection (PCI). PCI was achieved by intraperitoneal injection of a standardized and microbiologically characterized fecal slurry (0,7ml/kg bw).

The applied PCI sepsis model resulted in a mortality of 22% at 15h rising to 100% by 40 hours. At the time point of investigation (15h), animals displayed clinical and laboratory signs of multiple organ dysfunction, including liver injury. Sinusoidal perfusion was only moderately impaired while hepatocellular redox state was completely maintained. However, excretion into bile as a function of hepatic transporters were significantly delayed for the administered dye ICG (relative decrease of specific fluorescence 35 minutes after administration of ICG: 72,1 +/-3,1 % (sham) vs. 91,4 +/-3,4 % (sepsis) ; p<0.05).

The demonstrated hepatocellular accumulation of ICG as an example for potentially cytotoxic xenobiotics confirms and extends the differential susceptibility of the polar surfaces of hepatocytes during sepsis. Since a failure of specifically the canalicular transporters results in a hepatocellular accumulation, these findings have potential implications for understanding drug-induced liver injury often seen in septic patients and point out the need for monitoring excretory function, ideally including the canalicular pole. Introduction: During the last decades, fungi became important opportunistic pathogens causing life threatening infections, such as invasive mycoses and sepsis. Important human pathogens are Candida albicans, the most often from blood isolated fungus and Aspergillus fumigatus, the most common airborne fungal pathogen1. Research on them is done on various cellular levels, for example on transcriptome and proteome. Mostly, data from both levels are analyzed separately, although they are very much intertwined. 1Kullberg BJ, Oude Lashof AML (2002) . Epidemiology of opportunistic invasive mycoses. Europ J Med Res, 7(5), 183-191. Objectives: It is an important goal of Bioinformatics to combine data of transcriptome and proteome and to enhance analysis and comparison between them. Data integration requires data storage. There are about 1000 databases available via the internet that are of importance for molecular biology2. None of them includes transcriptomic and proteomic data of human pathogenic fungi and we want to fill this gap. 2Galperin M Y (2009). Nucleic Acids Research annual Database Issue and the NAR online Molecular Biology Database Collection in 2009. NAR, 37(Database issue), D1-D4 Methods: We established a data warehouse for the integrated storage, visualisation and analysis of genome and experimental transcriptome and proteome data of human-pathogenic fungi based on Protecs (Decodon GmbH, Greifswald)3. It uses the object relational database concept Caché and html with Java Script to visualize results. Furthermore R and Perl scripts are used to provide tools for data analysis. 3Albrecht D, Kniemeyer O, Brakhage AA, Guthke R (2007) Integration of transcriptome and proteome data from human-pathogenic fungi by using a data warehouse. J Integrative Bioinf 4, 52.

Results: The data warehouse stores images and corresponding data from microarray or 2D gel experiments and mass spectrometry data. Basic genomic data are derived from NCBI RefSeq and UniProt. All data are cross-linked. Access to the data warehouse is easy via internet at www.omnifung.hki-jena.de. We not only store transcriptomic and proteomic data but also provide some analysis tools. Just recently, DIGE analyser, a tool for the analysis of 2D DIGE data became publicly available. Furthermore, we offer FungiFun, a tool for functional categorization of fungal genes and proteins using FunCat, GO and KEGG. Additional tools, for example for promoter analysis or clustering are in preparation.

With the help of the data warehouse, transcriptomic and proteomic data of different experiments can be compared with only few clicks. Own experiments can be stored and experiments of other can be re-analyzed under new points of view without the need to re-do them. Pathogenic fungi can be compared with each other and with their non-pathogenic relatives. All this helps to get new insights into pathogenicity processes. Introduction: Dehydroepiandrosterone (DHEA) is the major circulating steroid hormone in humans. As an intermediate in the sex steroid synthesis it can be metabolized to both testosterone and estrogen. DHEA has been reported to exhibit predominantly estrogenic effects in a male hormonal environment. Estrogens appear to exhibit immunoprotective properties following trauma, shock and sepsis. Objectives: Therefore, we studied the effects of DHEA administration upon leukocyte activation and related parameters within the microcirculation in experimental sepsis (colon ascendens stent peritonitis -CASP) using intravital microscopy (IVM) of the wall of the terminal ileum. Introduction: Activated protein C (APC) is a physiologic anticoagulant, which has additionally multiple cytoprotective effects, including anti-inflammatory activity and may support endothelial barrier function. Recombinant human APC reduces mortality in severe sepsis patients. Encephalopathy is an early clinical indicator of sepsis. However, the changes within the cerebral microcirculation that may be responsible for the organ dysfunction are not yet completely elucidated and specific therapies are not established. Objectives: The goals of the study were to evaluate leukocyte-endothelial interactions, capillary perfusion and plasma extravasation within the pial microcirculation during experimental endotoxemia in rats and to study the effects of APC in this setting by intravital microscopy (IVM). Methods: A prospective, randomized, controlled animal study was conducted with 40 male Lewis rats. The groups consisted of the animals that (A) were untreated (controls), (B) had induced colon ascendens stent peritonitis (CASP; (4) ), (C) received endotoxin bolus (5 mg/kg lipopolysaccharide, LPS, i.v.), and (D) received endotoxin bolus (5 mg/kg LPS i.v.) accompanied by APC (2 mg/kg i.v.). After 2 hours of observation, IVM through two cranial windows (5) was performed and leukocyte-endothelial interactions, functional capillary density (FCD) and plasma extravasation were determined within the pial microcirculation. Results: During CASP sepsis and endotoxemia (groups B & C), leukocyte adherence and plasma extravasation in the pial microcirculation were significantly increased. FCD was significantly reduced because of a high number of dysfunctional or non-perfused capillaries (P<0.05, versus group A). The APC treatment (group D) reduced leukocyte adherence significantly as compared to untreated LPS animals (group C). In addition, a significant increase of the FCD was found in group D compared to group C (P<0.05).

The APC treatment reduced sepsis-associated impairment of the pial microcirculation in rats.

Patients with compensated liver cirrhosis are often admitted to the ICU because of an acute event like gastrointestinal bleeding complicated by sepsis or multisystem organ failure. Despite the reversible nature of this condition and the high potential for treatment of the primary event, the mortality is still very high. The currently available liver support systems failed to show benefits in terms of survival. Hepa Wash® is a newly developed liver support system based on the use of a recycled albumin dialysate.

Objectives: Aim of the study is to evaluate the safety and efficacy of Hepa Wash® in a newly developed animal model that simulates the clinical syndrome 'AoCLF' (acute-on-chronic liver failure). Methods: Eight female German Landrace pigs (~60Kg) were randomly allocated to the Hepa Wash (n=4) and control (n=4) groups. Animals were admitted to the operation room (on Day 0) for the surgical induction of ischemic and cholestatic liver injury. This was performed via a functional end-to-side portosystemic anastomosis and ligation of main bile ducts. Animals were returned then to the stall for observation. On the third postoperative day (Day 3), the animals were readmitted to the operation room and prepared for the induction of endotoxemia. The lipopolysaccharide (E.Coli O111:B4) was administered intravenously (after 90 minutes stabilization period) at an initial dose of 4 µg/kg/hr with a stepwise twofold increment every hour and stopped after 7 ½ hours. Treatment with Hepa Wash was started 2.5 h after endotoxemia and continued till the end of the 8-hour observation period. Results: On Day 3, animals (n=8) had hyperbilirubinemia (5.8 ± 0.3 mg/dl; mean ± SEM) and hyperammonemia (284± 44 µg/dl). After starting endotoxin infusion, there were signs of capillary leak, leukopenia, thrombocytopenia, respiratory distress, hyperdynamic/hypodanymic circulation and renal failure. All animals in the Hepa Wash group survived the 8-hour observation period. Animals in the control group died after (6.4 ± 0.4 h) though they received only one third of the total endotoxin dose on average. At the end of treatment with Hepa Wash, there were improvements of cardiovascular (e.g. mean blood pressure of 65 ± 10 vs. 30 ± 5 mmHg), cerebral, respiratory and renal parameters as well as the elimination of surrogate parameters as bilirubin (3.1 ± 0.5 vs. 5.4 ± 0.3 mg/dl) and ammonia (242 ± 75 vs. 1260 ± 553 µg/dl). We did not notice any adverse effects during treatment. The susceptibility of the intestinal barrier to platelet activating factor PAF in the isolated perfused rat small bowel Introduction: Liberation of inflammatory mediators like platelet activating factor (PAF) provoke barrier dysfunction, septic shock and multiple organ failure. Over the past years, the pharmacodynamics of PAF were decrypted, e.g. in the respiratory tract by use of isolated perfused lungs improving our understanding of pulmonary oedema formation.

Objectives: Surprisingly, research has not focused to the same extent on another important barrier organ, the intestine. We identified the dose-response relationships of PAF in the isolated rat small bowel. Methods: Small intestines were harvested from Wistar rats and perfused vascularly and luminally in a warm humidified chamber. After equilibration intestines were challenged vascularly with different doses of PAF (0.5, 5, 50, 150, 500 and 1500 pmol) . In one experiment, the PAF receptor antagonist ABT-491 was applied in parallel. Arterial, venous and luminal pressures as well as venous, luminal and lymphatic effluent flows were recorded. The transfer of FITC dextrane (150kD) from the vessels to the lumen and lymphatics was measured every 15 minutes. All parameters regarding stability and viability were recorded as described before (1). Results: PAF administration in increasing amounts from 0.5 to 1500 pmol evoked its typical transitory effects like vasoconstriction (Δ Pmax from 1.9 to 32 mmHg), loss of vascular fluid (from 0.8 to 27.6 ml/10min) and transfer of FITC dextrane from the vascular buffer to the luminal juice (from 1.7 to 373 µg/15min) and the lymph (from 6.4 to 191 µg/15min Introduction: One of the therapeutic essentials in septic shock is adequate volume replacement. The type of solution to be used as volume replacement remains controversial. Objectives: Using a porcine two hit model combining a haemorrhagic and septic shock we tested the effects of 6%HES 130/0.42 (HES130), Gelatine 4% (Gela) and 10%HES 200/0.5 (HES200) compared to Ringer Acetate (RAc) on inflammation and systemic haemodynamics. Methods: Prospective randomised, controlled animal study. 23 anaesthetised, ventilated pigs (28.2 ± 1.7 kg) (5 in each shock group, 3 in control group). Animals were bled until reaching half of their baseline mean arterial pressure (MAP) for 45 minutes. After hemorrhagic shock volume resuscitation started until baseline MAP was accomplished. As second hit sepsis was induced using an E. Coli bacterial clot into abdominal cavity 6 h after hemorrhagic shock. Infusion rate was titrated to maintain a central venous pressure of 12 mmHg. Systemic haemodynamics and cytokines were obtained before (bl) hemorrhagic and septic shock and every 2 hours after induction of septic shock. Statistics were performed using ANOVA. Results: 12h after sepsis induction MAP (mmHg) was lower in RAc group (59±5) compared to all other groups (HES200 70±21;HES130: 86±11;Gela: 71±26;Control: 96±7). CO [ml/kg] increased in all groups and was higher in colloid groups at study end (HES200: 143±48;HES130: 171±47;Gela: 160±42;RAc: 137±32;Control: 120±28). TNF-α Levels [pg/ml] Introduction: Sepsis remains one of the major reasons for death in intensive care units in industrialized countries around the world. The pathophysiology involves a vast network of pro-inflammatory cytokines such as tumor necrosis factor-(TNF-), interleukin-1β (IL-1β) and interleukin-6 (IL-6). This is accompanied by an up regulation of transcription factors such as nuclear factor-κB (NF-κB). Objectives: Previously it has been shown that the clinically used central acting alpha2 agonist clonidine is able to significantly reduce proinflammatory cytokines in surgical patients. We therefore hypothesize that central acting alpha-2 agonists, like clonidine or dexmedetomidine, have the ability to improve survival in experimental sepsis by inhibiting the sympathetic tone and consequently inhibiting pro-inflammatory cytokine release. Methods: We conducted a prospective, randomized laboratory investigation using a murine model of CLP-induced sepsis. Animals receiving pre-emptive injections were treated with either clonidine (5µg/kg) or dexmedetomidine (40µg/kg) 12 and 1 hours before the operation, as well as 1, 6 and 12 hours afterwards. Another group of animals only received clonidine (5µg/kg) 1h, 6h and 12h after the operation, while the pre-emptive injections were done with normal saline. The control groups received solvent injections at the respective time points. Results: Administering clonidine or dexmedetomidine pre-emptively significantly reduced mortality (clonidine: p=0.015; dexmedetomidine: p=0.029), while postoperative administration failed to prolong survival significantly. Moreover pre-emptive administration of clonidine significantly attenuated the cytokine response after CLP-induced sepsis (mIL-1β: p=0.017; mIL-6: p<0.0001; mTNF-α: p<0.0001), preserved blood pressure control (p=0.024) and down-regulated the binding activity of NF-κB. There were no changes in the pro-inflammatory cytokine response when peripheral blood was incubated with LPS alone compared to clonidine (10-4) plus LPS incubation. Conclusions: These results demonstrate that the pre-emptive administration of either clonidine or dexmedetomidine have the ability to successfully improve survival in experimental sepsis. Moreover there seems to be a connection between the central muscarinic network and the vagal cholinergic response. By down-regulating pro-inflammatory mediators sympatholytics may be a considered as a pre-emptive therapeutic option in high-risk patients undergoing major surgery. Conclusions: Our 2-D reference map of conidial proteins from A. fumigatus constitutes the first global view on the proteome of resting conidia of an Aspergillus species. Conidial germination in the lung is a key step in the establishment of invasive aspergillosis. The results of our detailed in-gel comparison of spore-, germling-and myceliumspecific protein expression will broaden our understanding of this process. We expect that our data will facilitate further comparative proteomic studies as well as immunoproteomic applications and thereby contribute to the identification of fungal allergens and virulence factors. Introduction: Thoracic epidural anesthesia (TEA) effectively reduces intraoperative sympathetic stimulation resulting from surgical trauma. TEA is therefore an integral part of a multimodal concept to accelerate rehabilitation after major abdominal and thoracic surgery and is increasingly incorporated into therapeutic strategies in critical care medicine. If TEA is also useful to accelerate recovery of high-risk patients with severe sepsis and septic shock is still not fully understood.

Objectives: Since pulmonary nitric oxide (NO) has been identified to play a critical role in septic lung injury (1), we hypothesized that TEA modulates endothelial dysfunction via a NO-dependent pathway. Methods: 28 Sprague-Dawley rats underwent sham laparotomy or induction of endotoxemia (Salmonella typhosa LPS i.p.). Septic animals were then treated with either bupivacaine 0.5% (15 µl×h-1, LPS + TEA group) or normal saline epidurally (15 µl×h-1, LPS group) for 6 h post injury. In addition, two sham control groups received either bupivacaine 0.5% (15 µl×h-1, SHAM + TEA group) or normal saline epidurally (15 µl×h-1, SHAM group). After 6 h exhaled NO (exNO) was measured in anesthetized and tracheostomized rats. Then bradykinin (BK)-induced pulmonary vasoconstriction (= surrogate marker of endothelial dysfunction) was investigated using an established model of isolated and perfused lungs. Therefore BK was injected into the pulmonary circuit in 3 different doses (1, 3 and 6 µg). Changes in perfusion pressure (Δp) were measured in mmHg. Differences between groups were evaluated with one-way ANOVA. Results: ExNO levels did not differ between SHAM (exNO 2.1 ± 0,2 ppb) and SHAM + TEA (exNO 3.1 ± 0.3 ppb). Endotoxemic rats (LPS) exhibited a distinctive rise in exNO levels (exNO 116 ± 10 ppb, p < 0.05 vs. SHAM). Treatment with TEA (LPS + TEA) resulted in a significant reduction of exNO levels (69 ± 12 ppb) when compared to LPS (p < 0.05) but remained elevated when compared to SHAM (p < 0.05). Pulmonary endothelial integrity was maintained in SHAM and SHAM + TEA as indicated by the absence of BK-induced pulmonary vasoconstriction (SHAM: BK 1µ 0 ± 0mmHg, BK 3µ 0.33 ± 0.2mmHg, BK 6 µg 0.33 ± 0.2mmHg and SHAM + TEA: BK 1µ 0.33 ± 0.7mmHg, BK 3µ 0.7 ± 0,3mmHg, BK 6 µg 0.8 ± 0,3mmHg Introduction: Septic acute lung injury is linked to elevated pulmonary nitric oxide (NO)-Synthase activity, which in turn alters hypoxic pulmonary vasoconstriction (HPV) (1) . Thoracic epidural anesthesia (TEA) has been shown to restore blunted HPV in acute pancreatitis in rats by suppressing exhaled nitric oxide (exNO) production (2). Objectives: We hypothesized that TEA improves HPV in isolated perfused rat lungs by affecting pulmonary NO production during endotoxemia. Methods: 32 Sprague -Dawley rats underwent sham laparotomy or induction of endotoxemia secondary to intraperitoneal injection of Salmonella typhosa lipopolysaccharide (LPS). Septic animals were then treated with either bupivacaine 0.5% (15 µl×h-1, LPS + TEA group) or normal saline epidurally (15 µl×h-1, LPS group) for 6 h post injury. In addition, two sham control groups received either bupivacaine 0.5% (15 µl×h-1, SHAM + TEA group) or normal saline epidurally (15 µl×h-1, SHAM group). After 6 h anesthesia and tracheostomy was performed. ExNO from rats` lungs was measured followed by isolation and perfusion of the pulmonary circuit. Angiotensin II (= surrogate marker for vascular smooth muscle cellfunction) was injected into the circuit to test vasoreactivity. Lungs were ventilated with a hypoxic mixture (3% O2) for 10 min. and then with a normoxic mixture (21% O2) for an equal period. Changes in perfusion pressure (Δp) were measured in mmHg. Differences between groups were evaluated with one-way ANOVA. Results: ExNO levels did not differ between SHAM (exNO 2.1 ± 0,2 ppb) and SHAM + TEA (3.2 ± 0.3 ppb). Endotoxemic rats (LPS) exhibited a marked rise of exNO levels (exNO 115 ± 9 ppb, p < 0.05 vs. SHAM). Treatment with TEA (LPS + TEA) resulted in a significant reduction of exNO levels (64 ± 10 ppb) when compared to LPS (p < 0.05) but remained elevated when compared to SHAM (p < 0.05). Endotoxemic rats exhibited a distinct HPV response (LPS: 19.2 ± 1.3 mmHg) in comparison to healthy animals (SHAM: 9.1 ± 0.9 mmHg, p < 0.05). Treatment with TEA resulted in a significant reduction of HPV in endotoxemic rats (LPS + TEA: 13,1 ± 2,3 mmHg, p < 0.05 vs. LPS) and healthy animals (SHAM + TEA: 5,7 ± 0,6 mmHg, p < 0.05 vs. SHAM). Angiotensin II induced pulmonary vasoconstriction was comparable among groups (SHAM: 2,2 ± 0,3 mmHg; SHAM + TEA: 1,7 ± 0,3 mmHg; LPS: 2,7 ± 0,6 mmHg; LPS + TEA 2,6 ± 0,3 mmHg).

We demonstrated that the implementation of TEA in endotoxemia resulted in a downregulation of the HPV response and a reduction of exNO levels. Since it is known that NO acts as a potent pulmonary vasodilator we conclude that TEA-related actions on HPV in endotoxemia are independent from exNO. The underlying mechanisms to explain the pulmonary vasoregulation following TEA under healthy and septic conditions needs further investigation. Introduction: Production of reactive oxygen species (ROS) by macrophages (MΦ) is an important mechanism to effectively kill microbes. However, this beneficial effect of ROS can be deleterious for the producing cell and the surrounding tissue. Therefore, intracellular defense mechanisms exist to detoxify ROS. One of them, heme oxygenase-1 (HO-1), affiliates to the intracellular protective systems, to increase MΦ endurance and to contribute to a confined pro-inflammatory response. Although versatile mechanisms provoking HO-1 expression in MΦ are mainly attributed to Nrf2-dependent gene induction, a role of PPARγ in regulating HO-1 expression remains controversially discussed.

Objectives: Therefore, we aimed at clarifying the role of PPARγ in HO-1 expression in MΦ. Introduction: Poly-traumatized patients who survive the initial insult are highly susceptible to secondary pneumonia, often progressing into sepsis and multi-organ failure. Severe trauma and hemorrhage has been shown to induce a strong initial inflammatory response followed by a sustained impairment of the host innate immunity. However, the underlying pathophysiological mechanisms of this immune suppression are poorly understood.

Objectives: This project aimed to investigate the role of MAPK signalling pathways for the development of impaired lung innate defence mechanisms in a two-hit model of acute trauma/hemorrhage (T/H) and subsequent pneumonia. Methods: Trauma was induced by midline laparotomy, followed by cannulation of femoral arteries and the right femoral vein to induce hemorrhage to mean blood pressure levels of 35.0 ± 5.0 mm Hg maintained for 60 min. Animals were resuscitated using Ringer s solution.

Mice were then infected with a pneumolysin-producing clinical isolate of S. pneumoniae. MAP kinase activation was measured in whole lung homogenates using Western blotting, and determination of bacterial loads was done in bronchoalveolar lavage fluids and lung parenchymal tissue. Organ dysfunction was determined via automated clinical chemistry. Phagocytosis capacities and cytokine responses by blood leukocyte subsets were assessed by FACS and ELISA techniques. Results: In the lungs of mice subjected to T/H, a significant increase of p38 MAPK activation was observed. In parallel, we observed increased bacterial loads in the lungs of T/H mice infected with S. pneumoniae, reflective of innate immune suppression in these mice. Interestingly, mice subjected to T/H followed by immediate inhibition of p38 MAPK activity responded with an increased bacterial clearance to infection with S. pneumoniae compared to control groups. Moreover, elevated levels of creatinine were also significantly reduced in T/H mice treated with p38 MAPK inhibitor. In addition, p38 inhibition in T/H mice significantly decreased the phagocytosis activity of granulocytes and reduced cytokine levels in plasma (IL-6, TNFα, IL-10). Conclusions: Our data show that induction of T/H leads to a strong p38 MAPK activation in the lung, which may contribute to post-traumatic immune suppression. Early p38 MAPK inhibition subsequent to T/H leads to decreased inflammatory responses, less organ damage in the kidney and an improved lung protective immune response to bacterial challenge. Objectives: This study aimed at defining the role of the alternative complement pathway for intact and controlled innate immune responses during the onset of experimental sepsis Methods: To investigate the effect of impaired activation of the classical/alternative complement activation pathway on outcome and innate immune functions during sepsis, we conducted CLP (cecal liga-tion puncture) experiments in C1q-/-and factor D-/-mice. Mice first were monitored for various signs of sickness for 7 days. Whole blood and plasma was collected to determine organ dysfunction via automated clinical chemistry, phagocytosis assay and measurement of various Cytokines via Cytometric Bead Array (FACS Analysis). Results: C1q-/-mice started to die after 24h very rapidly and after 54h none of the animals survived. Although factor D-/-mice survived longer the mortality also reached 100 percent after approximately 4 days. Wild type mice died only within the first 36h and the survival rate was approximately 58 %. Analysis for presence of various inflammatory mediators in the serum demonstrated a significant increase for IL-6, IL-10, MCP-1, and KC 6h after CLP in control mice. In factor D-/mice this increase was significantly higher for all depicted mediators when compared to the other groups. Investigation of phagocytosis activity in neutrophils showed decreased numbers of phagocytosing neutrophils. We analyzed organ specific indicators in the serum of septic mice. Factor D-/-and C1q-/-mice displayed increased values of urea and creatinine, AST and bilirubin at 24 h post CLP when compared to wilt type mice indicating elevated organ dysfunction of kidney and liver in the knockout mice.

Our results demonstrate an impaired survival capacity of the complement knock-out mice when compared to the wild type controls suggesting an important role for the classical and the alternative pathway for successful host response during sepsis. In addition, it can be hypothesized that factor D may have a controlling function for inflammatory responses during the onset of sepsis. Introduction: In healthy organisms the balance between pro-and antiinflammatory reactions functions as a prerequisite ensuring an appropriate immune response. The extent of the inflammatory response following an ischemic insult in the central nervous system influences the well balanced interplay of the nervous and immune system. Thereby, infections, i.e. pneumonia, are the leading cause of death in patients after ischemic insult due to CNS injury-induced immunodepression syndrome (CIDS). Furthermore ageing per se is associated with progressive incidence of immune dysregulation. Objectives: We focussed on the triggering receptor expressed on myeloid cells-2 (Trem-2) involved in the elimination of apoptotic cells without local inflammation to maintain brain homeostasis in healthy organisms. Since the role of Trem-2 during ageing and under ischemic conditions is still under discussion, there is increasing evidence that mutations in the gene coding Trem-2 are strongly associated with neurodegenerative disorder and infection/sepsis. Therefore, we study the role of Trem-2 as mediator of innate immune system in CNS after inflammatory insult during aging. Methods: Using qPCR we studied the gene expression pattern of Trem2 and Dap12, linked to Trem2, as well as mediators of inflammation (Tnfa and Il10) in mice brain at different age (2, 9, 15 sion of Trem2 and Dap12 declined. The expression rate of Tnfa increased during normal ageing (5 fold) as well as following MCAO (up to 17 fold). The up-regulation of Tnfa is attenuated in older mice following MCAO. Il10 is very low expressed in healthy brains and ageing per se shows no considerable effect to Il10 gene expression. Though, after MCAO Il10 was up-regulated in young animals but not influenced in elderly.

The up regulation of Trem2 as well as Dap12 after stroke attenuated with increasing age. Thus, we suggest that phagocytic activity is reduced, however inflammatory response is enhanced. In line with that, we showed that Il10 as one of the prototypic anti-inflammatory mediator is influenced to a higher degree than Tnfa as prototypic pro-inflammatory mediator after inflammatory insult at different age. Introduction: Thoracic epidural anesthesia (TEA) is an integral part of a multimodal concept for pain management in acute pancreatitis in selected cases. The impact of TEA on lung injury associated with inflammation is still unkown. We have previously shown that TEA prevented a rise in exhaled nitric oxide (exNO) levels and improved the impairment of pulmonary vasoreactivity associated with experimental necrotizing pancreatitis but had no effect on pulmonary endothelial dysfunction (1) . Conversely, pulmonary endothelial dysfunction was time-dependently modulated by TEA in experimental sepsis with improvement of endothelial integrity in hyperdynamic sepsis and deterioration of endothelial function in hypodynamic sepsis (2). Objectives: We hypothezised that TEA affects pulmonary nitric oxide production and modulates pulmonary endothelial dysfunction in mild edematous pancreatitis. Methods: 18 rats were instrumented with a thoracic epidural catheter (Th 7-9). Edematous pancreatitis (EP) was induced by intraductal injection of caerulein. Rats were randomly assigned to epidural infusion of NaCl 0.9% (EP, n = 6) or bupivacaine 0.5% (EP + TEA, n = 6) at 15 µl/h for 4 h. Healthy controls (SHAM) assigned to a third group received epidural NaCl 0.9% (SHAM, n = 6). 4h post injury animals were tracheostomized and exhaled NO (exNO) was determined. The pulmonary circuit was isolated and perfused. Then bradykinin-induced pulmonary vasoconstriction (= surrogate marker of endothelial dysfunction) was investigated using an established model of isolated and perfused lungs. Therefore bradykinin was injected into the pulmonary circuit in 3 different doses (1, 3 and 6 µg). Changes in perfusion pressure (Δp) were measured. Differences between groups were evaluated with one-way ANOVA. Results: ExNo levels did not differ between the 3 groups (SHAM 2,2 ± 0,3 ppb; EP 3,0 ± 0,4 ppb, EP + TEA 1,8 ± 0,5 ppb). Pulmonary integrity was maintained in SHAM animals as indicated by the absence of BK-induced pulmonary vasoconstriction (SHAM: BK 1µg 0 ± 0 mmHg, BK 3µg 0 ± 0 mmHg, BK 6 µg 0 ± 0 mmHg). Rats with EP exhibited a significant rise in bradykinin-induced pulmonary vasoconstriction (EP: BK 1µg 0,7 ± 0,3 mmHg; BK 3µg Introduction: Recent research has revealed an autonomic neural pathway that monitors and adjusts the inflammatory response, termed the inflammatory reflex. Sensory vagal afferent fibres are able to sense inflammatory mediators. The efferent arm of the inflammatory reflex has been termed the cholinergic anti-inflammatory pathway because acetylcholine (ACh) is the principle vagus nerve neurotransmitter. ACh is interacting with alpha7 cholinergic receptors expressed by macrophages and other cell types to inhibit production of inflammatory mediators, eg. cytokines and adhesion molecules (1, 2) .

The aim of the current study was to determine the capacity of the acetylcholine esterase inhibitor physostigmin (PhS), administered intravenously after endotoxin challenge, to inhibit endotoxininduced inflammatory responses in the rat intestinal microcirculation in vivo by intravital microscopy. Methods: A total of 40 male Lewis rats were randomly assigned to four groups (n=10): control, experimental endotoxemia (5 mg/kg lipolysaccharide from E. coli, serotype O26:B6), endotoxemia+20 mikrog/kg PhS (Anticholium®, Dr. F. Köhler Chemie, Germany), PhS alone. Following 2 hours of observation all animals underwent intravital microscopy of the intestinal microcirculation. Results: Endotoxemia resulted in a more than 60% reduction of the functional capillary density of the muscular and mucosal layers and a 4fold increase of the leukocyte adhesion in the submucosal venules of the intestinal wall. PhS administration completely reversed endotoxininduced disturbances of the functional capillary density. Leukocyte adhesion was completely abolished by PhS in the collecting venules of the submucosa and significantly reduced in the postcapillary venules compared to untreated animals (P<0.05).

The results suggest, that PhS protects intestinal microvascular perfusion as measured by functional capillary density and reduces leukocyte activation in rat experimental endotoxemia. Because of the importance of the intestinal microcirculation in the development of multiorgan failure in clinical sepsis PhS administration may have potential benefit for patients with sepsis. Introduction: Antibiotic therapy represents a mainstay of clinical sepsis therapy. It can be principally assumed that in parallel to the antibacterial effect, an improvement of the impaired microcirculation occurs. However, the toxin release during administration of antibiotics may also lead to an acute impairment of the microcirculation. Objectives: Therefore, the goal of our study was to investigate the acute effects of the new antibiotic substance tigecyclin (TIG) within the mesenteric microcirculation and on the cytokine release in experimental endotoxemia in rats.

A total of 40 male Lewis rats were randomly assigned to four groups (n=10): controls, LPS (15 mg/kg lipopolysaccharide iv.), TIG (Tigecyclin 5 mg/kg) and LPS+TIG. We determined leukocyte-endothelial interactions and plasma extravasation at 0, 1 and 2 hours following the administration of placebo, LPS and Tigecyclin by intravital microscopy (IVM). Cytokine plasma levels were measured at 0 and 2 hours. Results: Leukocyte adherence during endotoxemia was diminished significantly following TIG treatment (-42%) compared to untreated LPS animals. We measured also a significant decrease in the flux of the rolling leukocytes in both endotoxemic groups that was more pronounced in the untreated LPS group than in the LPS+TIG group. Endotoxin-induced plasma extravasation was significantly attenutated by TIG administration (P<0.05). The cytokine release was not affected by TIG.

Conclusions: In addition to the results of microbial sensitivity testing, a specific knowledge of the potential effects exerted upon the microcirculation is important in order to detect possible side effects of antibiotics. Acute tigecyclin administration did not worsen the mesenteric microcirculation or cytokine expression in experimental endotoxemia. In contrast, an improvement of the microcirculation could be observed. Therefore, tigecyclin treatment may be a preferred choice in the antibiotic therapy of severe sepsis. 

We established an oxygen-controlled chemostat, which enables accurate control and measurement of the oxygen partial pressure. A. fumigatus mycelium was cultivated at 100% and 1% atmospheric oxygen partial pressure at the same growth rate. After harvesting the mycelium, proteins were extracted and analysed by differential in gel electrophoresis (DIGE). Differentially expressed proteins were identified by MALDI-TOF/TOF analyses. Results: Proteins involved in glycolysis, amino acid biosynthesis, stress response and respiration showed an increased standard abundance under hypoxic conditions. In addition, a molecular oxygen incorporating monooxygenase of the ubiquinone biosynthesis pathway was upregulated as well. In contrast, proteins involved in sulphate assimilation and acetate activation were down-regulated. Strikingly, enzymes encoded by genes organised in a secondary metabolite gene cluster were also up-regulated. This finding was also confirmed on the transcriptional level and by HPLC analyses.

A. fumigatus adapts to low oxygen partial pressure by up-regulating respiration and by increasing the amount of enzymes involved in oxygen-consuming biosynthetic pathways. Furthermore, the production of secondary metabolites is induced upon hypoxic growth conditions. Introduction: Sepsis is a clinical syndrome that complicates severe infection and is characterized by systemic inflammation and widespread tissue injury. In face of extensive research in the past, the pathophysiology of sepsis in humans is still poorly understood. To study the underlying mechanisms of sepsis and the associated systemic inflammatory response, several experimental animal models have been developed. All models try to mimic pathophysiological changes typically seen in septic patients. Cecal ligation and puncture has become the most widely used model for experimental sepsis and is currently considered as the gold standard in sepsis research. To study the underlying mechanisms of sepsis in experimental settings CLP is considered to be a realistic model for the induction of polymicrobial sepsis.

Objectives: To present the CLP-sepsis model. Methods: For the CLP procedure in mice we use standardized procedures to induce acute sepsis. The mice are anesthetized, the cecum is exposed through a 2.5 cm abdominal midline incision and approximately 2/3 of the cecum is ligated. The ligated part of the cecum is then punctured through and through with a 21 gauge needle. After repositioning of the bowel, the abdomen is closed in layers and mice are monitored for various signs of sickness.

To determine the consequences of polymicrobial sepsis we typically analyze histological organ damage, quantify cytokine and chemokine levels in serum samples, analyze phagocytosis capacities of cells in whole blood, monitor organ dysfunction using automated clinical chemistry analyzer and we frequently determine coagulation abnormalities during sepsis by ROTEM analysis.

Conclusions: In this model, standardization of severity remains the most challenging part which is influenced by the needle size, the ligation area but also by the respective mouse strain under investigation. However, once established this model reaches a high reproducibility and simplicity and is, due its clinical relevance and widespread use, certainly a current gold standard. 

Peritonitis is a relevant model to study conditions associated with sepsis. The Cecal Ligation and Puncture (CLP) model is a widely accepted sepsis model mirroring the clinical situation in septic patients and is commonly considered as the gold standard. However, the CLP-model is also known for its difficulty to control the magnitude of septic challenge due to high variability in progression and survival depending on the applied technique, bacterial composition of the bowel content or surgical operator variability. These arguments cannot be neglected in a research setting in which many investigators apply sepsis models in different projects and intend to combine their findings to look at the big picture. The Peritoneal Contamination and Infection (PCI) model, originally described by Bauhofer and co-workers and modified by us, overcomes this issue representing a highly standardized, reproducible rodent sepsis model without the necessity for a surgical procedure. Objective: To present the PCI-sepsis model Methods: For PCI, standardized human stool inoculums are injected intraperitoneally into the pelvic region. For generation of the inoculums, a stool probe of healthy non-vegetarian humans is collected and processed for −80°C storage. Microbiological analysis determines the composition and number of cfu for the different pertinent strains. Results: Depending on the dose of the inoculum or accompanying treatments (fluid-resuscitation, antibiotics) the sepsis model can be modified in any order-starting from a sepsis model with acute onset and high mortality to a prolonged sepsis model with high survival rates. We established different sepsis models in mice (CL57B6, Balb/ c) and rats (WISTAR) and evaluated a variety of parameters describing many aspects of PCI-induced sepsis. Introduction: The function of the acute phase protein von Willebrand factor (VWF) is regulated by ADAMTS13-mediated proteolysis, which limits its multimeric size and ability to tether platelets. Therefore, the balance between VWF and ADAMTS13 is assumed to be involved in the development of sepsis-associated thrombotic microangiopathy (TMA). Objectives: We hypothesized that a decreased ADAMTS13-activity in a porcine 2-hit model of hemorrhage and sepsis is associated with laboratory and morphological findings of TMA. Methods: Animals (n=21, BW 24 kg) were subjected to hemorrhagic shock (45 min). After re-transfusion, sepsis was induced via intraperitoneal implantation of fibrin clot to restrain continuous release of 10 9 vital E.coli. Blood samples for determination of ADAMTS13-activity, VWF antigenconcentration as well as multimer pattern, for platelet count, creatinine and hematocrit (for normalization) were drawn. Directly postmortem we examined renal TMA development (CD61 staining) and ischemic acute tubular necrosis (epithelial brush border staining). Results: ADAMTS13 activity remained stable after hemorrhage, however, declined stepwise during sepsis accompanied by a strong drop off in platelet count (356 to 142 GptL-1). Moderate increase in VWF: Ag concentration was found. Densitometric analysis of VWF multimer composition revealed a shift to higher molecular weight VWF multimers, which may be a consequence of both, increased release of unprocessed protein from activated platelets and endothelial cells as well as of decreased proteolysis of circulating VWF by the protease ADAMTS13. Development of renal dysfunction was indicated by a slight, but significant increase in plasma creatinine concentration (107.0 vs. 141.0 µMolL-1, p<0.05). Characteristic findings for TMA with glomerular microthrombi in kidneys, fibrinoid necrosis of preglomerular arterioles and interlobular arteries with interstitial bleeding were observed. Glomerular luminal platelet activation and ischemic acute tubular necrosis were most prominent in septic animals in comparison to sham-operated ones.

Conclusions: Similar to patients with severe sepsis a decline in AD-AMTS13 activity in association with platelet consumption was observed in a septic porcine model whereas renal TMA was present in one animal. Therefore, we present an animal model to perform functional studies focusing on pathophysiological mechanisms of sepsisassociated TMA.

Infection 2009; 37 (Suppl. III): 21 mRNA coding the VWF cleaving protease is decreased under proinflammatory conditions -reversal by co-incubation with activated protein C and sodium selenite Introduction: In sepsis, the severity-dependent decrease of the VWFcleaving protease ADAMTS13 is a common phenomenon, which may contribute to aggregation of platelets/platelet consumption and the development of sepsis-associated thrombotic microangiopathy (TMA) and organ failure. Up to now, hepatic stellate cells (HSC) are considered to function as the primary source of ADAMTS13 protein. The underlying mechanisms of the decrease in sepsis remain unclear.

Objectives: Here we present data obtained in in-vitro experiments using cultured human HSC (LX2-line) and microvascular endothelial cells (HMEC) stimulated under proinflammatory conditions. Methods: Monolayers were exposed to i) cytokines known to be plasma abundant/relevant during systemic inflammation (TNF, IL-1beta, interferon gamma), ii) to bacterial endotoxin (100 ng/mL), iii) to a mixture of cytokines/endotoxin, or iv) freshly prepared serum obtained from patients (n=12) with severe sepsis/septic shock. Results: Both cell lines expressed ADAMTS13 mRNA as quantitated using qPCR normalized to a set of unvaried genes. Overall, incubation with cytokines resulted in a decrease of ADAMTS13 mRNA to different extents ranging between 40-80% of basal transcription rate in between 24 h. Furthermore, in endotoxin treated cells, ADAMTS13 declined around 60% of basal levels. This effect was more pronounced by the mixture of cytokines/endotoxin to levels of 55% (HSC) or 40% (HMEC). In monolayers treated with serum from patients with sepsis, only 10 % (HSC) or 49% (HMEC) of basal level was determined. Both, the trace element selenium and activated protein C, which are used in the supportive therapy of patients with sepsis, ameliorates the decrease in serum treated HSC cells and increased the level of AD-AMTS13 transcript in endothelial cells. Continuous infusion adapted to body weight also abolished the decrease of ADAMTS13 expression in hepatic tissues during the course of polymicrobial sepsis in mice.

In conclusion, we found that mRNA coding ADAMTS13 protein is also present in endothelial cells. Also we observed a marked decrease in both cell lines undergoing proinflammatory stimulation. This mechanism may contribute to the decline of proteolytic activity of ADAMTS13 in patients with sepsis and sepsis-associated TMA. Furthermore, the amelioration of this effect by selenate and APC may function as mechanisms resulting in a more favorable outcome observed in a number of clinical studies.

Infection 2009; 37 (Suppl. III): 21

Winning J Department of Anesthesiology and Intensive Care Medicine, University Hospital Jena, Germany.

In daily routine peritonitis often leads to abdominal sepsis with consecutive multiorgan dysfunction due to septic shock and impairment of micro circulation. The clinical course is influenced by the genetic background and the general condition of the patient, the virulence of the pathogen, the early identification and elimination of the septic focus, the early and effective antibiotic therapy, the appropriate fluid management in the context of a sophisticated therapeutic concept. Sepsis models will always fail to meet all these demands since they reflect only fragments of the interacting mechanisms. This however is a coexisting structural weakness and a benefit at the same time.

Whereas the transfer from experimental results into the clinical routine for example in the field of cytokines was disappointing, the focus on very little parts of septical pathways such as the induction via lipopolysaccharide (LPS) of SIRS as a part of Sepsis without continuous influence of persisting pathogens led to fundamental better understanding in endothelial function and its impact on microcirculation.

In general models of abdominal sepsis can be divided into three categories: -toxaemia models with exogenous administration of a toxin, such as lipopolysaccharide (LPS), exotoxins or zymosan bacterial infection models with exogenous administration of a viable pathogen such as bacteria and/or fungi (fecal peritonitis model) -host-barrier disruption models with alteration of the animal's endogenous protective barrier inducing colonic permeability, allowing bacterial translocation (caecal ligation and puncture (CLP)/ colon ascendens stent peritonitis (CASP) Depending on the question toxin models might be advantageous since they are simple, with little need for technical equipment and skills and due to the lack of additional trauma. The use of a fecal peritonitis model is technically equally simple, apart from the production of the biological agent. Beyond that different standardised bacterial-or fungi solutions can be used. They can be derived from human or animal faces or might contain isolated microbiological colonies. CLP/ CASP models are related to clinical needs since leakage of the gut is a common cause for abdominal sepsis in human patients. The animals used for experiments in septical research are usually pigs, rats, or mice. Especially in the last decade mouse models came to the fore due to their genetic variety, cost effectiveness and the progress to develop measure probes and surgical procedures that left almost nothing to be desired. Since many laboratories were upgraded to "MICU´s" continuous telemetric measurement of invasive blood pressure, heart rate, breathing frequency and activity of the awake mouse has become standard just as well as the measurement of cardiac output in mechanically ventilated mice. The oral presentation will give an overview on the techniques and significances of the abdominal sepsis models and will offer insight into the technical variety of monitoring, especially in mouse models. nohistological stainings of the kidneys were performed. The achieved blood concentrations of HES were comparable. To achieve the same hematocrit, significantly higher amounts of 6% HES 130 (1.9 ± 0.2 g/ kg) had to be administered compared to other groups (6% HES 200 1.5 ± 0.2, 10% HES 130 1.3 ± 0.5 and 10% HES 200 1.3 ± 0.2, P < 0.05). COP increased in all groups over time (P < 0.001), and was significantly higher in group 10% HES 200 (P < 0.05). There was no difference in viscosity of urine between groups. Over time, diuresis, creatinine clearance and sodium transport decreased significantly (P < 0.001), and to a similar extent in all groups, while N-acetyl-beta-aminoglucosidase (Beta-NAG) increased significantly (P < 0.001) in all groups. After 4 hours, Beta-NAG was significantly higher in the 10% HES 200 group compared to both 6% HES groups (P < 0.05), but did not differ to 10% HES 130. HES concentration in urine increased steadily severalfold over time (P < 0.001) and similarly in all groups. Osmotic nephrosis-like lesions were present in all groups to a similar degree. in 10% groups was higher compared to 6% groups (10% HES 130 8 ± 7.5, 10% HES 200 11.2 ± 6 vs. 6% HES 130 5.3 ± 1.4, 6% HES 200 5.1 ± 1.7), the difference reaching statistical significance between 10% HES 200 compared to 6% HES 130 and 6% HES 200 (P < 0.05). Conclusions: In this model of ex-vivo renal perfusion, all tested HES solutions led to a significant decrease in diuresis, creatinine clearance and impaired tubular reabsorption of sodium and starch irrespective of molecular weight and concentration of the administered HES solution. Despite the fact, that 6% HES 130 needed to be administered at significantly higher amounts, 6 % HES 130 as well as 6% HES 200 induced statistical significant less tubulointerstitial cell proliferation and induced less beta-NAG compared to 10% HES 200/0.5. Introduction: Monocyte/ macrophage desensitization contributes to immune paralysis during sepsis. This hypo-inflammatory sepsis phase is the main cause of death within septic patients. One characteristic feature of desensitized monocytes/macrophages is PKCα depletion. A crucial function of PKCα is activation of the NADPH oxidase system, which generates superoxide radicals. This oxidative burst is important for pathogen killing. Thus, preventing PKCα depletion in monocyte/ macrophage might be useful to improve septic outcome.

Objectives: Therefore, this study was designed to identify a pretreatment attenuating PKCα depletion during endotoxic shock in mice. Methods: Intra peritoneal application of lipopolysaccharide (LPS) for 24 h provoked PKCα depletion in CD11b+ splenocytes. In line, reactive oxygen species formation in response to a phorbolester (PMA) in vitro in peritoneal macrophages was abolished. Rosiglitazone pretreatment of mice for 30 min before LPS challenge, abrogates PKCα depletion in CD11b+ splenocytes and consequently restores the oxidative burst in response to PMA in vitro.

In Mac-PPARγ-KO mice, LPS injection causes PKCα depletion similar to PPARγ wild type mice. In contrast, rosiglitazone pretreatment did not prevent PKCα depletion in CD11b+ splenocytes derived from these Mac-PPARγ-KO mice. Hence, in vitro analyzed ROS formation was abolished. Conclusions: Our data provide evidence that pre-activation of PPARγ inhibits PKCα depletion in CD11b+ splenocytes and peritoneal macrophages and accordingly restored the oxidative burst in an endotoxic shock model. Preventing these established molecular markers of monocyte/ macrophage desensitization in sepsis might be useful to improve septic outcome with a special concern to immune paralysis. Introduction: Severe sepsis, septic shock, and resulting organ failure represent the most common cause of death in intensive care medicine, with mortality ranging from 40% to 70%. It is still unclear whether necrosis or apoptosis plays the predominant role in severe sepsis. Objectives: Determining the prevalent mode of cell death would be valuable, as new therapeutic agents (eg, antiapoptotic drugs such as caspase inhibitors) may improve unsatisfactory outcomes in patients with severe sepsis. Methods: In total, 147 patients (101 patients with severe sepsis, 28 postoperative patients after major abdominal surgery, 18 healthy volunteers) were enrolled. Baseline and clinical data were evaluated. Blood samples from patients with severe sepsis were collected at the time of sepsis diagnosis, and 48 and 120 hours later; samples from healthy volunteers were collected once, and from postoperative patients, once immediately after surgery. We measured caspase-cleaved and uncleaved Cytokeratin-18 (CK-18, intermediate filament protein) as a marker of cell death, isolated CK-18 fragments as a marker of apoptosis, as well as interleukin-6 (IL-6), soluble vascular cell adhesion molecule (sVCAM-1), and soluble intercellular adhesion molecule (sICAM-1). Results: Age and sex of patients with severe sepsis and postoperative patients were comparable, whereas healthy volunteers were significantly younger. In healthy volunteers, the mode of cellular turnover was primarily apoptotic cell death. Postoperative patients showed comparable levels of apoptotic activity, but necrotic cell death was markedly increased, probably due to surgical tissue injury. In contrast, patients with severe sepsis showed increased levels of markers for both apoptotic and necrotic cell death. Patients with sepsis who died showed more pronounced necrotic cell death at later stages. Conclusions: Loss of parenchymal cells due to necrosis appears to be the primary mode of cell death in patients who die from severe sepsis. This may limit possible therapeutic options. Introduction: Human-pathogenic fungi have developed various immune evasion strategies including the acquisition of complement regulators from human plasma, such as factor H (CFH), that may protect the fungi against the host complement system. The possible role of these proteins in facilitating host responses, however, got little attention to date. Objectives: Because CFH can bind to certain host cells, we analyzed the role of CFH and other CFH family proteins in neutrophil-yeast interaction. Methods: Candida albicans (SC5314) and blood-derived human neutrophils were used in these studies. Binding of CFH family proteins was determined by flow cytometry, Western blot and ELISA. For receptor binding studies, monoclonal antibodies recognizing CD11b, CD11c, CD18 and control antibodies were applied. Neutrophil adhesion and generation of reactive oxygen species were measured in fluorescence assays. Lactoferrin and IL-8 were determined by ELISA from supernatants of yeast-neutrophil co-cultures. Results: Both the opportunistic pathogenic yeast C. albicans and human neutrophils bound the CFH family proteins CFH, CFH-like protein 1 (CFHL1), and CFH-related proteins 1 (CFHR1) and 4 (CFHR4), both from plasma and as recombinant proteins. The main binding sites responsible for interaction with neutrophils were localized to the seventh and the two C-terminal domains of CFH, using recombinant CFH fragments. Related domains in CFHL1, CFHR1 and CFHR4 appear responsible for binding of these CFH family proteins to neutrophils. The integrin CD11b/CD18 was identified as the major cellular receptor for CFH, CFHL1 and CFHR1, but not for CFHR4. Adhesion of neutrophils to C. albicans was increased by CFH, CFHL1 and CFHR1. Furthermore, yeast-bound CFH and CFHR1 enhanced generation of reactive oxygen species and the release of the antimicrobial protein lactoferrin by neutrophils. In the presence of CFH, killing of C. albicans by neutrophils was increased. Conclusions: While pathogen-bound CFH, CFHL1 and CFHR4 may play a role in downregulating complement activation at the microbial surface, we identify CFH, CFHL1 and CFHR1 as three related proteins that enhance neutrophil adhesion via CD11b/CD18. Furthermore, the results show that CFH and CFHR1, when bound on the surface of C. albicans, enhance antimicrobial activity of human neutrophils. Therapy of infectious diseases with special regard to bacterial sepsis Introduction: In this preclinical project specially designed peptides (antimicrobial peptides, AMP) based on variations of amino acid sequences from binding domains of natural lipopolysaccharide-binding proteins are studied with respect to their suitability to be effective (i) against bacterial infectious diseases, in general and (ii) as anti-sepsis agents, in particular.

Brandenburg K (1), Schuerholz T (2), Hornef M (3)(1)

The main aim of this preclinical study is to design effective anti-endotoxin agents, which are able to neutralize isolated lipopolysaccharide aggregates with the aim to inhibit the LPS-induced inflammation reaction. These should therefore be able to act as newly developped medicament at least in the case of Gram-negative sepsis.

The study will consist of a comprehensive biophysical study of the AMP:LPS interaction, of pharmacological and toxicologic investigations of the peptides, of their antimicrobial action in vitro and in vivo against various bacteria including also resistant strains such as MRSA (methicillin-resistant Staphylococcus aureus), in animal studies regarding the transfer and size distribution of LPS aggregates in epithelial cells of the gut, and in in vivo models of peritonitis and the septic shock syndrom.

The feasibility of this approach has been proven in experiments regarding the effective action against the LPS-induced cytokine expression in an in vitro model of human mononuclear cells. Selected peptides suppressed the LPS-induced cytokine production already at an 3:1 molar ratio peptide:LPS. Furthermore,in an in vivo model of septic shock the survival rate was similarly high as for the gold standard polymyxin B Besides, some of the peptides were efficient in killing Gram-negative and Gram-positive bacteria, but were not cytotoxic and hemolytic.

The data so far are highly promising with respect to an effective inactivation of endotoxins and killing of Gram-negative and Gram-positive bacteria. The most suitable peptides will be tested according to GLP and GMP standards as prerequiste for a scheduled subsequent clinical study.

Infection 2009; 37 (Suppl. III): 24

Ion mobility spectrometry of human pathologic bacteria-metabolic profiling by volatile organic compounds Introduction: Suspected bacterial infection is empirically treated with broad spectrum antibiotics because definitive diagnosis and antibiotic sensitivities require time consuming cultures. These delays significantly increase patient mortality, while empiric antibiotics result in greater expense and increasing antimicrobial resistance. Appropriate, early antibiotic treatment results in lower mortality rates than when given once culture results are known. A multi-capillary column equipped ion mobility spectrometer (MCC-IMS) measures complex mixtures of gases regardless of the water vapour content in real time without sample preparation or preconcentration.

The study describes MCC-IMS and GC/MS identification of VOCs in the headspace above seven clinically important bacterial species (Klebsiella pneu., Pseudomonas aeruginosa, Serratia marcescens, Staph. aureus, Strept. Pneumoniae, E. coli) and human pathogenic yeast (Candida albicans, Aspergillus fumigatus) cultured on Columbia blood agar media. Methods: For detection of volatile organic compounds of bacteria a special measurement chamber, consistent of stainless steel heated to 37 °C, a gas inlet and a gas outlet was used. The gas outlet was attached to MCC-IMS or to adsorbtion tubes for GC/MS measurements. Tenax GA tubes were thermally desorbed and transferred using a cryotrap to a gas chromatograph/mass spectrometer. After blank measurement, a media with microbial mats (swap after 24h incubation) was introduced into a measurement chamber and a sample for IMS-measurement was drawn. Additionally to MCC-IMS measurement a sample for GC/MS measurement was achieved by preconcentration of 2L volume on Tenax GR. Results: As discriminating substances ethanol, 3-hydroxy-2-butanon, 2-methylbutylacetat, cyclohexanon, benzaldehyd, benzeneethanol and tricyclodecan-1-amin can be described by means of MCC-IMS. The pattern of VOCs determined with MCC-IMS is specific for the investigated germs. The MCC-IMS results differ from GC-MS analysis, as not all substances detected with GC/MS (e.g. alkene), are educible with MCC-IMS.

The major advantage of the MCC-IMS system might be the feasibility of measurements without preconcentration or preparation of samples and analysis of complex gas mixtures regardless water vapour content in an online setup. A discrimination of different germs by metabolic profile and therefore detection of VOC is feasible for the investigated germs. The detection of bacterial VOCs in breath of patients suffering infections might be a most promising approach to achieve a more early diagnosis of infection and might lead to an early therapy.

Infection 2009; 37 (Suppl. III): 24

The infection-associated EED1 gene encodes for a key regulator of filamentous growth and virulence of Candida albicans

Martin R, Jacobsen ID, Hube B

Introduction: Candida albicans frequently causes oral infections in even mildly immunocompromised patients. During this type of infection the fungus invades oral mucosa and persists within the epithelium causing superfical lesions. Switching from a yeast to hyphal morphology is an essential requirement for invasion. In previous studies we have identified a C. albicans unique gene associated with oral infections, EED1, which was proposed to be a regulator of morphology. In this study, the role of Eed1 in filamentation and virulence of C. albicans was analysed. Objectives: We have analysed the pathogenicity of the eed1Δ deletion mutant in different infection models, ranging from in vitro assays using oral epithelial cell-based invasion models to intraperitoneal infections of mice. Additionally, we aimed to identify downstream targets of Eed1 by using C. albicans DNA microarrays and quantitative RT PCR.

Methods: For the analysis of the interaction of eed1Δ and wild type strains with human oral epithelial cells we have used the TR-146 cell line in both monolayers reconstituted human oral epithelium (RHE) tissue models. For in vivo studies, female Balb/C mice were infected intraperitoneally with C. albicans strains and sacrificed after 24h. Dissemination of the different C. albicans strains was analysed by histology. Total RNA from C. albicans interacting with oral epithelial cells

was isolated to examine either the whole transcriptome with DNA microarrays or single gene expression with quantitative RT PCR. Results: Our studies show that Eed1 is essential for the maintenance of filamentous growth of C. albicans under all tested in vitro and in vivo conditions. Strains lacking Eed1 were attenuated in damage of epithelial cells and tissue and in an intraperitoneal mouse infection model. Transcriptional profiling of eed1Δ during RHE infections revealed the downregulation of hyphal associated genes as well as the downregulation of the important transcription factor Ume6. Remarkable, the eed1Δ mutant produces only transient filaments and switches back to yeast cell growth after filaments had been formed, leading to a pure yeast cell population. Ectopic overexpression of Ume6 in eed1Δ mutant cells prevented budding of yeast cells from filaments and restored hyphal growth. This bypass was also observed on the transcriptional level with the expression of hyphal associated genes at a level similar to wild type Conclusions: The unique C. albicans EED1 gene encodes for a central regulator of hyphal growth. Although it is not required for initial induction of filamentous growth it is essential for its maintenance and for full virulence in several in vitro and in vivo models. We could show that the key transcription factor Ume6 is a downstream target of Eed1, suggesting that both act in a novel pathway that regulates the maintenance of hyphal growth in C. albicans. Introduction: Blood is generally sterile and represents a hostile environment for microorganisms. However, the human pathogenic yeast Candida albicans can disseminate in the host via the bloodstream and cause life threatening systemic infections and sepsis in immunocompromised individuals. Our previous work has shown that blood components influence the morphology, viability and transcriptional response of C. albicans. Of the various blood components, neutrophils apparently have the strongest effect on C. albicans (Fradin et al., 2003; Fradin et al., 2005) . It has been shown that neutrophils discriminate between the morphological stages of C. albicans (Wozniok et al., 2007) . Nevertheless, the mechanisms by which neutrophils detect, inhibit and kill C. albicans remain poorly understood. Objectives: We aim to elucidate the C. albicans response during the interaction with neutrophils and to identify fungal components which are important to overcome the attack by this immune cells. Ongoing experiments concentrate on the analysis of single cells exposed to neutrophils using GFP reporter strains, the interaction of yeast and hyphal cells of wild type and mutant strains with neutrophils. Methods: Human neutrophils were isolated from peripheral blood of healthy donors using gradient centrifugation. To determine fungal cell damage by neutrophils, XTT assays were performed. Neutrophil migration towards Candida cells was measured via a transwell system. To investigate the interaction of our reporter strains with neutrophils, fluorescence microscopy was used and a differential staining method was established. Results: Single cell analysis approaches show that certain genes, like the ammonium permease MEP2 are expressed after 1 h of incubation with neutrophils indicating nitrogen starvation. Expression of MEP2 was contact dependent. The phagocytic index of C. albicans cells in contact to neutrophils was 95%. Deletion of MEP2 lead to increased susceptibility to neutrophils, which is not due to enhanced neutrophil attraction.

To analyse the role of either yeast or hyphal morphology during interaction with neutrophils, various mutants lacking genes associated with morphology were investigated. Several mutants lacking genes known to be key regulator of morphology showed less sensitivity towards neutrophil killing whereas others showed even increased susceptibility. In some cases a higher resistance towards neutrophil killing can be explained by a reduced attraction of neutrophils in the transwell system. Conclusions: C. albicans experiences contact dependent nitrogen starvation during interaction with neutrophils. Since 95% of C. albicans cells in contact with neutrophils were phagocytosed, it can be concluded that phagocytosis causes nitrogen starvation. In addition, factors regulating the yeast-to-hyphal transition of C. albicans directly or indirectly regulate fungal survival during the interaction with neutrophils. Introduction: Candida glabrata is, after C. albicans, the second most important human pathogenic yeast and an emerging pathogen , which is difficult to treat, particularly due to a high antifungal resistance of this fungus. Pathogenicity mechanisms of C. glabrata are largely unknown. However, immune evasion strategies may play key roles since C. glabrata causes very little inflammation in mouse models of infection despite tissue colonisation. Additionally it has been proposed that C. glabrata can withstand killing by macrophages and even replicates inside. Objectives: We aim to dissect the interaction between C. glabrata and human monocyte-derived macrophages (MDM) to identify the immune evasion strategies that enable C. glabrata to survive phagocytosis.

We studied different stages of the C. glabrata -MDM interaction including adhesion, internalisation and phagosome maturation, by immunofluorescence microscopy and transelectron microscopy. We used stage specific markers to analyse the phagosome maturation and examined the acidification of phagolysosomes by using the acidophilic probe Lysotracker-Red. The production of reactive oxygen species by MDMs was investigated by luminol-enhanced chemiluminescence and damage of MDMs was quantified by measuring lactate dehydrogenase (LDH) in the culture supernatant. With the help of a caspase 3/7 assay we also analysed the induction of MDM apoptosis by C. glabrata. Genome wide microarrays were used to monitor the transcriptional profile of C. glabrata during phagocytosis.

We observed that C. glabrata-containing phagosomes rapidly recruit the endosomal and lysosomal marker Eea1 and Lamp1, indicating a fusion with early and late endosomes and lysosomes. Transelectron microscopy confirms the existence of a phagolysosomal membrane. However, staining with Lysotracker-Red showed that phagolysosomes containing living C. glabrata, but not phagolysosomes containing heat-killed cells, lack acidification. Analysis of transcriptional profiles support the view that C. glabrata cells within MDMs were not exposed to an acidic pH. Despite significant intracellular proliferation of fungal cells, apoptosis or damage of macrophages was not apparent and the production of reactive oxygen species was scavenged. Introduction: Secreted aspartic proteases (Saps) are important virulence factors of the human-pathogenic yeast Candida albicans. They are involved in various aspects of infection such as epithelial surface interaction and immune evasion. Two members of the Sap family, Sap9 and Sap10 are, in contrast to other Saps, glycosylphosphatidylinositol (GPI)-anchored on the cell surface. We have previously shown that both proteases function in cell surface integrity and composition, fungal attributes with a major impact on host-pathogen interaction. Objectives: We aim to investigate the role of Sap9 and Sap10 in fungal-phagocyte interaction and to characterise potential protease targets on the fungal cell surface. Methods: C. albicans mutants lacking SAP9 or SAP10 were co-incubated with human monocytic cells (THP-1 cell line) and subsequently stained for immunofluorescence microscopy. Filamentation was monitored under different hyphae-inducing conditions. Cell wall proteins processed by Sap9 and Sap10 were identified via LC/MS/MS. Results: We found that sap9 deletion mutants expose beta-glucan. This cell wall component is a potent proinflammatory molecule that is normally masked by a mannoprotein layer. Recognition of betaglucan contributes to uptake and killing of the fungus by phagocytes. Indeed we could show that deletion of SAP9 causes a stronger ingestion of C. albicans by human macrophages.

Another key aspect during infection is hyphal formation. We found that a sap9 deletion mutant exhibits enhanced filamentous growth only when embedded in a semisolid matrix. Sap9 therefore may affect hyphal-mediated tissue invasion or escape following macrophage ingestion.

To analyse which C. albicans cell surface proteins are proteolytically digested by Sap9 and Sap10, we incubated isolated fungal cell walls with recombinant Sap9 and Sap10 proteases. We detected the release of peptides from seven GPI-anchored cell wall proteins. These potential target proteins have distinct functions from cell wall remodelling to iron acquisition and adhesion. An internal epitope-tagging approach is underway to verify the proteolytic cleavage on living fungal cells.

Conclusions: Our data provide evidence for an active role of Sap9 and Sap10 on the cell surface, regulating cell wall functions and host cell interactions by proteolytic processing events, thus contributing to pathogenicity of C. albicans. Objectives: To test the hypothesis that HES130/0.4 impairs coagulation to a lesser degree, we studied the effects of HES130/0.4, HES200/0.5 and normal saline on in-vitro hemostasis and pro-inflammatory platelet function. Methods: Human whole blood samples were anticoagulated with either sodium citrate or recombinant hirudin (cWB and hWB, resp.) and mixed with HES130/0.4 (Voluven 6%), HES200/0.5 (HAES steril 10%) or saline (0.9% NaCl; all Fresenius Kabi, Germany) to achieve a final haemodilution of 10% or 40%. Haemostatic capacity in cWB was characterised by ROTEM thromboelastography (Pentapharm, Germany). Pro-inflammatory platelet functions were characterised flowcytometrically in hWB by measuring the platelet activation markers CD62P as well as the formation of heterotypic platelet-leukocyte conjugates.

Results: Most significant effects of both HES130/0.4 and 200/0.5 in ROTEM were seen in the maximum clot firmness (MCF), in particular when the contribution of fibrinogen to MCF was tested (FIBTEM). In comparison to controls, HES130/0.4 reduced MCF by 30% and 65% at haemodilution of 10% and 40%, respectively. No significant differences were observed between HES130/0.4 and HES200/0.5 (Fig.  1A ). An effect of HES on FXIIIa (stabilising the fibrin network) as cause for the reduced MCF could be excluded.

Both HES130/0.4 and 200/0.5, at a haemodilution of 40%, significantly increase ADP-induced platelet expression of CD62P, an important receptor for platelet-leukocyte interaction. Interestingly, HES130/0.4 but not HES200/0.5 increased parallel formation of platelet-leukocyte conjugates (Fig. 1B Introduction: In inflammation and sepsis, fluid resuscitation with either natural/ artificial colloids or cristalloids is one part in first line therapy because of its high impact on an improvement of tissue perfusion. On the other hand fluid administration may also induce alterations of coagulation. Tissue factor (TF) as the initiator of the coagulation cascade in vivo is expressed by circulating monocytes in septic conditions. Objectives: The aim of our experimental study was to investigate the influence of different volume replacement solutions on intracellular TF expression of human monocytes realized in an experimental model of inflammation. Methods: Whole blood samples (200 µl) of healthy male volunteers (n=12) were stimulated with lipopolysaccharide (LPS) (final concentration 0,2 ng/ml) in cell culture medium and incubated with Normal Saline (NaCl 0,9%), Human albumine 5% (HA), Gelatine 4% (GEL), HES 10% (200/0,5) and HES 6% (130/0,42) at final concentrations of 10%, 25% and 40% of the original blood sample volume at 37°C and CO2 (5%) humified atmosphere for 3 hours. Intracellular synthesis of TF in human monocytes was measured using flow cytometry. Results: HA, GEL and HES 6% in concentrations of 10%, 25% and 40%, and HES 10% in concentrations of 25% and 40% induced a significant dose dependent increase of TF expression in LPS stimulated human monocytes (compared to control; measured as mean fluorescence intensity). NaCl 0,9% had no effect on the TF expression of LPS stimulated human monocytes. Conclusions: In this in vitro whole blood model of inflammation we were able to demonstrate that HA, GEL, HES 6% and HES 10% induce a significant increase of monocytic intracellular TF synthesis. These findings have to be verified by clinical investigation and may be of importance for clinical conditions with increased levels of LPS and/ or procoagulatory activity.

Infection 2009; 37 (Suppl. III): 27

Open-heart surgery with the use of cardiopulmonary bypass decreases blood levels of the essential trace elements selenium, copper and zinc -6] ) and glycocalix markers (syndecan-1, heparan sulfate). Three groups consisted of patients with severe sepsis or septic shock, patients after major abdominal surgery without systemic inflammatory response syndrome, and healthy volunteers. Blood was drawn, at the time of diagnosis or surgery, and 6, 24, and 48h later. We correlated these markers to each other and to clinically used inflammation markers. Results: Levels of inflammatory markers were markedly higher in patients with sepsis compared with patients after major abdominal surgery and healthy volunteers. After major abdominal surgery, glycocalix markers in human plasma were at levels comparable to patients with sepsis. In patients with sepsis, levels of IL-6 correlated with syndecan-1, ICAM-1, VCAM-1, and lactate, while ICAM-1 furthermore correlated with CRP and lactate levels.

Conclusions: High levels of glycocalix markers indicated that significant flaking of the endothelial glycocalix occurred in patients with sepsis, and to a lesser extent in patients after major abdominal surgery. This novel finding could explain the nonspecific capillary leaking syndrome of patients with sepsis and after major abdominal surgery, and may identify new targets for treating those patient populations. Introduction: Despite the progress in modern medicine, sepsis is still a major health problem challenging fast diagnostics and effective therapies. New approaches for diagnostics and therapy were developed in the past but were not yet able to significantly decrease overall sepsis mortality, which still reaches rates of up to sixty percent. To improve prospective patient outcome, new comprehensive diagnostic approaches, which allow for faster diagnosis should be implemented into clinical practice to enable an early and effective sepsis therapy.

The clinical gold-standard in identifying pathogens and their resistance pattern was pioneered in the early 20th century and is based on microbiological blood culture. This process is time-consuming and limiting an early and precise anti-infective therapy. In clinical investigations however, early and especially goal-oriented therapy has proven essential for the survival of sepsis patients, implying the need for a rapid and sensitive detection of the responsible pathogens. Methods: To overcome the limitation in terms of diagnostic speed, we develop a molecular approach based on DNA microarray technology to rapidly identify and characterise sepsis-related pathogens. The choice of diagnostic determinants was carried out in close clinical cooperation to develop a comprehensive diagnostic tool. Starting from complex blood samples, clinically relevant information like pathogen identity and resistance patterns is available. Results: More than thirty sepsis-related pathogens can be classified in parallel in less than six hours. Resistance detection in the first stage will cover five important target genes to determine resistance profiles.

Here we present the preliminary data of our diagnostic tool. A subsequent validation of the system in the clinical setting will be performed to demonstrate the usefulness of our tool for molecular sepsis diagnosis.

The resulting improvement in the time course of sepsis therapy is promising to increase patient survival and to improve overall patient outcome and should increase the clinical acceptance of molecular sepsis diagnostics.

Infection 2009; 37 (Suppl. III): 28

Autophagy as a unique biosignature in hemophagocytic cells of patients after major trauma Lorenz MR (1), Walther P (2), Weiss ME (1) Introduction: Severe trauma and SIRS are associated by a so-called cytokine storm. This pathology may lead to macrophage activation syndrome (MAS) characterized by erythrophagocytosis and phagocytosis of myeloid cells (Kuwata K et al, 2006) as described for natural killer cells function deficient patients with HLH (Schneider EM et al, 2003) and may condition the manifestation of immunesuppression during sepsis.

Objectives: The aim of this study was the comparative analysis of the ultrastructural characteristics of antigen presenting cells (APC) derived of patients with sepsis and HLH with conventional and high pressure freeze substitution transmission electron microscopy (TEM). Methods: Ficoll-isolated peripheral blood or bone marrow derived mononuclear cells were depleted from non-adherent cells following 2h of plastic-adherence. Following 4 weeks of cell culture, hemophagocytes were fixed for TEM. Chemical fixation and high pressure freezing method were used alternatively (Buser et al, 2008) . A total of 6 patients (3 sepsis) and 3 HLH were studied. Results: Ultrastructural studies identified phagocytosis of autologous erythrocytes and fully viable leukocytes, including other hemophagocytes itself by cultured hemophagocytes of both, pediatric genetic and non-genetic HLH as well as secondary HLH/MAS in patients with severe sepsis and septic shock. Phagocytosis appears to co-exist with impressive autophagy and impaired phago-lysosome fusion, mitochondrial degradation and multivesicular body formation. The autophagic signatures are entirely identical between HLH and sepsis associated hemophagocytosis but, the shape and flexibility of the nucleus is distinct in that pediatric HLH nuclei of hemophagocytes are polymorph in shape and highly flexible, whereas sepsis associated hemophagocytes are predominantly round and rigid. High pressure freeze fixation techniques further revealed the asymmetric double membrane of autophagic membranes and illustrates two types of vesicle fusion mechanisms, the one occurring by digestion of the inner lipid bilayer of each of the fusion membrane partners and the second by engulfing the vesicle fusion partner without membrane degradation. This latter process explains the occurrence of as yet multiple double layer membrane containing vesicles. Conclusions: The present study illustrates novel aspects of APC pathology to explain immunedysfunction against virus reactivation and bacterial infection. The induction of autophagy may impair antigen presentation. Whether autophagy itself may explain the prominent hemophagocytosis of autologous and viable leukocytes remains an issue of further investigation. Splenectomy is not protective in abdominal sepsisa retrospective study Introduction: Infections are a major cause of mortality and morbidity in ICU. Respiratory tract infections, followed by intraabdominal infections (IAI) and primary bloodstream infections were most common. Considering septic patients after general surgery, IAI is the predominant focus (85%). The mortality of septic patients remains unacceptably high. Objectives: The proinflammatory cytokine imbalance during severe sepsis can be reduced by the nervous system via a reflex of the vagus nerve that is termed the cholinergic antiinflammatory pathway (CAIP). The spleen plays an important regulatory role in the innate immunology. In addition, it has been postulated that the spleen is an essential target of the CAIP. Therefore, a splenectomy might attenuate the limiting effects of CAIP. However, recent experimental findings revealed that a splenectomy could be protective in septic IAI. Thereby, the systemic release of high mobility group box 1 (HMGB1) triggered by splenic apotosis was significantly reduced after splenectomy. HMGB1 is described as a crucial mediator in lethal sepsis. Therefore, we evaluated mortality and morbidity of surgical ICU patients with proven IAI depending on whether they did undergo former splenectomy or not. Methods: This is a retrospective, non-randomized, single-center. All patients were treated for severe sepsis or septic shock due to a surgically proven IAI. Splenectomized patients served as study population in comparison to non-splenectomized controls. Survival was defined as the main outcome criterion. Time to death, ICU length of stay, hospital length of stay, duration of mechanical ventilation, need of renal replacement procedures, and need of re-laparotomy served as secondary outcome criteria. Results: 284 patients were included. 27 (9.5%) did underwent splenectomy before the development of sepsis symptoms. Both populations were well balanced due to gender, age, biometric characteristics. In the control population more patients with chronic obstructive pulmonary disease were observed whereas in the study group more subjects suffered for diabetes mellitus. There was no significant difference in mortality between both groups ( Objectives: The aim of the study was to compare the phagocytic function and the kinetics of phagosome-lysosome fusion in leukocytes from patients with sepsis, septic shock, before and after G-CSF (Neupogen) with the activity exhibited by healthy donors. Methods: After taking whole blood with 50IU sodium heparin, the sample was diluted 1:5 with RPMI1640, 25mM Hepes and antibiotics. A total of 109 Eos-P transfected E. coli were added and incubated at 37°C for various time points. At the end of incubation time treating with BD lysising buffer and 2 washing steps followed, cells were measured by flow cytometry.

Lichtenstern C (1), Zimmermann J (2), Weismueller K (1), Hofer (2), Walter V (2), Haag S (2), Weitz J (3), Weigand MA (1)(1)

The phagocytic activity was determined by mean fluorescence index (MFI): Over time (30', 60', 120' and overnight incubation), healthy donors showed quite a constant Eos-FP-E. coli-phagocytosis, as 526(30'); 540(60'); 563(120'); 549(o/n); (median values) whereas the septic patients started with lesser activity at 30' (compared to the controls) but already after 60', the septic patients had a higher MFI: 472(30'); 564(60'); 698(120'); 1209(o/n) respectively. The phagosom-lysosome fusion event was determined by a decline of red fluorescent light emission, quantified in green/red-ratio. Here the majority of the septic were heterogenous reducing red fluorescent much faster with 2,3(30'); 3,6(60'); 6,1(120'); 29,6(o/n), (median values) than the control group with 1,0(30'); 1,4(60'); 2,0(120'); 10,3(o/n), respectively. But also a few septic patients were as low as controls. In the G-CSF-treated group characterized by an impaired increase of circulating leuocytes despite sepsis, there was a strong impact of G-CSF substitution to upregulate both: phagocytosis and digestion of the Eos-FP E. coli.

The EOS-FP E. coli whole blood phagocytosis/digestion assay is useful and easy to handle. The faster lysososome phagosome fusion event in circulating leukocytes of patients with sepsis is not observed in every septic patient. Therefore this assay is capable to selectively address a minority of patients lacking functionally active PMN despite sepsis, possibly predestinating for septic shock. Introduction: Arginine vasopressin (AVP) as the strongest endogenous vasopressor in terms of maximal contractile effect induced and its duration has found growing interest in the therapy of vasoplegic states, e.g. patients with septic shock. In these patients a decrease of a metalloprotease (ADAMTS13) with inactivating activity against the ultra-large, highly thrombogenic form of Von Willebrand Factor (UL-VWF) has been described. Subsequent formation of VWF-rich microthrombi impairs the microcirculation and administration of a stabilized analogue of AVP results in a decrease of ADAMTS13/ appearance of ULVWF in plasma. Objectives: We prooved the hypothesis whether AVP-administration has an effect on VWF/ADAMTS13 balance in plama in patients with septic shock. Methods: Twenty patients with septic shock (12 male, 8 female) were enrolled. AVP was administered in a dosage of 2 IU/kg/h over 2 hours. Hemodynamic and haemostaseological parameters, platelet count and urine output were analyzed. Oral mucosal tissue oxygen saturation (SO2), microcirculatory blood flow and flow velocity were measured with a laser Doppler flowmetry and remission spectroscopy system (O2C). ADAMTS13 activity was determined using the Kokame method.

Results: AVP administration did not change cardiac output, but resulted in significant decrease of oral mucosal oxygen saturation, blood flow and a significant decrease of flow velocity. Also we analyzed a significant decline of ADAMTS13 activity (0.92; 0.69-1.15 U/mL vs. 0.51; 0.39-059 U/mL) and of platelet count after 4 hours (75; 64-100 vs. 71; 55-87 Gpt/l). Furthermore, urine output increased (125; 20-480 vs. 210; 30-690 ml/h). AVP administration causes a deterioration of oral mucosal blood flow independent of macrocirculatory conditions. This was accompanied by a further decrease of ADAMTS13 activity from declined baseline levels in patients with septic shock, which may result in secondary AVP induced platelet aggregation, collateral formation of VWF-rich microthrombi and subsequent occlusion of microvessels, which may be reflected by platelet consumption and impaired microcirculation. Conclusions: Therefore, besides its beneficial vasoconstrictive effects, AVP administration may be reconsidered due to potential aggravation of organ dysfunction subsequent to triggering thrombotic-microangiopathic complications. Methods: In order to investigate whether bck1, mkk2 and mpkA, are involved in CWI signalling, we generated deletion mutants by replacing every gene with a resistance marker gene. The obtained mutants were phenotypically characterised and challenged against cell-wall acting compounds using a growth extension assay. Induction of pyomelanin formation was performed by culturing the mutant strains in presence of 10 mM L-tyrosine. Pigment formation was spectroscopically measured at a wavelength of 405 nm. Formation of 4-maleylacetoacetate was spectroscopically measured in vitro and HmgA activity consequentially deduced. Results: Deletion of bck1, mkk2 and mpkA resulted in severe sensitivity of the mutants against cell wall disturbing compounds and drastic alterations of the fungal morphology. Nevertheless, no influence on virulence of the mpkA deletion mutant was observed in a murine infection model. Pyomelanin formation was significantly increased in the bck1, mkk2 and mpkA strains, and the total HmgA activity was significantly reduced compared to the wild type.

We functionally characterized bck1, mkk2 and mpkA in A. fumigatus confirming their role for CWI. It is interesting to note that activation of the MapK module leads to specific reduction of HmgA activity. Therefore, it is likely that CWI-MAPK signalling directly or indirectly represses formation of pyomelanin by preventing accumulation of HGA and therefore pyomelanin formation. Reduced heart rate complexity using poincaré plots parallels an increase in both pro-and anti-inflammatory cytokines during severe septic shock Introduction: Different models of septic shock have demonstrated an intense elevation of pro-inflammatory cytokine serum levels, followed by an anti-inflammatory response. Furthermore, reduced heart rate variability (HRV), which is the variability of R-R in the electrocardiogram (ECG) and complexity, have been associated with worse outcome in patients with severe sepsis and multiple organ failure.

Objectives: In this observational study, we tried to evaluate longitudinally over time the systemic cytokine response in a cohort of surgical critically ill patients, diagnosed with septic shock, and correlate different patterns of inflammatory biomarkers with measures of complexity of heart rate signals. Methods: We daily assessed the two values of standard deviation (SD1 and SD2), as indicators of the dispersion of R-R points of the ECG obtained from the Poincaré plot, since SD1/SD2 is an indirect measure of complexity of a signal. We also measured on an every day basis C-reactive protein (CRP), pro-inflammatory (TNF-α, IL-6) and antiinflammatory (IL-10) cytokine serum levels, in twenty surgical patients admitted to our Intensive Care Unit with a primary diagnosis of septic shock. Patients with a previous history of arrhythmia, trauma brain injury and immunodeficiency were excluded from the study. The ECG signal was daily recorded for 10 minutes under sedation and mechanical ventilation from a standard lead II ECG, obtained with monitors (Marquette 8000, GE, Milwaukee, USA). For off-line biosignal measurements we used an advanced analysis tool for Windows, developed from the Department of Physics of the University of Kuopio, Finland. In addition, we measured every day Specific Organ Failure Assessment score (SOFA) and ΔSOFA (SOFA maximum-SOFA admission) as an index of severity of illness. Linear regression and correlation analysis with Pearson's test were performed on log-transformed SD1/SD2, SOFA, ΔSOFA, CRP and cytokine data to assess whether different biomarkers were independent predictors of SD1/ SD2 and for evaluation of trends over time.

Results: SD1/SD2 exhibited significant positive correlations with mean values of IL-10 (r=0.85, p<0.01), IL-6 (r= 0.68, p<0.05), TNF-(r=0.74, p<0.01) and CRP blood levels (r=0.66, p< 0.05) and with ΔSOFA (r= 0.88, p< 0.01). Mean SOFA score was positively correlated with IL-10 (r= 0.64, p< 0.05) and CRP (r=0.83, p< 0.01). ΔSOFA, TNF-α and IL-10 were the most significant predictors of increasing SD1/SD2 (p<0.01 for the 3 comparisons). Conclusions: Our data suggest that an increase in both pro-and antiinflammatory cytokine serum levels is associated with a loss of complexity of heart rate signals, particularly in surgical septic patients with increased morbidity, supporting the hypothesis of a possible pathophysiologic link between autonomic dysfunction and inflammatory response. Introduction: Sepsis is the presence of organisms with the blood, it has a high prevalence within patients presenting at Emergency Departments (ED) and if untreated can progress to life threatening severe sepsis or septic shock. In recent years there has been research into predictive tools regarding mortality in septic patients in adult EDs. Recent work has focused on models that can provide quick assessment using information that is commonly collected in the ED. The Mortality in Emergency Department Sepsis (MEDS) score has been found to be the best performing models in predicting 28 day mortality. The original US based MEDS scoring system has been abbreviated by Vorwerk et al in 2009 making it applicable for UK ED use. Objectives: To retrospectively assess the abbreviated MEDS scoring system and so either confirming or refuting reports of it as an accurate method of predicting mortality in adult septic patients within the ED. To assess the potential of the abbreviated MEDS scoring system as an integrated element of routine assessment of adult septic patients presenting at the ED. To establish whether any differences in management exist between high risk surviving and non-surviving septic patients who present at the ED. If identified, make recommendations for future management changes within the ED for the management of sepsis. Methods: An interval sample of 3 months starting from 1st January 2009 was chosen for selection of patients. 101 patients were identified through a search of the NGH patient administration system for key words sepsis or septic, of these 99 cases were available for viewing, a further 13 were excluded as they had been inappropriately coded. To assess the abbreviated MEDS score for the 72 septic cases, information was obtained from patient ED cards and blood results recorded on the hospital ICE system. Treatment of the high risk surviving and non-surviving septic patients who presented at the ED was compared relative to the initial resuscitation bundle recommended as part of the 2008 Surviving Sepsis Campaign guidelines. Results: The data required to formulate an abbreviated MEDS score for patients with sepsis was generally present for all patients. Only 6 cases were excluded because of missing information, these were all because of no recorded Glasgow Coma Score (GCS). Of these 66 patients the average patient age was 72.5 years (95% CI 76.1 -68.9 years). The oldest effected patient was 96 and the youngest 23. Fifty percent of patients were female and 50% male. The 28 day mortality of the study population was 21.2%. At 28-days, fifty two of the patients had survived and 14 had not. The abbreviated MEDS score ranged from 0-16 (95% CI 6.7 -8.4) for the surviving patients and from 5-13 (95% CI 9.0 -10.0) for the non-surviving group. A P-value of 0.0063 shows that the abbreviated MEDS score is an accurate tool for identifying septic patients at higher risk of death within 28 days. Categorisation of patients into low (0-4 points), moderate (5-12 points) and high (>12 points) risk groups on the basis of their abbreviated MEDS scores found that 15.1% of the patients were in the low risk group (28-day mortality 0%), 77.3% in the moderate risk group (28day mortality 24%) and 7.5% in the high risk group (28-day mortality 20%). Comparison of high-risk surviving (abbreviated MEDS score >11) and non-surviving patients shows differences in management. Of significance: The sensitivity and specificity of neutrophil gelatinase-associated lipocalin (NGAL) in acute kidney injury -a systematic review and meta-analysis Objectives: The present study attempted to identify the diagnostic significance of investigated markers in acute abdominal conditions, preoperatively, as well postoperatively during the first 3 days. Methods: This was prospective clinical study on 98 operated patients with acute abdominal conditions who have preoperatively met the established criteria for SIRS, 58 of which had positive bacteriological findings from peritoneal cavity and were included in the sepsis group, while 40 were included in non-infectious SIRS group. The investigated biomarkers were observed from preoperative period and during the first 3 postoperative days. Results: Preoperative PCT concentrations were significantly higher within the sepsis group than they were within the SIRS group (median (interquartile range) 2.32 (7.41) ng/ml vs. 0.45(2.62) ng/ml). A cut-off value of 1.1 ng/ml yielded 72.4% sensitivity and 62.5% specificity. (AUC=0.740; PPV=73.7%; NPV=61.0%). Postoperatively, AT III (AUC=0.677; Sens.=70.2%; Spec.=67.5%) and Protein C (AUC=0.669; Sens.=70.2%; Spec.=67.5%) shoved significant accuracy. The other parameters did not identify abdominal sepsis in early period. Conclusions: PCT was the only marker that can be helpful in preoperative diagnosis of abdominal sepsis. PCT was the only marker that showed diagnostic significance, both preoperatively, and postoperatively, on all days of measurement. Diagnostic significance that was reached by AT III and Protein C postoperatively was reached by PCT preoperatively. Conclusions: Prove-it TM Sepsis was considered to be a fast, robust, and high performance diagnostic platform which is easily implemented into everyday laboratory workflow. Both study sites identified patient cases where timely information provided by Prove-it TM Sepsis would have significantly improved patient management. Examples here include more rational management and antibiotic choice subsequent to earlier differentiation of 'Gram-positive cocci in clumps' into MRSA, MSSA, or coagulase negative staphylococci, and earlier speciation of Gram-negative organisms. After the performance evaluation study, the pathogen panel of Prove-itTM Sepsis is further configured for detection of Candida spp. and new bacterial targets, i.e., Neisseria non-meningitidis, Kingella kingae, and Propionibacterium acnes. The assay identifies now 60 out of the 302 samples that were not covered during the performance evaluation, increasing the pathogen coverage from 86% to 89%. Prove-it TM Sepsis has also been evaluated with highly promising results for the diagnostics of bone and joint infections using bone biopsy and articular fluid as sample types. These evaluation studies have demonstrated excellent performance for this DNA-based diagnostic platform for various diagnostic applications. The earlier speciation provided by Prove-it TM Sepsis could contribute to faster, more evidence-based patient management and positive outcomes. Introduction: Fungal blood stream infections (BSI) especially candidemia remain a major cause of morbidity and mortality in the intensive care setting. US data report an increasing incidence and a changing epidemiology of fungal infections in the intensive care unit (ICU). Objectives: To analyze the secular trends in the epidemiology and the microbiology of ICU-acquired primary BSI with a focus on fungal infections and to evaluate demographic and patient outcome variables. Methods: CDC standard definitions were used for diagnosis of ICU acquired laboratory confirmed primary BSI and these data were re- Conclusions: Contrary to reports from the US, in German ICUs there was neither a significant change in the incidence of fungal nosocomial BSI nor in the distribution of pathogens (gram-positive, gram-negative and fungal). ID of C.albicans BSI was only a fifth of the ID in US American ICUs (0.04 versus 0.27). Therefore, it seems most worthwhile to consider the regional situation. C. albicans is nonetheless the sixth most frequent pathogen in nosocomial BSI and has a crude ICUmortality of 23%. 

Introduction: S. aureus carriers have an increased risk of developing nosocomial S. aureus bacteremia with their own strain. Interestingly, their prognosis is much better than that of noncarriers who develop exogenous bacteremia. We propose that carriers raise an antibody response against their colonizing strain, which (partially) protects them from the complications of S. aureus infection. Objectives: We aimed to elucidate how anti-S. aureus antibody responses are generated. In view of the pronounced strain variability of S. aureus this required a comprehensive integrated analysis of the strain-specific anti-S. aureus antibody response to colonization and infection.

We first colonized 16 healthy volunteers with S. aureus strain 8325-4, which was selected for safety reasons because of its low virulence, and obtained serum samples before and four weeks after colonization. Second, more than 1000 hospital patients at high risk of infection were screened for nasal colonization in a prospective study. 15 patients later developed S. aureus bacteraemia. The infecting strains were isolated and patients' sera obtained before onset, at diag-nosis and during the course of infection. The secreted staphylococcal proteins from the colonizing and infecting strains were separated by two-dimensional gel electrophoresis and quantitative immunoblots with the human sera were performed. Results: In the healthy volunteers there was a large inter-individual variability in spot patterns and spot intensities already before experimental colonization with S. aureus 8325-4. Only rarely we observed additional spots or increased spot intensities after experimental colonization with S. aureus, mostly in carriers. Bacteremia, in contrast, elicited a strong humoral immune response to the infecting strain with characteristic differences between endogenous and exogenous infection. Before onset of bacteremia, S. aureus carriers exhibited much stronger antibody binding to antigens of the infecting strain than did non-carriers. The complex binding patterns of carriers were largely conserved during infection, but spot intensities increased up to 45fold. Exogenous S. aureus strains, however, elicited antibody binding to large numbers of novel antigens. Conclusions: Minor infections rather than epithelial colonization are the likely triggers of humoral anti-staphylococcal immunity in the healthy population. The protective potential of these antibodies in bacteremia remains to be determined. Introduction: Septifast® is a multiplex real-time PCR assay for detection of bacteria and fungi DNA in blood. Blood culture remains the gold standard but has several limitations as a large time needed, false positive results due to contaminations and false negative results due to antibiotic treatment Objectives: Our aim is to check up on the presence of concordance between the results of PCR technique and standard BC in septic and non septic patients. We also want to verify a correlation between the resistance to the empiric antibiotic therapy of detected microorganisms and the presence of concordance between the two methods. Methods: A total of 42 episodes (combined BC and PCR samples) were collected from 38 patients: 27 of them were considered as cases (according to ACCP/SCCM sepsis criteria); 15 were considered as controls. All patients were treated with empiric antibiotic therapy at the time of testing. We obtained 21 ml of blood from each patient using sterile technique; 7 ml were inoculated into each Bactec aerobic, anaerobic, fungi blood bottle. Immediately after, 5 ml were obtained and inoculated into EDTA tube as PCR sample. Both procedures were started at the same time. If positive, Bactec was followed by standard microbiological identification protocol and antibiogram. Results: Septifast was positive in 20 cases and negative in 7 cases. Bactec was positive in 21 cases and negative in 6 cases. In controls, Septifast was never positive, Bactec was positive twice and negative 13 time Septifast detected 40 microorganisms totally (16 Gram+, 22 Gram-, 2 fungi) instead of 29 microorganisms We considered also different kind of concordances between the two methods for both cases and controls, according to microbiological identifications. A full-positive concordance resulted in 4 cases and 0 controls (9,5%) whereas a partial positive concordance detected in 8 cases and in 0 controls (19%). Negative concordance resulted in 1 case and 13 controls (33,5%) while no concordance was founded in 13 cases and 2 controls (36%). Each time there was a full positive or partial positive concordance (12 cases) we demonstrated the presence of resistance to the antibiotics admin- Introduction: Bloodstream infection is a life-threatening condition with a high mortality rate, especially in intensive care and neutropenic patients. Standard diagnostics is based on blood culturing (BC). However, limitations of BC include relatively low sensitivities and a long time-to-result for the identification of the pathogen, generally over two days and more. Molecular methods are discussed as an alternative to BC, mainly because of its rapidity and higher sensitivity. Objectives: A new commercial PCR test for direct detection of bacteria in whole blood was compared to BC in terms of sensitivity, specificity, and predictive values of bacterial infections of the blood stream of critically ill patients.

The test, SepsiTestTM (Molzym, Bremen), comprises the extraction and 16S rDNA PCR detection of bacterial DNA in whole blood samples. Bacteria in positive samples were identified by sequence analysis of the amplicon. In a prospective, multicentre study 342 blood samples from 187 patients with systemic inflammatory response syndrome (SIRS), sepsis, or neutropenic fever were included. Results: Compared to BC, the diagnostic sensitivity and specificity of PCR/sequencing was 87.0% and 85.8%, respectively. The sample positivity rate of PCR/sequencing (25.7%) was higher than BC (15.8%). Of 31 PCR/sequencing-positive, BC-negative patients, most of whom received antibiotics, the PCR results of 25 were judged as true or possible to bacteraemia.

The PCR approach facilitates the detection of bacteraemia in blood samples within a few hours. Despite the indispensability of BC diagnostics, the rapid detection of bacteria by SepsiTestTM appears to be a valuable tool, allowing earlier pathogen-adapted antimicrobial therapy in critically ill patients. Introduction: The incidence and prognosis of septic shock in cardiac surgical patients is poorly characterized. Objectives: In this study we aimed to report the incidence of septic shock in the cardiac surgical setting in relation to the evolution of postoperative multiple organ dysfunction. Therefore, an observational cohort study with prospective data collection was started in a seven bed ICU of the a tertiary care, university affiliated hospital. Methods: Among 1910 consecutive adult cardiac surgical patients admitted to the ICU, all those (n=183; 9.6%) with a complicated postoperative course were considered, i.e. those requiring postoperative ICU stay > the 90th percentile. The severity of organ functional im- Table. Estimates of unadjusted hazard ratio of septic shock for mean total SOFA score and for individual organ system scores.

SOFA, Sequential Organ Failure Assessment; HR, hazard ratio; CI confidence interval; neuro, neurological. Number refers to the increment in hazard for an increment of one point in SOFA score, numbers in parenthesis are the 95% confidence limits for the hazard ratios

In conclusion, after cardiac surgery, septic shock is a late event complicating early perioperative cardiogenic shock and multiorgan failure. Among patients with postoperative complications, those with septic shock have higher mortality rates and septic shock could be predicted by increased severity of disease, ongoing cardiac failure, thrombocytopenia and an increasing number of failing organs. In the period preceding septic shock, the presence of sepsis or severe sepsis could not be consistently ruled out. Gram-negative organisms were the most frequently isolated pathogens, although documentation of infection could not be achieved at all times. 

The measurement of indocyanin green elimination from circulation is an outstanding non-invasive way for estimation of hepatosplanchnic hemodynamics. It immediately indicates the worsening liver function in contrast with statical parameters. Objectives: We explored a possible correlation between direction and amplitude of ICG-PDR and outcome of critical illnesses. We examined furthermore the relationship between ICG-PDR and the changing of usually used statical liver function parameters. Methods: We performed an ICG measurement before, and after haemodynamic stabilization in the 0. and the 6th hours. We registered conventional hemodynamical and volumetric parameters, serum lactate and ScvO2 for estimating tissue oxygenization, and statical liver function parameters as serum bilirubine and prothrombin. We defined a "decreasing" (1) and an "increasing" (2) ICG groups. We created furthermore an ICG-PDR<8 (A) and ICG-PDR>16 (B) groups.

We found a 69% mortality in group 1 in contrast with 9,6% in group 2. The mortality was significantly higher in group ICG-PDR<8 than in group ICG-PDR>16 (53,8% versus 11,1%). We mea-Clinical Sepsis Research -Diagnosis sured significantly higher serum lactate and billirubine levels in case of decreasing ICG-PDR than in "increasing" patients (4,306mmol/ l/2,43mmol/l versus 3,43mmol/l/47,8mmol/l). We found a significant difference in serum billirubine and lactate levels (3,43mmol/ l/36,4mmol/l versus 2,51mmol/l/25,94mmol/l). The difference was not significant in ScvO2 levels.

The direction and absolute value of ICG-PDR change has a prognostic value in critically ill patients. The decreasing ICG elimination is an indicator of splanchnic hypoperfusion. The low absolute ICG-PDR level is in good correlation with statical liver function parameters. Introduction: There are no published data describing the pattern of central venous oxygen saturation (ScvO2) changes during and after major general surgery or any relationship with outcome. Objectives: We examined a probable correlation between perioperative ScvO2 changes, frequency of postoperative complications and outcome of illness in high risk surgery patients. Methods: We included 50 patients undergoing high risk surgery longer than 90 minutes. Inclusion criterias were: ASA > II and at least two points of following: age>60 years, prior cardiovascular history, malnutrition. Our patients were ranked to a patient group with complications (A) or to a complication-free control group. We measured preoperative (basic) ScvO2 value, intraoperative and postoperative ScvO2 values for 12 hours continously. We determined the relative time under 95% of preoperative ScvO2 level in intra-and postoperative period. We additionally recorded pre-and postoperative hemoglobine values, SAPS, ICU and total LOS and outcome in the time of discharge. Introduction: Sepsis is associated with disturbances of haemostasis which may result in hypo-or/and hypercoagulability, i.e. in bleeding and/or disseminated intravascular coagulation and/or thrombotic microangiopathy. ROTEM is a point-of-care method that reflects more closely than classical coagulation tests the in vivo haemostasis and the contribution of fibrinogen and platelets to clot formation. Objectives: We want to test whether ROTEM can be used to identify hypo-and hypercoagulability in patients with severe sepsis/septic shock. Methods: So far 17 patients of a surgical intensive care unit were included within 24 h after they had met the criteria of severe sepsis or septic shock. ROTEM (Pentapharm, Munich) was performed according to the manufacturer's instruction on four consecutive days. Other laboratory variables of the haemostatic system were obtained from the patient records. Data are given as median (interquartile range). ROTEM data from 11 healthy volunteers were used as controls.

Results: At day 1 prothrombin time, aPTT and plasma fibrinogen level in septic patients amounted to 70 (60-81) %, 49 (42-58) sec and 6.4 (3.4-8.9) g/l, respectively, and did not significantly change during the next 3 days. Platelet count was significantly decreased at day 2 and 3 (2.1 (1.7-3.0) and 1.9 (1.5-3.1) Gpt/l, respectively) when compared to day 1 (2. Septifast® allows more rapid detection of liver transplant patients being at risk of sepsis than blood culture or inflammatory parameters Introduction: Early detection of sepsis in immunocompromised or even immunosuppressed such as liver transplant patients is a challenge for the intensivist.

Objectives: The utility of the newly available Septifast ® -a multiplex real-time PCR-based assay -which detects 25 clinically important pathogens -19 bacteria, 6 fungi -from whole blood within 6 hours [1] was evaluated in comparison to standard blood culture usually lasting 48 hours and inflammatory parameters in an observational study. Methods: After approval by the local ethics committee and having obtained informed consent in 48 patients, scheduled for orthotopic liver transplant, 195 paired blood sampels were taken under sterile conditions before induction of anaesthesia, on arrival at the ICU, and on day 1, 2, 3, 4, 5. Blood samples were taken for Septifast ® (PCR), blood culture, PCT, CRP, IL-6, LBP. Patients received standard treatment -including antibiotics -, standard microbiological screening (swaps etc.) and were clinically evaluated for inflammatory episodes. 

In cardiac surgical patients a severity score is needed to standardize reports in order to improve the understanding of the course of disease and to allow evaluation of the impact of new treatments on outcome. Objectives: The purpose of this study was to evaluate the usefulness of repeated measurements of the SOFA score in terms of survival in cardiac surgery patients. Methods: This prospective study consisted of all consecutive adult patients undergoing cardiac surgery with cardiopulmonary bypass between January 2003 and October 2005. The SOFA score was calculated daily until discharge. Initial SOFA score, maximum SOFA score, and delta-SOFA scores (differences between subsequent scores) were calculated and their correlations with mortality were assessed. Results: A total of 2372 patients with a mean age of 66,2 +-11,2 years were admitted to the ICU after cardiac surgery. The operations performed were 1518 (64 %) isolated CABG, 332 (14 %) isolated valve surgery, 237 (10 %) combined CABG with valve surgery, 95 (4 %) surgery of the thoracic aorta, and 190 (8 %) other procedures. The overall mortality rate was n=85 (3,6 %). The mean stay on the ICU was 3,0 +-6,1 days. The receiver operating characteristic (ROC) curve of the maximum SOFA score was excellent with a value of 0,97. When analyzing trends in the SOFA Score during the first 96 hours (1038 patients), regardless of the initial score, the mortality rate was 30,6 % when the score increased, 17,2 % when it remained unchanged, and 2,0 % when it decreased. Conclusions: The highest SOFA Score during the ICU stay showed an excellent discrimination. Serial evaluation of the SOFA Score during the first 96 hours is a good indicator of prognosis in cardiac surgical patients. Introduction: In the commonly used 1992 ACCP/SCCM consensus conference definitions for sepsis, severe sepsis is defined as sepsis plus organ dysfunction or hypoperfusion. However, besides lactic acidosis and oliguria, limits for organ dysfunction variables have not been given in the 1992 publication. Later on, it has been recommended to define organ dysfunction, and thus, severe sepsis by exceeding two points in at least one organ system using the "Sequential Organ Failure Assessement" (SOFA) score. However, it has not been validated how the prevalence of various stages of sepsis and mortality rates differ in the same patient population, if organ dysfunctions are defined with cut-offs at 1, 2, 3, or 4 points of the SOFA score in one organ system. Objectives: The present study was performed to compare the prevalence of severe sepsis and ICU mortality rates, and to find out the case mix within the same collective of critically ill postoperative/posttraumatic patients applying the sepsis definitions with increasing cut-offs of organ dysfunction points in the SOFA score. Methods: Retrospective observational single-centre study in surgical critically ill patients admitted to an University adult ICU. From 01/2007 to 12/2008, 1355 admissions were surveyed daily computer-assisted with respect to organ dysfunctions defined by the SOFA score and to different stages of sepsis, defined by the 1992 ACCP/SCCM sepsis definitions. Organ dysfunction was defined as lactic acidosis or oliguria, or if patients reached 1 -4 points in at least one organ system (lung, coagulation, liver, kidney) using the SOFA score. For each patient, the first day with the worst degree of sepsis severity defined with a cut-off of 1 point in the SOFA score in at least one organ system during his/her ICU stay was compared with the corresponding scores defined with a cut-off of 2, 3 or 4 points on the same day. Results: Within the same patient collective, applying a cut-off of 1, 2, 3 or 4 points in the SOFA score, resulted in a decrease of cases classified as SIRS or septic shock, i.e., from 642, 535, 357 to 218 out of 1355 cases, however an increase in mortality rate from 19% (124/642), 22% (115/535), 26% (91/357) to 33% (71/218), respectively.

The prevalence and mortality rates of SIRS and septic shock differ using different cut-offs in the SOFA score. Using greater than 2 points in the SOFA score to define organ dysfunctions may under-classify patients with SIRS and septic shock. Clinical utility of endotoxin measurement using the endotoxin activity assay as an example (2)). This definition uses a fixed time interval (48 hrs) in which AKI may develop.

Objectives: Aim of our study was to investigate characteristics and outcome of AKI in a large cohort of patients with severe sepsis and septic shock.

Methods: Data from the multicenter VISEP trial (Brunkhorst et al. NEJM 2008; 358: 125-39) were analyzed for the first 48 hrs after enrolment of patients. Complete data about the new AKI definitions were available in 521/537 of patients. Patients were divided into (1) no AKI, (2) AKI I, (3) AKI II and (4) AKI III. AKI I was defined as an increase in serum creatinine (S-Cr) from baseline > 0.3 mg/dl, a percentage increase in S-Cr of > 50%, or a reduction in urine output of less than 0.5 ml/kg/h for > 6 hrs. AKI II was defined as an increase in S-Cr from baseline of > 200-300%, or a reduction in urine output of less than 0.5 ml/kg/h for > 12 hrs. AKI III was defined as an increase in S-Cr from baseline > 300% or a rise > 4mg/dl, or a reduction in urine output of less than 0.3 ml/kg/h for > 24 hrs or anuria for 12 hrs, or the need for renal replacement therapy. Results: 227/521 (44%) patients were classified as AKI and 294/521 (56%) as non-AKI. Patients with AKI were older, had a higher APACHE II score, higher lactate levels and a lower platelet count. Mean arterial pressure (MAP) and CrP levels were not different (Table) . After multivariate regression 28-and 90-day mortality and mean SOFA scores were significantly higher in patients with AKI and increased with increasing severity of AKI ( 

The term metabolome denotes the collection of all metabolites in a biological organism, metabolomics is the systematic study of the chemical signatures that specific cellular processes and states of tissues, organs and organisms leave behind. For biomarker discovery, analyzing metabolites is of great interest due to its high potential. However many metabolomic strategies to date have focused mainly on metabolomic profiling, which is disadvantaged by lack of metabolite identification. In contrast to this approach, the targeted quantification of metabolites offers the unique opportunity for direct translational biomarker discovery and validation. In addition it enables immediate biochemical interpretation and therefore functional annotation. Thus, the technology can easily be used to improve the understanding of diseases and of toxicological effects on the metabolic level. In principle, it has a high potential for biomarker development and for diagnostics. Objectives: The metabolomics technology will be introduced and data of case studies from complex diseases presented.

We are performing high-throughput metabolomics applying mass spectrometry (FIA and LC-MS/MS methods) to run at least 80 samples per day and to analyse hundreds of metabolites from (but not limited to) the following types of molecules: amino acids, acylcarnitines, carbohydrates, lipids, phospholipids and lysophospholipids, sphingo(phospho)lipids, oxysterols etc. Results: Comprehensive metabolomic characterization of diseased states and selected case studies for application in biomarker identification will be presented. 

The potential and possible utility to future sepsis diagnostics will be assessed. Objectives: The goal of this study was to evaluate the potential use of this marker in critically ill patients with either sepsis or systemic inflammatory response syndrome after coronary artery bypass grafting (CABG). Methods: A total of 126 patients (65 patients with severe sepsis and 61 patients CABG) have been included in this study. Plasma samples have been collected daily in the sepsis group or during ICU stay after surgery in the CABG-group. 1,3-beta-glucan level was measured using a calorimetric assay (Wako Pure Chemical Ind.). A cut-off level of 1,2 pg/ml was adopted. Results: Beta-glucan serum levels were significantly elevated after uncomplicated CABG compared to the severe sepsis group. These levels progressively decreased during ICU and were comparable between CABG group and severe sepsis group by day 5 (until day 4 p<0.03). However, patients with sepsis show elevated levels during the whole observation period. Interestingly, patients after CABG without heart-lung-machine (HLM) showed significantly lower 1,3-beta-Dglucan levels compared to patients undergoing HLM (p<0.03).

Conclusions: This observational study demonstrated the potential clinical usefulness of serum beta-glucan measurements in different populations of critically ill patients. Furthermore, the reliability for diagnosing fungal infections is inconclusive. While bacterial translocation has been suspected as reason for SIRS after CABG, this has never been associated with fungemia. Our present findings support this hypothesis. Fungal pathogen associated molecular pattern might contribute to the inflammatory response to surgery. Our data suggest that the use of HLM may be associated with elevated 1,3-beta-D-glucan level. (1), Alvarez J (2), Hunfeld KP (3), Goglio AP (4), Kost GJ (5), Louie RF (5), Raglio A (4), Regueiro BJ (6), Wissing H (7), Stueber F (1) (1) Anaesthesiology and Pain Therapy, Inselspital, Bern, Switzerland, Introduction: Sepsis is still a leading cause of death in ICUs. Early diagnosis, followed by prompt implementation of adequate antimicrobial treatment improves the prognosis of septic patients. PCR may shorten the time to pathogen identification, thus decreasing the time of potentially inadequate empiric antibiotic treatment. However, data for the clinical utility of PCR in microbiological testing for sepsis are lacking.

Objectives: Timeliness of antimicrobial treatment may be improved by utilizing a new multiplex PCR assay that identifies sepsis-relevant microorganisms in blood.

Methods: Blood samples for multiplex PCR analysis were taken in parallel with the blood culture of each sepsis episode. The potential impact of reporting microorganisms by PCR on adequacy and timeliness of antimicrobial therapy was analyzed. The number of sepsis episodes and potential days gained of adequate antimicrobial treatment attributable to PCR findings was assessed in a model analysis.

Results: 465 episodes with severe sepsis were included in the study. 99 sepsis episodes were BC positive (BC+) after median reporting time of 72 hours. Initial antimicrobial therapy was modified in 125 episodes, in 88 episodes without any BC+ evidence. 131 sepsis episodes were PCR positive (PCR+). Utilization of PCR+ findings could accelerate antimicrobial modification in 46 episodes of observed antimicrobial change. This calculates to a potential sum of reduction of 106.5 inadequate treatment days, or 2,3 days per episode. The ratio of gainable early adequate treatment days to number of PCR tests done is 22.8 days/100 tests overall (CI 15 -31). Conclusions: Rapid PCR identification of microorganisms may supplement conventional BC testing and thus contribute to a reduction of early inadequate antibiotic treatment in sepsis. Introduction: The significance of detection of fungal microorganisms in septic patients remains controversial. A recent trial in 142 patients with sepsis comparing detection of pathogens via blood culture or multiplex PCR revealed in 8 patients fungal DNA while blood cultures of paired samples remained frequently negative. 7 of these 8 patients died. These findings suggest a high mortality of a hitherto underestimated problem in critically ill patients.

Objectives: To further evaluate possible impact of fungal DNAemia in critically ill patients Methods: Retrospective analysis of multiplex PCR assay (VYOO, Sirs-Lab GmbH, Jena, Germany) results with respect to detection of fungal DNAemia in patients with signs of inflammation treated on a 50 bed university hospital ICU; test results between January and March 2009 were evaluated. Results: 98 PCR assays of whole blood samples were performed during the study period. In each case a blood culture was drawn at the same time. 11 samples in 10 patients revealed a positive PCR result for fungal DNA (11%), while corresponding blood cultures were negative with respect to fungal pathogens. In 7 of the 10 patients other microbial specimens like BAL, wound smear or urine had positive results for different fungal species. Patients with a positive PCR result had an overall mortality rate of 50%; 4 patients were treated with antifungal substances (3 fluconazol, 1 voriconazol), 6 patients were untreated, with a mortality rate of 25% and 67%, respectively.

In accordance with a previous study we could detect fungal DNA in about 10 % of whole blood samples of critically ill patients with clinical signs of inflammation, while corresponding blood cultures were negative. However, microbial specimens with positive fungal results were common in these patients. Untreated patients had a high mortality rate. These results confirm and extend previous data that fungal infection might still be an underestimated problem in frequency and severity in critically ill patients. Impact of PCR-guided antifungal therapy in the early therapeutic window requires prospective testing. Introduction: Assessment and aggressive treatment of central venous pressure (cvp), mean arterial blood pressure (RRmean), central venous oxygen saturation (ScvO2) and hematocrit (hct) are cornerstones in the therapy of severe sepsis and septic shock. Whether one of these or all of these parameters are crucial is not known nor is the impact of cardiac dysfunction (lvef) or myocardial ischemia (TnI). Objectives: To study the feasability and influence of left ventricular function assessment with transthoracic echocardiography in septic shock and determine the influence of myocardial ischemia with measurment of TnI plus the standard hemodynamics (see above). Prospective observational study, 9-bed conservative intensive care unit of a university hospital. 31 consecutive patients (pt) with septic shock as defined in the actual guidelines. Differences in 28 day-mortality. Methods: Assessment of cvp (mmHg), RRmean (mmHg), ScvO2 (%), hct (%), lvef (%) and TnI (ng/ml), normal range < 0,015 ng/ml, on day 1 and day 7 and 28 day-mortality. Transthoracic echocardiography is performed in each subject. Documentation of baseline characteristics, medical history and therapeutic interventions. Results: 31 pt, mean age 71 years±7,22, 18 male (58%), 18 pt with known coronary artery disease (58%), mean APACHE II-score 35,9±6,8. 17 pt died within 28 days (52%). Most often isolated species (specimen from blood culture, urinary sampling, tracheal suctioning) are E. faecium (16,5%), E. coli (16,1%), MRSA (12,9%), P. aeruginosa (9,7%), S. epidermidis (12,9%), S. aureus (9,7%) and others. Transthoracic echocardiography could be performed in 29 pt (94%). Mean cvp 9,5±5,2, RRmean 72,6±14,3, ScvO2 60,1± 22,5, hct 29,8±,9,3, trop i 2,7±9,2, lvef 39,5±21,7. Influence of initially reduced hemodynamics at day 1 on 28 day-mortality are as follows: 21 pt (68%) with cvp<8 vs. 10 pt (32%) with cvp>8 (p=ns), 12 pt (39%) with RRmean<65 vs 19 pt (61%) with RRmean>65 (p=0,03), 17 pt (61%) with ScvO2<70 vs 11 pt (39%) with Scvo2>70% (p=ns), 13 pt (42%) with a hct<30 vs hct>30 (p=ns), 18 pt (67%) with TnI>0,015 vs 9 pt (33%) with TnI<0,015 (p=0,013), 8 pt (26%) with lvef<40 vs 23 pt (74%) with lvef>40 (p=ns).

In patients with septic shock early assessment of the standard hemodynamic parameters is indispensable for a consistent and aggressive treatment. Markedly diminished hemodynamics are associated with an increased mortality rate. Especially this applies to reduced arterial blood pressure and elevated TnI as a marker of myocardial ischemia. Assessment of global left ventricular function with echocardiography is feasible even in mechanically ventilated pt and provides additional useful information. Which therapeutic consequence is to be drawn remains unclear. Introduction: Time to result of culture based methods for detection of microbials is too long to guide empiric antimicrobial therapy in sepsis. Furthermore, blood cultures often yield negative results despite presence of sepsis. PCR based methods may help to overcome this problem since PCR results are available within 6-8 hours.

Objectives: To report the performance of PCR based pathogen detection compared to blood culture in ICU patients with suspected infection. Methods: Patients treated on an interdisciplinary ICU were included into this observational study if a blood culture (BC) was drawn on discretion of the treating physician. Blood cultures and EDTA-blood were taken by sterile venous puncture. The EDTA-blood was processed with a PCR-based assay (VYOO, SIRS-Lab GmbH, Jena), which detects a panel of 34 bacterial and 6 fungal pathogens as well as five antibiotic resistances. Data are given as median and interquartile range. Results: 64 patients were included into this study. Age was 68.0 (55.5; 74.0) years, APACHE II-score was 17 (13; 23), and ICU mortality was 33.3%. 92 pairs of BCs and PCRs have been drawn. 42.1 % patients had at least one positive PCR and 12.3 % patients had at least one positive BC. 7 (8 %) BCs and 29 (32 %) PCRs were positive. At study inclusion, 5 % of the BCs and 35 % of the PCRs were positive. 3 out of 9 pathogens detected in the BC were also reported by the PCR. However, 4 BCs where contaminated with Staph. epidermidis which was not detected by the corresponding PCR. Fungi were detected in 10 of the positive PCR compared to 1 candida spp. detection in BC. 50 % of the patients with positive for fungi in the PCR died. Likewise, 50 % of patients (2 out of 4) with more than one positive PCR result died, as well. None of the differences in mortality rates reached statistical significance.

The PCR based assay resulted in considerably higher amount of positive results than the blood culture in this group of high risk patients. Furthermore, the data suggest that PCR can detect a high risk group of patients with positive results for fungi. The fact that repetitive positive PCR results were associated with a high mortality rate further suggests the clinical relevance of microbial DNAemia. Results: Patients (n=303) received prophylaxis (n=29), empiric therapy (n=140), pre-emptive therapy (n=85) or definitive therapy (n=49). Major risk factors for fungal infection included recent use of broad spectrum antibiotics (95%) and central venous catheters (97%). A total of 407 mycology results were available from 251 patients. Candida was the most common isolate (393/407 isolates, 97%). Candida albicans was present in 190/407 isolates (47%), non-albicans Candida in 92/407 isolates (23%), and non-specified Candida in 111/407 isolates (27.3%). A total of 298 patients (98.4%) initiated IV fluconazole as first line therapy. Complete response was evident in 66% of prophylactic group, 55% of empiric group, 45% of pre-emptive group, and 43% of definitive group patients. The overall mortality rate was 42%. Age ≥65 years, number of non-IV fluconazole treatment cycles, colonization with Candida not specified as C. albicans, and continuous renal replacement therapy including hemodialysis (CRRT) were the strongest predictors of treatment failure in univariate analyses overall. The presence of Candida not specified as C. albicans, prior fungal colonization and CRRT were significant negative predictors of empiric therapy treatment response in the multivariate analysis. The mean length of hospital stay (LOS) for patients from initiation of IV fluconazole therapy to ICU discharge was 12.7 ± 16.1 days; the mean LOS from initiation of IV fluconazole therapy to hospital discharge was 37.9 ± 46.9 days. Conclusions: Our study informs treatment decision makers that approximately 23% of the identified isolates within participating sites belonged to non-albicans Candida, highlighting the importance of appropriate therapy. Introduction: The development of resistance by bacterial species is a compelling issue to reconsider indications and administration of antibiotic treatment. Adequate indications and duration of therapy are particularly important for the use of highly potent substances in the intensive care setting. Until recently, no laboratory marker has been available to differentiate bacterial infection from viral or non-infectious inflammatory reaction, however, over the past years, procalcitonin (PCT) is the first among a large array of inflammatory variables that offers this possibility. Objectives: The present study aimed to investigate the clinical usefullness of PCT for guiding antibiotic therapy in surgical intensive care patients. Methods: All patients requiring antibiotic therapy based on confirmed or highly suspected bacterial infections and at least 2 concomitant systemic inflammatory response syndrome criteria were eligible. Patients were randomly assigned to either a PCT-guided (study group) or a standard (control group) antibiotic regimen. Antibiotic therapy in the PCT-guided group was discontinued, if clinical signs and symptoms of infection improved and PCT decreased to <1 ng/ml or the PCT value was >1 ng/ml, but had dropped to 25-35% of the initial value over 3 days. In the control group antibiotic treatment was applied as standard regimen over 8 days. Results: A total of 110 surgical intensive care patients receiving antibiotic therapy after confirmed or high grade suspected infections were enrolled in this study. In 57 patients antibiotic therapy was guided by daily PCT and clinical assessment and adjusted accordingly. The control group comprised 53 patients with a standardized duration of antibiotic therapy over 8 days. Demographic and clinical data were comparable in both groups. However, in the PCT group the duration of antibiotic therapy was significantly shorter than compared to controls (5.9±1.7 vs. 7.9±0.5 days, p<0.001) without negative effects on clinical outcome. Conclusions: Monitoring of PCT is a helpful tool for guiding antibiotic treatment in surgical intensive care patients. This may contribute to an optimized antibiotic regimen with beneficial effects on microbial resistances and costs in intensive care medicine. Introduction: The incidence and clinical relevance of rare invasive fungal infections as a cause of sepsis is increasing worldwide. Immunosuppressed patients, such as those with haematological malignancies are at a particular risk. Reliable information on their clinical course, diagnosis and treatment is scarce. Objectives: To determine the clinical pattern of disease, to describe and improve diagnostic procedures, and therapeutic regimens, as well as to facilitate the exchange of clinical isolates, we are coordinating a global registry for rare invasive fungal infections. Methods: Patients with cultural, histopathological, antigen, or molecular biologic evidence of invasive fungal infection may be included into the study. Those with infections due to Aspergillus spp., Candida spp., Cryptococcus neoformans, Pneumocystis jiroveci or any endemic fungal infection, such as coccidioidomycosis or histoplasmosis, as well as colonization or other non-invasive infections are excluded. Data entry is accomplished via a web-based electronic case report form under www.fungiscope.net Results: By now, 90 patients with rare invasive fungal infections from a wide variety of pathogens have been included. The most common underlying conditions were chemotherapy for a hematologic malignancy (23 %, n=21), diabetes mellitus (23 %, n=21), hematopoietic stem cell transplantation (18 %, n=16), and/or stay at an intensive care unit (16 %, n=14). The lungs were the most common site of infection (34 %, n=31), followed by paranasal sinuses (17 %, n=15) and soft tissues (16 %, n=14). 22 patients displayed disseminated disease. At the latest assessment, complete response to antifungal therapy was observed in 41 % (n=37). The crude mortality rate was 23 % (n=26). 5 patients (8 %) were lost to follow up and in 7 patients (6 %), final evaluation of clinical evolution is still pending.

The clinical relevance of rare invasive fungal infections is increasing steadily. In a short period of time, current cases from Europe, Asia and South America could be documented. Further investigators and coordinators are cordially invited to contribute to Fungiscope. Introduction: Disease severity varies widely in patients with severe sepsis. Previous trials (IL-1RA, TNF-sR p55, antithrombin, and drotrecogin alfa activated [DAA]) suggest that more severely ill patients benefit most from treatment. Objectives: The aim of this study was to evaluate the efficacy of eritoran for interaction effects with baseline illness severity.

Prospective covariates from a randomized, double-blind, placebo-controlled, phase 2 trial were analyzed for treatment interaction measured by 28-day mortality. Breslow-Day and multiple logistic regression (LR) were used to assess categorical (CAT) and continuous (CONT) treatment by severity-of-illness interactions. Results: Modified intent-to-treat population (N=292) all-cause 28-day mortality was: placebo, 33.3% (32/96); total eritoran 45 mg/105 mg, 29.6% (58/196). LR analysis identified Acute Physiology and Chronic Health Evaluation (APACHE) II scores, Predicted Risk of Mortality (PROM) scores, IL-6, age, sex, race, and eritoran as associated with survival outcomes. Significant treatment interactions were observed (eritoran vs placebo) for baseline covariates: APACHE II (CAT, P=0.059; CONT, P=0.035); PROM scores (CAT, P=0.028; CONT, P=0.008); number of organ failures (CAT, P=0.079); international normalized ratio (CAT, P=0.05); and acute physiology score (CONT, P=0.039). No significant treatment interactions were observed with age, sex, shock, DAA use, infection site, microorganism type, platelets, IL-6, or endotoxin levels. Interaction results were similar for eritoran 105 mg only vs placebo. Conclusions: Potential survival benefits of eritoran in severe sepsis patients may be associated with high severity of illness. Treatment by disease severity interaction will be further explored in a phase 3 trial.

Infection 2009; 37 (Suppl. III): 47

Glycemic profile in septic patients: Is daily glycemia level variation connected with poor outcome?

Szrama J, Smuszkiewicz P, Trojanowska I Department of Anesthesiology, Intensive Therapy and Pain Management, University Hospital Poznan, Poland.

The beneficial effects of tight glycemia control on mortality and morbidity have been widely proved. However, intensive insulin therapy which is recommended to control the glycemia in septic patients, can induce hypoglycemia. Objectives: The goal of the study was the evaluation of the glycemic profile of patients with severe sepsis and septic shock treated with a nurse-managed intensive insulin therapy protocol.

The study was conducted in two groups of patients: I -included nonsurvivors (n=16), II -patients who survived (n=61). Glycemia was measured every 4 hours, and the dose of insulin infusion was adjusted to maintain glycemia of 4.4 mmol/L to 8.3 mmol/L. We analyzed glycemia levels and daily variations, insulin dose, episodes of hypo and hyperglycemia. Results: Preexisting diabetes and corticotherapy in the treating process of sepsis included a comparable percent of patients in both groups. Mean glycemia levels (7.38 ± 0.98 mmol/L vs 7.08 ± 0.83 mmol/ L; P = .20), mean insulin requirements per day (35.9 ± 30.4 units/d vs 26.8 ± 22.5 units/d; P = .22), and per hour (1.9 ± 1.2 units/h vs 1.6 ± 0.9 units/h; P = .25) were similar in both groups. Mean daily glycemia variation differed significantly between the groups (nonsurvivors, 5.00 ± 1.65 mmol/L vs survivors, 3.35 ± 1.8 mmol/L; P < .001). Episodes of severe hypoglycemia (18.8% vs 3.3%; P = .02), spontaneous severe hypoglycemia (12.5% vs 0%; P = .006), spontaneous hypoglycemia < 3.3 mmol/L (31.3% vs 4.9%; P = .003) occurred more frequently in nonsurvivors. Episodes of hyperglycemia > 8.3 mmol/L occurred at statistically similar levels in the two groups. Episodes of hyperglycemia > 10.0 mmol/L (87.5% vs 57.4%; P = .03) were more frequent in the nonsurvivors group. Conclusions: Patients who died due to severe sepsis and septic shock, compared with patients who survived, had more episodes of spontaneous severe hypoglycemia (< 2.2 mmol/L and < 3.3 mmol/L), episodes of hyperglycemia > 10.0 mmol/L and higher mean daily blood glucose level variation. It cannot be judged whether hypoglycemia, hyperglycemia, and mean daily blood glucose variation were factors that in-

creased mortality in patients with severe sepsis and septic shock or whether they were only markers of an increased risk of death. Mean blood glucose level is not a reliable parameter to evaluate glycemic homeostasis in patients with sepsis and daily blood glucose variation should be included in the analysis of the glycemic profile. Introduction: Recommendations from the 2008 Surviving Sepsis Campaign suggest using a validated protocol for insulin dose adjustment and targeting glucose levels to < 8.3 mmol/L. However hypoglycemia related to intensive insulin therapy occurs 4 to 7 times more frequently in patients treated with strict glycemic control, and sepsis itself may be a risk factor for severe hypoglycemia < 2.2 mmol/L. Objectives: The goal of the study was to evaluate safety and effectiveness of a nursing-controlled insulin therapy protocol in patients with severe sepsis and septic shock.

The study included 77 patients with severe sepsis and septic shock. Tight glycemic control was maintained by continuous intravenous infusion of short-acting insulin. Glycemia was measured every 4 hours, and the dose of insulin infusion was adjusted by the nursing staff according to a validated protocol to maintain glycemia of 4.4 mmol/L to 8.3 mmol/L. If the blood glucose level exceeded the recommended range, additional samples were taken every 1 or 2 hours, until glycemia level returned to desired range. If the blood glucose level was > 21.1 mmol/L or < 4.4 mmol/L an intensivist was called. The effectiveness of nurse-managed intensive insulin therapy protocol was evaluated by analyzing the percent of all blood samples and blood glycemia levels within the 4.4 mmol/L to 8.3 mmol/L range. The safety of the protocol was examined by analyzing the percent of blood glycemia levels < 2.2 mmol/L (severe hypoglycemia) and < 3.3 mmol/L (hypoglycemia). Results: A total of 5393 blood samples and blood glucose levels were examined. Only 3 of 77 patients (3.9%) experienced one insulin-induced episode of severe hypoglycemia, and 3 of 5393 blood glycemia samples (0.05%) matched criteria for severe insulin-induced hypoglycemia. Mean glucose level maintained by intensive insulin therapy was 7.14 ± 0.87 mmol/L, and 4013 samples (74.4%) were in the recommended 4.4 mmol/L to 8.3 mmol/L range. When a higher upper range of blood glucose concentration of 10.0 mmol/L was allowed over 90% of analyzed blood samples matched the desired range.

The study confirmed the effectiveness (75% of the samples met the recommended range) and safety (only 3.9% of the patients experienced an episode of severe insulin-induced hypoglycemia) of the nurse-managed intensive insulin therapy protocols. ) or 5% human albumin (HA) were administered to 50 pts per group over 30 minutes. Hemodynamic measurements were taken regularly but in this analysis the baseline values, the maximum effects and the 120th min values of infusions as well as the AUCs of the hemodynamic parameters during 120 min were considered. Ten hemodynamic parameters were altogether analyzed in septic (n=94) vs non-septic pts (n=106).

Results: There were no significant differences in age, gender and type of operation however septic pts were more severely ill (severity scores, morbidity and mortality data) than non-septic pts. The baseline, the maximum and the 120th min' values did not differ significantly in sepsis vs non-sepsis relation.

In intergroup relationship, the colloids had more pronounced maximum effects than LR in central venous pressure (CVP), cardiac index (CI), stroke volume variation (SVV), central venous O2 saturation (ScvO2) and extravascular lung water in septic pts, while the systemic vascular resistance (SVR) decrease was more significant in non-septic pts after GEL, HES volume loading. The colloids, especially HES showed a more significant 120th min' effect than LR in mean arterial pressure, CVP, global end-diastolic index (GEDVI), SVV and ScvO2 in septic pts. Moreover, the effect of HES in GEDVI was significantly larger than GEL in septic pts. There were significant differences in the AUCs after HES and HA administration in CVP, CI and SVR in septic vs non-septic pts. The intergroup analysis revealed a more pronounced AUC increasing effect in CI, GEDVI, SVV, and O2 delivery index after HES and lesser degree after HA administration in septic pts.

In postoperative septic pts the colloid infusions, especially HES and HA caused more significant hemodynamic changes than LR in the studied dose. At 120th min LR had no demonstrable hemodynamic effect either in septic or in non-septic pts. The adsorber was initially evaluated in two randomized safety studies including cardiac surgical patients undergoing cardiopulmonary bypass. No side effects were observed in any of the patients (n=18 in the adsorber arm).

In an initial study of seven patients with septic shock blood endotoxin was reduced from 1.44 EU/ml before treatment to 0.40 EU ml/ml after treatment with a concurrent reduction of dopamine (-73%), procalcitonin (-64%) and relevant cytokines. In addition improvement in hemodynamics and oxygenation index (98%) was observed. Efficacy was also explored in an observational study with five patients in the adsorber arm and ten historical controls, all with septic shock due to intra-abdominal infection. The main duration of vasopressor infusion was 46 hours shorter in the adsorber group. Furthermore SOFA score decreased substantially 24 hours after treatment (-3.4±1.7) as well as mean LPS and PCT values. Moreover the average length of hospital stay was 3.4 days shorter in the adsorber group. Conclusions: The Alteco® LPS Adsorber is based on a new promising technology ensuring a safe and efficient design. Together with the positive initial clinical data and easy handling these features makes the Alteco® LPS Adsorber a natural choise in order to reduce the endotoxin load in the patient with septic shock or the potential septic patient. Introduction: Therapy of sepsis continues to be largely supportive and new therapeutic interventions with clinical outcome benefits are clearly needed. Nevertheless, an improving understanding of sepsis pathophysiology has created new opportunities for advances in the clinical management of patients with this syndrome. Since inflammatory mediators play a major pathophysiologic role, their modulation Clinical Sepsis Research -Therapy by extracorporeal dialysis-based modalities may be a rational approach. In this regard, although conventional continuous renal replacement therapy (CRRT) techniques have little ability to influence mediator concentrations, modified approaches may be beneficial. One such modified approach is a highly permeable CRRT membrane having pore dimensions that allow for substantial removal of solutes with molecular weights in the inflammatory mediator range (approximately 5 kDa to 50 kDa). As such, this high-cut off (HCO) membrane, manufactured by Gambro (Hechingen, Germany), extends the molecular removal spectrum beyond that which occurs in conventional dialysis techniques. Objectives: The purpose of this presentation is to discuss the clinical results reported from several studies in which the Gambro HCO membrane has been applied in septic patients. Methods: The review will focus on the effect of HCO therapy on plasma cytokine clearance and concentrations, immunologic and hemodynamic parameters, and safety-related parameters reported from the studies. Results: The review will present data demonstrating that HCO membranes effectively remove inflammatory mediators from blood and that treatment using HCO membranes has beneficial effects on immune cell function. Clinical data from both single-center and larger multi-center trials regarding the effect of HCO membranes on plasma cytokine levels and the need for vasopressor therapy will be presented. Finally, the safety profile of the HCO membrane based on the clinical experience to date will be discussed.

The HCO membrane represents a potentially promising therapy for critically ill patients with sepsis syndromes. Additional studies are needed to define the patient populations which may benefit from this novel therapy. Introduction: Therapy of sepsis continues to be largely supportive and new therapeutic interventions with clinical outcome benefits are clearly needed. Nevertheless, an improving understanding of sepsis pathophysiology has created new opportunities for advances in the clinical management of patients with this syndrome. One pathophysiologic aspect that continues to be a focus of both experimental and clinical studies is the role of endotoxin in sepsis. Clinical sepsis is characterized by the episodic appearance of endotoxin in the blood and an unpredictable profile of endotoxin fragments that activate inflammatory pathways. However, therapies focused on endotoxin removal may have a beneficial impact in septic patients. The purpose of this presentation is to discuss a new membrane (Oxiris) manufactured by Gambro (Lyon, France). The substrate of the Oxiris technology is the AN69 membrane, which has unique properties allowing for the adsorptive removal of inflammatory mediators and other low-molecular proteins during conventional continuous renal replacement therapy (CRRT). The endotoxin-removing capabilities of Oxiris are derived from the application of positively charged polymer to the AN69 base. The effective creation of a positively charged surface creates an environment favoring the removal of negatively charged endotoxin fragments during CRRT with this membrane. Moreover, this polymer application allows for the binding of heparin during the manufacturing process, resulting in a membrane with immobilized heparin. Finally, neither the polymer application nor the heparin immobilization negatively influences the inherent mediator removal capabilities of the base AN69 membrane. In fact, due to the larger surface area (1.5 m 2 ), mediator removal is enhanced relative to standard AN69 membranes used for conventional CRRT.

Objectives: The purpose of this presentation is to discuss the experimental, animal, and clinical results reported from several studies in which the Gambro Oxiris membrane and its precursor products have been used. Methods: The presentation will address the following: 1) blood-membrane interactions during CRRT, with focus on protein and peptide adsorption; 2) principles of the AN69 surface treatment underlying preparation of Oxiris membrane; 3) endotoxin and inflammatory mediator removal by the Oxiris membrane Results: The presentation will discuss data demonstrating that surface treatment results in a stable layer immobilized heparin with no loss of adsorptive function of the base AN69 membrane.

The Oxiris membrane represents a potentially promising therapy for critically ill patients with sepsis syndromes. Additional studies are needed to define the patient populations which may benefit from this novel therapy. Introduction: Local antibiotic treatment using a temporary antibioticimpregnated PMMA spacer for severe bacterial joint-infections or infected orthopedic implants is currently well accepted. But the longterm exposure to low doses of antibiotics after an initial release of high doses is also seen critical by many. Persistence of bacterial growth on the foreign body and development of bacterial resistance are possible dangers.

Objectives: The objective of this study was to determine possible risks of antibiotic loaded PMMA-Spacer.

We examined 56 patients treated in the years 2006-08 with regards to different types of bacteria, local and systemic antibiotic medication, times of revision, final outcome and patients condition before the treatment. Results: The most common bacteria detected were of staphylococcal (64%) and enterococcal (11%) origin. Other bacteria or mixed infection played only a less important role. Multiresistant organisms (e.g. MRSA) complicated the treatment in 8 patients. Five of these patients acquired the infection with MRSA during the treatment with a gentamicin-vancomycin-loaded spacer. These patients mostly had severe comorbidities and needed multiple revisions after the spacer implantation. This group of patients had a high risk for the development of sepsis and fatal ending.

Our results indicate that in vivo growth of multiresistant specimen on gentamicin-vancomycin-loeaded bone cement is possible. Long-term low dose local antibiotic treatment seems to favor the development of bacterial resistance. Multiresistant bacterial strains and a compromised immune system highly endanger the patient. It has to be evaluated in following studies if other treatment options or the modification of the existing protocol can be helpful particularly for patients with a reduced immunity. Introduction: Patients with septic shock and relative adrenal insufficiency are at increased risk of death, but there remains clinical equipoise regarding the role of short term, "low dose" hydrocortisone (HC) in this population. Furthermore, the incidence of HC induced metabolism dysregulation especially hyperglycaemia had been largely underreported (1) . In a recent large multi-center randomized placebocontrolled trial (CORTICUS) HC did not reduce mortality in patients with septic shock, regardless of their response to a corticotropin stimulation test. Moreover, in the per protocol analysis of safety endpoints, there were significant increases in the rate of hyperglycaemia, defined as glucose concentration >150 mg/dL (RR 1.18, 95% CI, 1.07-1.31). It has been hypothesized, that the increased glucose levels in the HC group may have contributed to increased mortality (2). Objectives: To determine the occurrence of hyperglycaemia (>150 mg/dL) in patients during the first week of the CORTICUS trial and its association with mortality at 28 days. Methods: Glucose values available on study days 0,1,2,3 and 7 were analyzed. Patients were stratified by treatment group (HC 200 mg/day administered as a 50-mg intravenous bolus every 6 hours for 5 days or placebo) and response to a 250-µg ACTH -adrenocorticotropic hormone stimulation test -(responder or non-responder, defined as an increase in cortisol level < 9 µg/dL from baseline ). Introduction: The risks of anemia and blood transfusion have never been investigated in surgical intensive care patients. In addition, transfusion medicine is evolving and leukoreduction has been more widely implemented nowadays. We investigated the epidemiology of anemia and blood transfusion in a large cohort of surgical ICU patient and their relation to outcome in these patients. Clinical Sepsis Research -Therapy

In this group of surgical ICU patients, anemia was common and was associated with higher morbidity and mortality rates. Higher hemoglobin concentrations and blood transfusions were independently associated with low hazard of in-hospital death. The groups more likely to benefit of blood transfusion were the elderly patients with higher severity of illness and considerable organ dysfunction.

Infection 2009; 37 (Suppl. III): 53

Hemodynamic stabilization and reduction in inflammation in the brain-dead organ donor by methylprednisolone administration Introduction: Animal experiments and clinical studies have shown that irreversible loss of cerebral function leads to an increase in the proinflammatory cytokines in the blood and tissue, which may contribute to cellular lesions in the donor organs. Further, this cytokine elevation reduces the response of the peripheral vascular flow areas to endogenous and exogenous catecholamines and augments the circulatory impairment already caused by the lack of central sympaticoadrenergic circulatory regulation. Objectives: We aimed to investigate whether high-dose methylprednisolone administration in organ donors improves the circulation, reduces the noradrenalin requirement and lowers the proinflammatory cytokine level.

We performed a randomized prospective study of 100 organ donors. Fifty (group I) were treated with methylprednisolone and 50 (group II) were not treated. Methylprednisolone was given as a bolus followed by 100 mg per hour until organ explantation. Donors hemodynamics were monitored using the the Picco system (Pulsion Medical Systems Inc., Munich) and the following parameters were recorded: heart rate, mean arterial pressure, central venous pressure, intrathoracic blood volume index, cardiac index, systemic vascular resistance index and extravascular lung water index. Measurements were made every 2 hours; blood was sampled directly after the first measurements and before the donor left the intensive care unit. S-cortisol, CRP, PCT, Il-6, Il-8, Il 2R, TNF alpha, FT3 and FT4 were documented. Results: There were no differences between the two groups in age, gender, cause of brain damage, intensive care unit stay, number of multiorgan donors or number of transplanted organs per donor. The time between first diagnosis of loss of brain stem function and collection of the first blood sample was 42.8 h in group I, only slightly longer than in group II (38.4 h). Time between the first and second blood sample did not differ significantly between the groups (9.2 vs. 10 h). The methylprednisolone treatment led to a significant increase in mean arterial pressure and cardiac output index (p<0.05) and a significant reduction in noradrenalin requirement (p<0.05). Il-6, Il-8, Il 2R und TNF alpha were also significantly reduced (p<0.01). In the group without treatment the cytokines rose significantly (p<0.01); the mean arterial pressure fell and the noradrenalin requirement rose (both without statistical significance).

Our results show the advantages of methylprednisolone treatment. This should be begun in organ donors as soon as brain death is diagnosed. Introduction: Liver failure remains associated with high mortality.

Besides patients with acute-on-chronic liver failure due to, e.g., bleeding episodes or acute infections there are patients with MODS in whom acute liver failure persists or aggravates despite successful treatment of underlying causes such as sepsis or shock. As liver transplantation is rarely an option in these patients, extracorporeal liver support therapy (ELS) may be considered as temporary emergency measure until liver function is restituted ("bridging to recovery"). Advantage of ELS with the Prometheus ® system is the simultaneous removal of both albumin-bound and water-soluble metabolic waste products and toxins. HES was administered to patients between day 0 and day 63 after start of sepsis (Fig. 1) . Leucine enkephalin as treatment of septic polyneuropathy and encephalopathy

Niehaus I Kiel, Germany.

The neurotransmitter and delta-opioid receptor agonist leucine enkephalin (LE) is a pentapeptide with the amino acid sequence tyrosine, glycine, glycine, phenylalanine, leucine. LE protects lipopolysaccharide (LPS)-stimulated dopaminergic neurons and reduces anti-LPS-antibody production of LPS-stimulated B-cells. LE increases cerebral blood and lymphatic flow. The activity of primary afferent nerve fibers of the spinothalamic tract is elevated by LE. Acute and long-term neurological sequelaes of sepsis occur in up to 70% of the patients. Objectives: Description of the permanent positive effects of LE in a single case study of LPS-induced polyneuropathy, encephalopathy and parkinsonism 14 yrs after sepsis. Methods: LE is used in oral doses of 1 micromol every third day in the patient.

Results: Electrophysiologically examinations showed results typically for critical illness polyneuropathy like missing F-waves in the N. peronaeus and sensory polyneuropathy with conduction blocks. The sensitivity of all nerves but especially in the legs is regained dose by dose with ability to walk up to 3 km and even running for short distances. Parkinsonism-related stiffness, rigidity and hypokinesia were markedly improved. The ability to concentrate improves. Chronically elevated IgM-antibodies in the blood of the patient were reduced. Conclusions: LE shows excellent anti-parkinsonian and anti-inflammatory effects with stimulation of afferent nerve fibers, which might be partly blocked by LPS binding to the alpha2-delta2-subunit of voltage-gated calcium channels in delta-opioid receptors. LE stabilizes the reduced glucose and dopamine metabolism in the brain of the patient by increased blood flow. Clinical trials in parkinsonian and septic patients with neurological damages with LE should be considered. Introduction: Sepsis is the third most frequent cause of death after acute myocardial infarction in Germany. Lack of information about the patient's medical history and the exact circumstances of the cardiac arrest might results in efforts to resuscitate a patient.

Objectives: This report describe the case of 76 year old female patient with cardiac arrest, who received a successful initial cardiac resuscitation outside and inside hospital (3 mg epinephrine, epinephrine-infusion, nitro-infusion and 1 mg atropine). Methods: case report Results: The initial diagnosis (echocardiography, ecg, troponin I 0,236 ng/ml, CK-MB 1,62 µmol/l, lactat 11, 5 mmol/l, leucocytes 19,1 Gpt/l, C-reactive protein 52,5 mg/l) was cardiogenic shock. Procalcitonin (PCT) was 10 ng/ml. After reading medical history with the family about the acute myocardial infarction 3 months ago and bronchitis 1 month ago, we started with PICCO-monitoring and early goal directed therapy. The recovery was effective in few hours after start with infusions, hydrocortison, tazobactame, dobutamine and norepinephrine. The patient was catecholaminfree and extubated within 48 hours. Conclusions: Sepsis is the most important challenge in intensive care medicine, associated with significant morbidity and mortality. Procalcitonin levels correlate with the organ dysfunction away from the site of infection and should be used for differential diagnosis in all shock cases. Introduction: Hemorrhagic cystitis (HC) is a common complication after hematopoietic stem cell transplantation (HSCT). In the majority of cases the cystitis is BK virus (BKV) associated. The management is difficult. Many treatment options exist, but they all have a weak base of evidence, or are potentially toxic. Objectives: We report an 18-year-old adolescent with systemic mastocytoses and associated myelodysplastic syndrome. 25 days after hematopoietic stem cell transplantation the first symptoms of HC dysuria, pollakisuria and hematuria occured. BKV DNA was detectable in blood and urine. Our treatment with cidofovir, hyperhydration, estradiol and oxybutynin was not successful. The blood coagulation in the urine increased. Therefore catheterisation and bladder irrigation with 0,9% saline was initiated. After bladder tamponade urological intervention was necessary and a peridural catheter for pain management was implanted. The symptoms did not improve. Catheter coagulation, pain attacks and hematuria were still present. Bladder irriga-tion with cidofovir repeatedly over 5 days showed temporarily response 49 days after HSCT the patient developed acute GVHD of the skin and prednisolone treatment was initiated. Interesting, after the start of prednisolone we observed improving symptoms of the HC. Methods: A case report Results: HC is a possible life threatening complication and the management is difficult. Acute GVHD as a trigger of the HC was described earlier and must be discussed in this case. Conclusions: In many cases treatment options show no significant improvement. Future researches are necessary for preventive and therapeutic strategies which include the role of the acute GVHD. Introduction: Sepsis is a significant cause of morbidity and mortality in children, accounting for 7% of all childhood deaths. In the neonatal population, sepsis is responsible for 45 % of late deaths in the neonatal intensive care unit. Objectives: Low protein C plasma levels in septic neonates have a strong positive predictive value for death. There have been several studies testing interventions with Protein C. Whereas recombinant activated protein C (aPC) showed no benefit but increased bleeding complications in septic children especially in those less than 60 days of age, plasma-derived human non-activated protein C concentrate (hPC) has been successfully used in pediatric and neonatal patients as well as in adults at high risk of hemorrhage. We report on the use of human Protein C concentrate in low birthweight preterm infants with disseminated intravascular coagulation (DIC), septic shock and purpura fulminans.

Methods: Retrospective analysis of preterm neonates with DIC, septic shock and severe impairment of microcirculation with incipient purpura fulminans according to nosocomial culture-proven gram-negative sepsis. The patients received intravenous bolus administration of 200 IU/kg human Protein C Concentrate (hPC, Ceprotin®, Baxter Vienna, Austria) followed by a daily bolus administration of 50 IU/ kgq4 hPC additively to conventional treatment for sepsis and septic shock. Results: 4 preterms with a median birth weight of 1410 g and a gestational age of 29 weeks were assessed. Protein C activity prior to treatment was 12 %, predicting a mortality of 80 %. Predicted mortality according to NEOMOD-Score was nearly 70 %; however, all patients survived. Fast improvement of microcirculation, DIC and septic shock was observed in all patients. Purpura fulminans regressed and all patients recovered completely. No amputation of affected extremities was necessary. Neither de novo intracerebral bleeding nor aggravation of a pre-existing prematurity associated intraventricular hemorrhage was observed. No acute adverse events or bleedings, thromboembolic complications during a follow up period of 6 months were observed.

In this small group of preterms with severe sepsis and severe microcirculatory impairment, human protein C concentrate rapidly restored the microcirculation, improved sepsis and patients' outcome without acute adverse effects like bleeding complications nor any other other drug related complications during a follow up period of 6 months. Our current pathogenetic understanding is that Shiga toxins cause endothelial injury, leading to thrombotic microangiopathy . Renal failure is the predominant manifestation of the disease. The prognosis of HUS has improved in recent years due in part to peritoneal dialysis. Although the chance for recovery of renal function is generally favourable, there is still a five percent rate of mortality particularly caused by cerebral involvement. Objectives: As human protein C (PC) concentrate may anticipate thrombotic microangiopathy and facilitate fibrinolysis in this disease, we report the effects of substitution with PC in 8 patients with EHEC. Methods: 8 EHEC-positiv patients with HUS, shigatoxin 2 and severe neurological symptoms (seizures and coma) were treated with human protein C concentrate (Ceprotin, Baxter). PC was administrated in a dosage of 100-200 U/day as a bolus. Results: 7 of them suffered from a multiorgan failure. 1 patient was resuscitated as a result of a myocardial microangiopathy.The symptoms of the multiorgan failure improved rapidly and the seizure were terminated. The duration of the PC-therapy depended on persisting of clinical and chemical signs of the HUS. All patients showed signs of a disseminated intracvascular coagulation. The activity of Plasminogen-Activator-Inhibitor 1 (PAI-1) and D-Dimers were significantly elevated. They rapidly decreased in the first 48 hours of the therapy especially in those patients, who showed already high levels at the beginning of the desease. A peritoneal dialysis was administered in all cases. The renal function of all patients recovered during the hospital stay. CCT were performed to detect HUS typical alterations of the brain. Conclusions: Mortality in HUS accompanied with cerebral microangiopathy is high and difficult to alter. This is the first trail of human protein C concentrate administration to anticipate thrombotic microangiopathy in HUS. All of our patients showed rapidly clinical improvement after PC administration. A significant Protein C deficiency was not existing, but all patients showed signs of a disseminated intravascular coagulation. All patients survived the severe cerebral form of the HUS. 6 of them were discharged with a restitutio ad integrum. Two girls suffered from severe neurological residues. The containment of the severe neurological involvement in our patients yield hope, that PC treatment may be an effective therapy regimen in the treatment of severe cerebral HUS.

trations when neutrophils were in a passive state. The neutrophil elastase concentrations were not different from neutrophils of neonates and adults. The mentioned PDE-inhibitors significantly increased the deformability of activated PMN. Significant differences between the deformability of PMN in neonates and adults were not found. Conclusions: PDE III/IV inhibitors lead to similar improvement of mechanical properties of stimulated neutrophils in neonates and adults despite of the functional differences of neutrophils in neonates. These drugs may ameliorate impaired microcirculation also in neonates during inflammation. Introduction: Invasive meningococcal infections are an uncommon but redoubtable cause of sepsis. In 2008, the incidence amounted to 453 cases in Germany (0,55/100.000) with an overall mortality of 8%. The disease pattern ranges from overwhelming sepsis to purulent meningitis and nearly asymptomatic bacteraemia. The understanding for these differences still is rudimentary, even though a multitude of information is available about virulence factors of the pathogen and specific causes for host susceptibility.

Objectives: The aim of our network is to aggregate and combine information about molecular epidemiology and strain-specific virulence factors with disease severity and risk factors for host susceptibility, in order to explain the different disease courses and outcomes. Methods: Since 2008 we record nationwide all cases of invasive meningococcal disease in children and adolescents by a voluntary reporting system where all children's hospitals in Germany are asked two times yearly to notify invasive cases by a case report form and to send in blood samples. Disease severity is calculated by a score based questionnaire. Host susceptibility is examined by tests for functional asplenia, defects in complement cascade (CH50, mannose binding lectine, properdine) and a screening for single nucleotide polymorphisms in molecules of the innate immune system. The reported cases are matched with those of the National Reference Centre for Meningococci (NRCM), where epidemiologic and genetic characterisation of the meningococcal isolates is performed. Spatiotemporal cluster analysis provides information about epidemiology and outbreak of hypervirulent meningococcal strains. Whole genome analysis and comparison with a definded "meningococcal pathogenome" investigates virulence of the specific strain. Results: Our return rate is 10% of all cases in Germany, and approximately 50% of all cases in the paediatric age group, with a matching rate of 97% for our returns with the NRCM. Severe sepsis made up 30%, 13% requiring ventilator therapy, 16% vasopressors, respectively. Overall mortality was 8.5%. Petechiae proved to be the cornerstone to raise clinical suspicion of the disease. In our immunological screening we found about one third of patients with at least a preliminary suspicion for underlying immunological disease.

The German Meningococcal Network shows to be a since 2008 well established tool to investigate interrelationship between bacterial and host mechanisms of disease severity. Continuation and further evaluation is encouraged as well as participation of all colleagues -including those dealing with adult patients -faced with treatment of acute invasive meningococcal disease. Copeptin and Mr-proANP concentrations also correlated with the first measured mean arterial pressure (MAP) after birth. The groups with a MAP of < 30mmHg, 30-34mmHg, 35-39mmHg and >40mmHg had decreasing Copeptin levels with Medians of 1270 pmol/l, 245 pmol/l, 106 pmol/l und 135 pmol/l. Analogous the Mr-proANP levels had Medians of 1110 pmol/l, 419 pmol/l, 573,5 pmol/l and 408 pmol/l. Due to the extent scatter range no significant difference could be found. No significant correlation between concentrations of Mr-proADM and MAP, GA and birthweight (BW) could be found. No significant difference could be found between healthy infants and those developing an early-onset sepsis in all three biomarkers. Conclusions: Ct-proAVP and Mr-proANP concentrations in term and preterm infants depend highly on GA and BW. Term neonates had 10 times higher and preterm neonates even 100 times higher copeptin concentrations in comparison to healthy adults. This shows the ability of term and preterm newborns to regulate their blood pressure via AVP and might be an indication that AVP is a major factor in blood pressure regulation. Additionally Mr-proANP -levels in term and preterm infants had significantly higher concentrations in comparison to healthy adults.

A case report with special clinical decisions Objectives: To evaluate and to discuss the therapy strategy of paediatric sepsis with use of ECMO Methods: A fourteen year old girl was transferred by the emergency team of our university Paediatric Intensive Care Unit (PICU) from a local hospital. The transfer was realized 8 hours after the initial admission. The patient was presented with septic multi organ failure and Purpura fulminans. ECMO was necessary because of heart failure and failure of circulation and showed no major complications. The child had wide spread dermal necrosis within the face and all extremities. Because of arterial ischemia amputation of the left leg at tibia level was necessary. This was followed by recurrent surgical debridement of necrotic tissue and constant vacuum therapy of necrotic lesions. Later on transplantation of autolog skin was performed by plastic surgery. A multidisciplinary treatment of early rehabilitation with physiotherapy (including chair of Bipedestation, mobilisation in CO2 water and ergotherapy), local treatment by Plastic Surgery and intensive care treatment by our PICU was very effective. Local pain therapy after end of systemic treatment was helpful. Results: The patient had been transferred to a neurophysiological rehabilitation hospital 3 month after admission. In a 24-month evaluation she showed excellent neurological rehabilitation, with small deficits in the area of concentration. The local findings at left leg were without any signs of infection. Problematic is still re-integration in normal school, despite of legislation for children with special needs in the normal school. Conclusions: ECMO was successful in a paediatric septic shocked patient despite of severe local infection and the need of lower leg amputation. An excellent multi disciplinary cooperation coordinated by PICU between different departments was the key of early discharge and successful rehabilitation. The outcome could have been better by fastening treatment access inside a regional paediatric intensive care network with very early implementation of treatment. Unified integrated clinical pathways from emergency department to reintegration in normal life should be designed and supervised. Centralized control of outcome by coordinated social, educational and medical services is needed. Introduction: For intensive care patients particles in infusion solutions imply a potential health risk. As part of a clinical trial (ClinicalTrials. gov; ID NCT 00209768) we assessed the exposure of particles to our patients and their inflammatory effects in vitro. Objectives: To investigate the nature of infused particles in vivo we examined the physical properties and chemical composition of particles captured by inline micro-filters used in the treatment of critically ill children. We analysed the inflammatory and cytotoxic effects of particles on human umbilical vein endothelial cells (HUVEC) and murine macrophages (RAW 264.7) in vitro. Methods: 22 filters (Pall NOE96E/ELD96D, pore diameter 0.2µm) used in the treatment of critically ill children were analysed. Each filter was analysed by electron microscopy, and the chemical composition of random particles was determined by energy dispersion spectroscopy (EDX).

In the in vitro model, HUVEC and RAW 264.7 cells were incubated with different solutions of glass particles for up to 24 hours. This was conducted in the presence or absence of lipopolysaccharide (LPS) to mimic severe sepsis in vivo. Levels of interleukin-1beta (IL-1β) and IL-8 were measured in the HUVEC model; IL-6 and TNF-alpha in the RAW-model (ELISA). Results: Average particle density found on the surface of a used filter membrane was 542 per cm2, compared with 48 per cm2 on the negative controls. They varied considerably in shape and size. 54% of the detected particles were between 5-15µm, whereas only 5% were above 50µm. Overall silicon was the most abundant element. When HUVEC and RAW 264.7 cells were incubated with glass particle solutions we found that cytokine levels were significantly modulated depending on time of incubation, particle load and stimulation with LPS. Secretion of IL-1β and IL-6 was significantly suppressed in the presence of glass particles; in the case of IL-6 suppression only occurred after previous stimulation with LPS. Levels of IL-8 were also suppressed after incubation with glass particles in the presence of LPS. Conclusions: Inline-filtration prevents the infusion of potentially harmful particles. Our findings confirm that micro-particles are capable of modulating cytokine secretion by endothelial cells and mac-rophages in vitro. In this way they may exert harmful effects which are beyond simple mechanical obstructions, and their immunosuppressive effect may specially deteriorate the clinical course of septic patients. Introduction: Particulate contamination of infusion solution may potentially contribute to a clinical deterioration of intensive care patients especially on this certain background of debilitation and impaired host responses. Particles have been shown to induce thrombogenesis, deterioration of microcirculation and modulation of immunoresponse.

We assessed the effect of in-line filtration on the reduction of major complications in critically ill children (Clinical Trials.gov ID NCT 00209768). Objectives: Primary objectives were a reduction in the incidence of sepsis, thrombosis, systemic inflammatory response syndrome (SIRS) or organ failure (liver, lung, kidney, circulation). Methods: In a randomised, prospective trial paediatric patients admitted to the interdisciplinary PICU of a tertiary university hospital were assigned to either control or interventional group the latter receiving in-line filtration (infusion filter Pall ELD96LLCE/ NOE96E, Braun Intrapur Lipid/ Intrapur Neonat Lipid) throughout whole infusion therapy. Prior to this study, infusion regiment was optimised to prevent precipitation and incompatibilities of solutions and drugs. Results: Interims analysis of 398 children (171 female, 227 male, mean age 72 months) revealed a heterogeneous background of underlying diagnoses and a Gaussian distribution to either control (208 patients) or in-line filtration group (190 patients) . First analyses demonstrated a significant reduction in the incidence of SIRS for the interventional group (95% CI, 44 to 68, P< 0.035).

The occurrence of sepsis, SIRS, thrombosis or organ failure often complicates the course of disease in critically ill patients. Inline-filtration is most effective reducing the incidence of SIRS. Additional analyses are expected to confirm the preliminary results as well as to further identify the influence of inline-filtration on other complications like sepsis.

Organisational Aspects emergency surgery. Patients were equally distributed in all hospital size strata, or in surgical or medical ICUs. Conclusions: Although the risk of EOL-D is increased in patients with sepsis, post-hoc analysis of a representative observational prevalence database showed that frequency and ICU mortality of EOL-D was not higher in patients with severe sepsis in German ICUs than in all ICU patients in a prospective European multi-center study. Systematic educational program improves compliance with sepsis protocols Objectives: To determine whether, in a university hospital ICU setting, continuous educational program, according to the recommendations of the Surviving Sepsis Campaign, improves diagnostics and therapy of severe sepsis and septic shock, and reduces hospital mortality.

The study was executed following a pre-and post-interventional cohort design. The intervention consisted of implementation of a systematic educational program for nurses and physicians on early detection of sepsis. In addition, the ICU staff was trained in the state of the art therapy interventions which consisted in the awareness of the six-hours resuscitation bundle criteria: maintenance of MAP>=65 mm Hg, CVP>=8mm Hg and ScvO2>=70%, serum lactate < 4mmol/l (through improving oxygen delivery), blood culture before antibiotics, and antibiotic compliance (administration by 1 hour following sepsis diagnosis). Sepsis-themed posters supporting the campaign were displayed in various hospital departments, personnel were given pocket card instructions about the diagnosis and treatment of sepsis, and the intranet was updated to include important information and links concerning sepsis. A qualified study nurse screened all ICU patients daily following the SIRS criteria and enrolled them if the consulting physician confirmed the clinical presence of infection, thereby determining time zero for the diagnosis. The data for the preinterventional cohort was collected during a period of 22 months (2006 -2007) . The data for the post-interventional cohort was collected over 13 months (2007 -2008 Introduction: Severe sepsis and septic shock are among the most common diseases in intensive care units and are associated with a high mortality.

Most patients information is stored in the Patient Data Management Systems (PDMS). This database is unfortunately too large so as to facilitate sufficiently well for fast diagnostics of sepsis. Indeed it has been proved that early begin of treatment combined with goal-directed therapy improves outcome of the disease significantly, while for early beginning of treatment an early diagnosis is necessary. Objectives: To develop software tool that would permit early diagnosis by detecting automatically from the already existing data in the PDMS, the presence of severe sepsis or septic shock and to test its potential for early diagnosis of the condition. Methods: A tool, the Sepsis-Tool (ST), was developed which screens automatically every 8 minutes the data in PDMS and issues a warning as soon as the criteria (according to the ACCP/SCCM Consensus Conference for SIRS, one or more organ failure and/or shock) are met. The ICU staff is then immediately prompted to verify the existence of concomitant infection in order to establish a diagnosis. Consecutive Patients admitted to the ICU of university hospital were entered in the study using a prospective cohort design. The patients were permanently screened by the ST, which was followed only by a study nurse who established the diagnosis and, on the following day, compared the finding of the ST with these of the ICU physician who were left unaware of the ST warning. Results: At present, from 176 ICU patients, the ST identified 70 as SIRS, one or more organ failure and/or shock, from which only 24 were identified by the ICU staff (study nurse) as severe sepsis or septic shock. The ICU physician, who did not use the ST, was also able to identify the same 24 patients as severe sepsis or septic shock, although 12 from them he identified with 2.5 hours (median) delay. Conclusions: An automatically screening tool in addition to PDMS could assist the ICU staff in early identification of septic patients. These findings should be further verified in a larger sample of the ICU patients. Introduction: Patients with severe sepsis and septic shock still have a high mortality rate, despite improvements in intensive care therapy.

We assessed the impact of a standard operating algorithm adjusted on international treatment recommendations in patients with severe sepsis and/or septic shock, e.g. volume resuscitation, hemodynamic control, glycemic control, substitution of selenium and/or hydrocortisone and using adjunctive therapies like recombinant human activated protein C (rhAPC) on the outcome. Objectives: Is there a benefit Introduction: Critical Care is poorly developed and resourced in Malawi. Setting: The intensive care ward at Queen Elizabeth Central Hospital has 4 beds for a 1000 bed hospital and admits all patients from neonates to the elderly from all specialities. The hospital has high dependency units, but these are not adequately staffed, or resourced. Results: We are battling against multiple problems. Poor infrastructure produces erratic supplies of essential drugs, limited qualified personnel and little attention of educators to the triage and optimisation of the critically ill. This leads to delays in referral and advance disease at presentation, both at referral heath centres and within the tertiary hospitals. The situation is compounded by the twin epidemics of HIV/ AIDS and road traffic accidents, with approximately 30% of our admissions being HIV positive and 30% of our workload is in the management of severe head injury. As Malawi tertiary care develops, more trained doctors are returning with an increasing vision for possible medical interventions. Such increasing sophistication inevitably requires a higher degree of support. Unfortunately critical care is perceived as a luxury that cannot be afforded in a poor country, and as a consequence the country's investment in new specialists will never be fully realised. Due to delays in patients seeking treatment and referrals, we perform many laparotomies on sick patients. If ventilation is not available due to the limitations outlined above, patients usually miss out on critical care. We are convinced that many patients die as a direct consequence of inadequate fluid monitoring post-operatively on over-stretched wards. Due to lack of resources ICU outreach is not a realistic possibility, so alternative solutions must be found. Resource utilisation is a challenge worldwide, not just in Africa, although clearly more pronounced here. Conclusions: Several new technologies have been developed to stratify and optimise care for sick patients,one of which is the oesophageal Doppler. We validated a protocol for intra-operative fluid replacement guided by the oesophageal Doppler, due to its non-invasiveness and ease of use. This approach has been shown to reduce mortality in the elderly population in Europe following a fractured neck of femur. We will establish whether we can reduce mortality of similar high risk patients denied critical care in the African setting. We believe this will be the first such study undertaken in such a resource poor country. 

Infection 37 · 2009 · Supplement III © URBAN & VOGEL 110

Peri-phagocytic reactions of monocytes after infection: Contribution to inflammation in neonatal sepsis?

Spring B (1), Leiber A (1), Dreschers S

Spies M (3), Radtke C (3), Danne F (1), Kuether

Clinic of Plastic Surgery, MHH Medical School Hannover. Infection 37 · 2009 · Supplement III © URBAN & VOGEL Organisational Aspects 027

Prevalence and mortality of infections on the interdisciplinary Intensive Care Unit in Mali

Department of Anesthesiology and Intensive Care Medicine

Schrappe M (3), Kern W (4), Gastmeier P

Methods: A calculation of the amount of preventable NIs was performed by analyzing the data of epidemiological studies (1-5) published in the literature during the last years. The data of two large independently performed studies (the intervention study NIDEP 2 (2) and the prevalence study of SepNET (3)) were utilized to estimate the number of preventable death cases due to NIs. Results: Taking into account that all in all 20 to 30 % of NIs are preventable about 80 000 to 180 000 NIs are avoidable annually in Germany. This proportion may be even higher in hospitals with infection rates high above the average. In German hospitals mortality due to NI is estimated with 7500 up to 15 000 cases per year. 20 to 30% preventable cases in this context result in 1500 to 4500 avoidable death cases

Methods: Data collection was performed using the Medline database. 23 studies were identified , 6 studies were considered suitable with a total sample size of 107 patients requiring a renal replacement therapy for acute or chronic renal failure. Mean decrease in blood pressure, systemic vascular resistance and cardiac output was calculated in patients dialyzed with acetate (35 mmol/l) compared to other buffers like bicarbonate or citrate. Results: There was an overall 24% (range 15-34) decrease in blood pressure decrease in patients dialyzed with acetate in comparison to 10% (2-17) in the acetate free group. A 4% higher drop in systemic vascular resistance was calculated for patients exposed to acetate. A 24% (19-29) lower cardiac output was found during acetate dialysis compared to 8% (3-12) in the acetate free group . Conclusions: Acetate containing volume solutions may lead to hemodynamic instability especially in critical ill patients. However, data about adverse effects of acetate containing volume solutions are missing

Counseling interviews for patients, relatives and health care workers in the field of severe sepsisexperiences of the German Sepsis Aid

Brunkhorst FM

Results: More than 1,500 nationwide counseling interviews were performed by e-mail, phone or mail. In principal, interviews or frequent asked questions (FAQs) can be divided in three categories: (1) uncertainty regarding the definition of sepsis, an about 20%; (2) counselling for acute problems, an about 30% and (3) counselling for subacute/ chronic problems, an about 50%. The most frequent FAQs in category (2) were: uncertainty regarding diagnosis of sepsis and focus of infection. Procalcitonin measurements were helpful in some cases. The most common overseen infections were pneumonia, endocarditis and spondylodiscitis. In category (3) FAQs were: physical and mental long-term sequalae of severe sepsis survivors, need for experienced rehabilitation and chronic care facilities after ICU-care, mourning after the death of relatives and problems in understanding the reason for death by sepsis, and finally need for help due to medical errors. Conclusions: There is an urgent need for the support of self

Introduction: Enterobius vermicularis ('pinworm') is extremely rarely found outside the gastro-intestinal tract. We describe a case of extraintestinal hepatic pinworm abscess associated with a severe hepatic failure and a severe septic shock . A brief review of the literature is given and possible mechanisms of tissue invasion are discussed. Haematogenous spread and direct penetration of the liver preceded by either invasion of the peritoneum through unhealthy or traumatized intestinal tissue, or from migration up the genital tract are the infective mechanisms considered. Objectives: We report the case of an hepatic pinworm granuloma, localised in the right hepatic lobe of an elderly male diabetic immunocompetent subject, nowadays dead. The patient is admitted for septic shock and severe hepatic failure, and a right hepatectomy is done. The anatomo-pathological examination put in obviousness a parasitic granuloma and presence of eggs of Enterobius vermicularis. In the present case direct passage of the nematode through damaged intestinal tissue seems unlikely and no embolism in portal vein was demonstrable. Key words: hepatic pinworm granuloma, severe hepatic failure. Methods: A case report Results: Diagnosis and Observations: A 67 years old male, with several pathological antecedents (COPD, hypertensive cardiopathy, ischemic stroke, epilepsy) is admitted in ICU for septic shock and severe hepatic failure. The CT to Scan shows a right hepatic liquid mass of 10 cms of main line, in contents multiple subdivisions. The MRI showed within the right hepatic lobe the presence of the voluminous cystic heterogeneous lesion about 14 cms of main line. After collective discussion a right hepatectomy is proposed by respecting the segment 7 to guarantee a hepatic function. The suppuration state of the right lobe required a right complete hepatectomy. The anatomo-pathological examination put in obviousness a parasitic granuloma and presence of eggs of Enterobius vermicularis. An antihelmintic albendazole treatment was started. No other site of infestation was found. Unfortunately the patient dies within the month of a respiratory sepsis with an important degradation of his hepatic function. Conclusions: Granulomas are focal accumulation of modified macrophages. They are thought to form when macrophages ingest poorly soluble antigens. These can be viral, bacterial, fungal, parasitic or unrelated to infection or infestation; sarcoidosis and tuberculosis are high on the list in many series. Enterobius vermicularis (syn. Oxyurus vermicularis), also known as pinworm or seatworm, is the causative agent of human enterobiasis (oxyuriasis). The disease is more prevalent in temperate regions and is facilitated by factors such as overcrowding in schools and family groupings, as well as inadequate personal and community hygiene. In the great majority of cases, enterobiasis is asymptomatic. One common symptom is intense pruritis ani that in some patients can lead to insomnia, restlessness and irritability. Scratching may cause skin irritation, and in more serious cases, eczematous dermatitis, haemorrhage or secondary bacterial infections. Ectopic migration of E. vermicularis often results in pinworm infestation of the female genital tract often causing granulomas of the uterus, ovary and the fallopian tubes and pelvic peritoneum. Enterobius vermicularis ('pinworm') is extremely rarely found outside the gastro-intestinal tract especially in males. Far fewer peritoneal pinworm lesions have been found in males due to the lack of a convenient entry site. Peritoneal lesions in males have been attributed to penetration through unhealthy intestinal mucosa (ulcerations, tumors) . Less understood is the migratory route of pinworms found in peritoneal organs (liver, ovary, kidney, spleen) because buccal apparatus of Enterobius is unsuitable for invasion of healthy tissues. Therefore, several invasive routes have been suggested: for ovary -penetration through ruptured graafian follicles (Beckman & Holland), for kidney -by ascending the urinary tract (Symmers), for liver -haematogenous spread (Little & al) . Slais reported a case of hepatic granuloma with embolism in portal vein. Multiple intraperitoneal granulomas were discovered in a 31-year-old woman after surgical excision for a perforated appendix. A case of omental enterobiosis was reported by Reyes and colleagues in a 62-year-old woman after ruptured sigmoid diverticulitis. However, in the present case no colonic tumor or perforation was demonstrable. Therefore, direct passage of the nematode through damaged intestinal tissue seems unlikely. Introduction: Organ scarcity in Germany is still present and leadsbecause of raising waiting lists-to acceptance of marginal organs. It is not possible anymore to reject organs from elder patients, with elevated parameters or infections; the borders are marcerated. We want to introduce and follow a case of organ donation from a septic donor. Objectives: A 43-year old man came to the emergency room with a femoral abscess. It was opened and a grave impairement of the infectional siuation followed: He suffered a cardial arrest and reanimation for 5 minutes and complicating aspiration. This situation led finally to brain death and organ donation. Methods: At the time of operation a moderate catecholamine therapy was necessary: norepinphrine 0,10 µg/kg /min, dobutamine 1,96µg/kg/ min. Antibiotical therapy was initially metronidazol 1 g/day and ceftriaxon 2 g/day, after 6 days it was adjusted in flucloxacillin 6 g/day, clindamycine 1,8 g/day and meropenem 1,8 g/day according to antibiogramm. Results: In the initial femoral scrape test grampositive diploccae were found. After aspiration, bronchial lavage showed Staphylococcus aureus (SA) and a multiresistent SA. In blood culture no bacteria were found during antibiotic therapy. White blood cells rose from 21 Gpt/l to 41 Gpt/lin 1 week, CRP was elevated during the whole stay (286 to 216 mg/l). Finally only the kidneys were allocated; heart, lungs and liver were excluded for medical reasons. Both kidneys were transplanted, during the operations no complications occurred. Under standard antibiotic therapy (ceftriaxon and sulfamethoxacole / trimethoprime) there was no incidence of any infection. Kidney function was appropriate and patients could be discharged in time. Conclusions: In times of organ scarcity marginal organs cannot be refused. Though infectious problems and septic clinical picture with Staphylococcus aureus and MRSA, an organ transplantation was possible. With targeted monitoring also a standard antibiotic therapy was sufficent. So this case shows that also marginal organs can be transplanted successfully, if there is an experienced center with well-rehearsed settings. An organ transplantation is possible without endangering the recipient. 

We show the effect of a network for the treatment of pediatric septic shock, especially for children with septic shock. In 2003 we founded a pediatric intensive care network with 26 childrenhospitals (PIN). The primary aims of the network were the standardisation of clinical therapies, implementation of certified training programs and the installation of an emergency system in the region. Objectives: The first project was the implementation of a common sepsis bundle. The coordination center was the tertiary children hospital of the Medical University School of Hannover. Methods: The different parts of the sepsis bundle are described in the following : (i) Task force: A workgroup with participation of all hospitals was established. The task force comprised of the majority of the chief pediatric intensivists from the different clinics. They discussed and decided the therapy algorithm and operated as the primary contact person and multiplier of the task force decisions. (ii) Symposium: The implementation of the sepsis bundle started with a central symposium. The symposium informed of pathopysiology, epidemiology, therapy and prevention of pediatric sepsis. The symposium was supported by members of the german sepsis society and the german society of pediatric infectiology. (iii) Local lectures and instructions of the decided therapy strategies were followed. The university childrens hospital provided continuous pediatric intensive care workshops for sepsis therapy and diagnostic, management of circulation and organ failure as soon as instructions in technical skills. The university childrens hospital of Hannover offered a 24 h emergency and transportation support. If necessary, the team of the tertiary center assisted in the therapy in the primary clinic till the patient was out of danger or they transfered the child in the university hospital for further treatment. The PIN established a 24 h consultation service with experienced specialists of the university pediatric intensive care center. All cases were registrated at the university hospital.Therapy and Outcome were analysed. Results: In the first 6 years 31 cases of patients with septic shock were registrated in the network. 26 patients were treated with the support of the sepsis emergency team or the consultation service. 24 Patients were transferred to the university hospital. 26 children survived with Restitutio ad integrum. 2 Patients had skin necrosis or amputation and 3 died. One child died directly after admission in the primary hospital. In the other cases with complications, the gap of the recruitment in the network was too long. After the initial training period, the sepsis bundle worked very effective. Conclusions: Every child with immediatly support of the task force survived in good conditions. A time delay of patient recruitment in the network treatment seems to be a negativ prognostic factor. As a result of the optimized algorithm the medical costs could be reduced significantly. Effects of phosphodiesterase (III/IV)-inhibitors and cytokines on mechanical properties of neutrophilic granulocytes in neonates and adults Ruef P, Craciun E, Altfelder F, Simon C, Poeschl J Department of Pediatrics, Division of Neonatology, University of Heidelberg, Germany.

Introduction: Sequestration of activated PMN and enrichment in tissues play a key role in tissue damage during septicaemia and after ischemia/reperfusion. Objectives: Since polymorphonuclear neutrophilic granulocytes (PMN) of term neonates show various functional differences compared to PMN in adults (decreased chemotaxis, decreased intracellular killing, decreased adhesion), we studied the influence of IL-8, TNF-α and fMLP on the reduction of deformability of PMN in neonates and adults. Phosphodiesterase (PDE) -Inhibitors were used to ameliorate the reduction in deformability when the PMN were stimulated with fMLP or IL-8: Enoximone, Milrinone (PDE-III inhibitors), Pentoxifylline and Piclamilast (PDE-IV inhibitors). Methods: Cord blood samples from 20 healthy neonates and blood samples by venipuncture of 20 healthy adults were taken. The micropipette technique and the cell transit analyzer (filtration technique) were used. Aspiration times into micropipettes with an internal diameter of 5µm, transit times through 8µm filter pores and neutrophil elastase concentrations were determined Results: Despite of the functional differences of PMN in neonates compared to adults the significant decrease of deformability of PMN activated with cytokines compared to passive PMN was not different in both groups. The neutrophil elastase concentrations reflect the rigidity of the neutrophils: highest concentrations during activation, decreased concentrations due to PDE-inhibitors and lowest concen-Introduction: Neonatal sepsis-related inflammatory sequelae involve destructions in brain and lung. Phagocytosis and intracellular degradation of bacteria by monocytes are early events in pathogen-host interaction, resulting in pro-inflammatory activation. Phagocytosis induced cell death (PICD) and clearance of apoptotic cells, e.g. neutrophils are essential for resolution of inflammation. Autopsies of neonatal, pediatric and adult septic patients revealed extensive apoptosis of lymphocytes and dendritic cells. An imbalance between host cell survival survival and apoptosis would either lead to immune paralysis due to effector cell loss or to an exaggerated, prolonged inflammatory reaction. Monocyte PICD may play a role in limiting inflammatory reactions and tissue destruction. Objectives: We studied phagocyotis-associated reactions and compared cells of newborns to those of healthy adults.

We applied flow cytometry, ELISA, PCR-techniques and used gfp-labelled E. coli and group B streptococci. Results: Although neonatal monocytes from cord blood (CBMO) were equivalent to monocytes of of adults (PBMO) in phagocytosis and degradation of bacteria (E. coli and group B streptococci) and in ROS-production, we found profoundly reduced PICD in CBMO as compared to PBMO with only minimal up-regulation of caspases-8 and -9, CD95L and CD95L-mRNA. Post-phagocytic reactions of CBMO differed significantly to PBMO with respect to a stronger and more sustained IL-8 response and the capability to stimulate other immune cells, e.g. T cells with a polyclonal T cell mitogen. Furthermore, and in contrast to their equal bacterial phagocytic indices, CBMO were impaired to phagocyte apoptotic granulocytes, paralleled by a down-regulation of receptors of their phagocytic synapse.

Our results indicate that CBM are less sensitive towards PICD, produce a different pattern of T cell response and are impaired to eliminate apoptotic granulocytes. Taken together these abberant reactions may impair CBMO to terminate inflammatory responses and play a role in their perpetuation, leading to organ destruction in preterm infants. 

In both groups was no difference in initial APACHE II-Score and clinical baseline characteristics. With implementation of the SOP, use of volume in the first 6 hours (1506 vs. 2154 ml p<0,05) and in the first day (4005 vs. 6122 ml p<0,001) significantly increased. Furthermore, treatment with hydrocortisone, selenium and insuline increased after implementation of the SOP significantly. Catecholamines and rhAPC were unaffected. Most outstanding fact was the singificant descenting of mortality between the groups, from 57% (without SOP) to 38,5% in the intervention group with SOP (p < 0,05). Conclusions: Due to the implementation of a standard algorithm significant increase in volume therapy and adjunctive sepsis therapy were realised more frequent compared to patients treated without standard operating procedure and could be associated with a lower mortality rate in patients. The realisation of an evidence based standard operating procedure in daily practice in patients with severe sepsis and/or septic shock is effective, shown in alteration of treatment practice and especially the reduction of mortality. Introduction: Since the 1960s invasive infections by encapsulated bacteria have been recognized as a serious complication in patients with anatomical or functional asplenia. However, earlier studies have critical limitations, and evidence regarding pathogenesis, incidence and outcome of overwhelming post-splenectomy infection (OPSI) is insufficient. Objectives: We made attempts to assess the current epidemiology of infectious complications after splenectomy in Germany. Methods: Incidence and prevalence of asplenia and infections after splenectomy where searched in MEDLINE. The annual rate of splenectomy in Germany was calculated from DRG statistics. Databases for invasive pneumococcal disease (IPD) of the national reference center for streptococci (NRZ Aachen) and for pediatric invasive meningococcal infections (Meningokokken-Netz, Freiburg) were searched for asplenia as underlying disease. Results: According to DRG statistics, 8012 splenectomies were performed in Germany in 2007. The prevalence of asplenia in Germany is unknown, but it was estimated ~9 -10 per 10.000 population in the United Kingdom. Assuming similar frequencies, we estimate the prevalence of anatomical asplenia to be ~80.000 persons in Germany and this number does not even take patients with functional hyposplenia into account. Thus, asplenia is a frequent immunodeficiency. Earlier studies estimated the annual risk for OPSI to be in the range 0,25% -0,45%. Under these assumptions we can expect > 200 to 360 OPSI cases per year in Germany resulting in > 100 to 180 deaths due to septic shock. Studies published in the literature consistently report pneumococci as the number one pathogen in OPSI, accounting for 50 to 80% of the cases, while the role of meningococci in OPSI has remained controversial. Surprisingly few cases of pneumococcal OPSI (11 cases of more than 15.000 isolates in IPD between 1996 to 2009), however, are being reported to the NRZ Aachen. On the other hand, 5 of 46 (11%) of cases of meningococcal sepsis in children in the context of hypo-or asplenia were recorded in Meningokokken-Netz since 2007. Conclusions: Asplenia is a common but underrecognized immunodeficiency in Germany. The rates of pneumococcal and meningococcal OPSI notified in Germany are in poor agreement with the frequencies found in retrospective studies. We believe there is an urgent need for prospective analysis of asplenia as a risk factor for septic shock and invasive pneumococcal and meningococcal disease. (1), Studart FS (1) Introduction: In the USA more than a quarter of hospital pharmacists work as ward pharmacists. Many studies there have shown that pharmaceutical care has a positive influence on the quality of drug therapy. In Germany this concept has so far not been considered to the same extent, because it is not as well known there how much influence pharmaceutical interventions exert on the quality of pharmacotherapy in the German setting. Objectives: To determine the kind of pharmaceutical interventions for the improvement of drug therapy. Methods: For the period of March 1 to March 31, 2008, all clinical pharmaceutical interventions in an intensive care unit with 26 beds were documented and classified. During the time examined, a total of 153 patients were attended to. The interventions took place following an examination of the electronic patient records. Therefore, the pharmacist had access not only to the entire medication, but also to all laboratory and patient data. After investigating the medication and the determination of pharmaceutical clinical problems the pharmacist intervened and the therapy was changed by the medical staff. All such interventions were classified and evaluated.

The patient population's average age during the study time was 56.57 years with an average of 19.65 medicaments per day. The time spent in the intensive care unit was on average 13.9 days. A total of 397 pharmaceutical interventions were recorded. The most frequent reason for an intervention was the change from intravenous to oral administration of medication (59%). Additional reasons concerned dual prescriptions (20%), clear indication for a drug without appropriate prescription (6%), as well as the wrong dosage (8%) and taking the drug at the wrong time (7%).

The results on hand indicate that the introduction of a clinical pharmacist can also contribute to an improvement in drug therapy in Germany. Included in the scope of duties of a clinical pharmacist are, among others, the examination of the drug therapy with respect to side effects, interactions, dosage, the adjustment of the doses related to kidney function and consideration of plausibility, since most drug-related problems occurred in these areas. As a result of such early detection and intervention a topical qualified consultation with the physicians and nursing staff for the solution of drug-related problems is made possible. In this way the work of a clinical pharmacist represents an important contribution toward ensuring the quality of pharmacotherapy. Introduction: Structures in intensive care medicine comprise human as well as material resources, health care organization and management and may thereby be related to processes and subsequent patients' outcomes. Objectives: Utilizing a unique data base obtained from a representative random sample of German intensive care units (ICUs) we sought to relate the existing structural elements to recommendations in the literature.

The study was carried out by the German Competence Network Sepsis. Data was prospectively collected by a trained physician on a cross-sectional (one-day) basis in a representative random sample of German hospitals utilizing a structured questionnaire. Among others, staff presence and qualification was evaluated. Structures were related to ICU mortality of patients with severe sepsis or septic shock. The sample was subdivided in 5 strata according to hospital size.