key: cord-0007030-bonq48bq
authors: nan
title: 9th International Congress “Sepsis and Multiorgan Dysfunction”
date: 2019-07-30
journal: Infection
DOI: 10.1007/s15010-019-01341-2
sha: 65ffcbccc69446d371ffe8e9701362a7b6222a4e
doc_id: 7030
cord_uid: bonq48bq

nan

Introduction: Sepsis is a known colossal challenge in the clinical setting and experimental models have proven to be instrumental to emulate and understand clinical conditions better. Previous studies performed in sepsis patients and murine endotoxic shock models have shown that increased systemic pancreatic proteases are a cause of ensuing organ dysfunction (1, 2) . Objectives: Therefore, we wanted to look into the intestinal mucus composition after experimental sepsis to evaluate if the local impact of a heightened proteolytic activity modulated initial immune responses. Methods: Sham or Cecal liagtion puncture (CLP) was performed in male C57bl6 mice aged [8] [9] [10] [11] [12] week old. Animals were euthanized after 24 h and mucus from small intestine (jejunum) and large intestine (colon) were obtained. A soluble fraction of the mucus was prepared by adding Phosphate Buffered Saline to the mucus, with or without protease inhibitors, sonicated and supernatant was collected for measurement of complement factors and proteases respectively. Results: After confirming through ELISA, we used western blot analysis to look into C5 and C3 complement fragments, which were copiously generated in the jejunum of CLP mice compared to sham mice. However, this effect was not completely the same for colon mucus samples of CLP mice. As these cleavage products could be due to proteolytic activity, we measured non-specific total protease and found that CLP mice had a higher protease content in their mucus. As commercially available chromogenic substrates were unreliable for protease activity measurements, we established the use of chargechanging peptides as an analytical tool to measure specific protease activity. We found MMP2/9 and elastase activities significantly higher and no difference in trypsin activity in CLP jejunum mucus versus sham mucus. Conclusions: Therefore, during sepsis, locally generated complement fragments could be due to higher protease content and may have further immunopathological implication. Acknowledgement: We are thankful to Prof. Schmid-Schoenbein and Dr. Elaine Sworonski for helping us transfer their novel technique of charge changing peptide substrates to test for proteolytic activity from San Diego to Ulm. (1), Plehutsa MD (2), Sydorchuk AR (1) (1) Bukovinian State Medical University, (2) Acute and Emergency Hospital Chernivtsi

Introduction: This experimental issue deals with microbiological investigation of quantitative and qualitative composition of luminal colonic microbiome in albino rats with abdominal sepsis within 6 h of its initiation.

Objectives: The aim of the study is to clarify the early changes of luminal colonic microbiome in experimental abdominal sepsis Methods: Case-control experimental study conducted on 25 albino rats with weight from 200 to 220 g: Ten intact animals (control group) and 15 rats in basic group with abdominal sepsis. The content of large intestine served as research material for microbiological investigation with obtaining pure cultures of microorganisms. Population level of microflora of large intestine content displayed in logarithm of colony-forming units (lg CFU/g). In the current study, few important indices had used for evaluation of character of microbiota violations-constancy index, Margalef index, Berger-Parker index, Simpson index. Results: It had been observed the reduction of dominant Bifidobacteria by 33.5%, Lactobacilli by 36.72%, Peptostreptococci by 2 times and Enterococci by 2.5 times in the large intestine cavity of experimental animals with abdominal sepsis within 6 h. However, quantitative dominance in microbiocenosis of large intestine cavity increases for opportunistic Bacteroides by 38.55%, Peptococci by 2.97 times, E. coli by 9.17% and Proteus by 59.37%. Significantly increased role of opportunistic Enterobacteriaceae in the formation of microbiota of large intestine cavity in animals with abdominal sepsis after 6 h: Bacteroides-by 42.86%, Peptococci-by 3 times, E. coli-by 10.53%. Opportunistic pathogenic Enterobacteriacea (genera Klebsiella, Edwardsiella), which contaminate and colonize the large intestine cavity, reach a moderate population level and microecological indexes.

Conclusions: The study of qualitative and quantitative composition, micro-ecological indexes and coefficients that demonstrated interactions and co-existence of main, additional and accidental gut microbiome of albino rats with abdominal sepsis established minor violations of species composition within 6 h after modelling: deficiency of Bifidobacteria, Lactobacilli, Peptostreptococci on the background of the increased number of conditionally pathogenic bacteria. By constancy index, the frequency of occurrence, Margalef index, Berger-Parker index and Simpson index, and by quantity domination the role of Bifidobacteria, Lactobacilli, Peptostreptococci and Enterococci in formation of microbiocenosis had evidently decreased, meanwhile domination role of conditionally pathogenic Enterobacteria, Escherichia, Bacteroides and Peptococcus had evidently increased. (1) (1) Bukovinian State Medical University, (2) Emergency Hospital Chernivtsi

Introduction: Epithelial biofilm plays the role of a protective barrier that inhibits the pathogenetic translocation of microorganisms, toxins, antigens and other harmful substances of exogenous and endogenous origin into the internal environment of an organism. So, disbalance of it can lead to further complications of initially serious case of abdominal sepsis.

Objectives: The aim of the study is to evaluate changes of epithelial colonic biofilm after 12 h of experimental abdominal sepsis Methods: The experimental study was conducted on 25 male unbred albino rats [15 intact animals (control group) and 15 rats in basic group with abdominal sepsis]. Animals with experimental abdominal sepsis were withdrawn from the experiment after 12. During laparotomy, visual inspection of the organs of the abdominal cavity and the collection of material for microbiological examination were performed. Under sterile conditions, about 3 cm of the distal part of the colon was taken, which was used to isolate mucosal microflora with its further identification.

Results: In the epithelium colonic biofilm of animals with experimental abdominal sepsis, after 12 h of the modeling, a marked deficiency of Bifidobacterium is formed with a decrease in their population level by 54.20%, and Lactobacillus by 62.53%. In this case, the number of intestinal E. coli increases by 49.30%, Enterococci-by 22.46%, Clostridia-by 14.21%. In addition, microorganisms that have contaminated the mucosa include Enterobacteriaceae of the genus Proteus, Klebsiella, and also P. niger: Staphylococcus and yeast-like fungi of genus Candida, that reach a moderate and high population level. The changes in the biotope leads to the reformatting of the role of each taxon in the system of intestinal microbiocenosis. The role of the most important in the composition of mucosal microbiota, as well as according to the multifunctional value in this biotope of bacteria of the genus Bifidobacterium, is reduced by 2.20 times, Lactobacillus-by 2.15 times. Also, the role of bacteria of the genus Peptostreptococcus is reduced by 91.63%, Enterococcusby 84.44%. The role of conditionally pathogenic Enterobacteriaceae grows substantially by 61.59%, Clostridia-by 5.56 times, Bacteroides-by 6.26%. The main microflora of the large intestine includes anaerobic bacteria of the genus Bifidobacterium, Lactobacillus, Bacteroides, Peptostreptococcus and non-pathogenic species of Clostridium. Escherichia, Enterococcus form the additional microbiota. In intact animals (control group), the ratio of anaerobes to aerobes is 3.21 units, while in the experimental group of animals, the ratio of anaerobes to aerobes is 0.78 units.

Conclusions: This is evidence of a deep violation of not only the qualitative but also the quantitative composition of the mucus microbiota of the colon.

of the intestinal epithelium facilitate essential tolerance signals to commensal microbiota of the gut flora and regulate an adapted immune response against invading pathogens. Systemic inflammation is typically associated with a dysregulation of this adapted immune response and is accompanied with a disruption of the epithelial and endothelial gut barrier which enables dissemination of pathogens within the human body.

Objectives: To understand the pathophysiological mechanisms underlying the inflammation-associated gut barrier breakdown, it is crucial to elucidate the complex interplay of the host and the intestinal microbiome. We established a microfluidically perfused three-dimensional gut-on-chip model to emulate these processes in the presence of immune cells and commensal bacteria. Methods: MOTiF (multi organ tissue flow) biochips made from polystyrol were used for the establishment of the gut model. The biochips are composed of two chambers separated by a micro-porous membrane. Each chamber is connected to inlet and outlet channels allowing independent perfusion of the individual channels and application of microfluidic shear stress. Human umbilical vein endothelial cells (HUVECs), monocyte-derived macrophages and intestinal epithelial cells were assembled on the biochip membrane. Following 7-14 days of growth in presence of physiological flow conditions, the epithelium was colonized with the commensal bacterium Lactobacillus rhamnosus, while the endothelium was perfused with peripheral blood mononuclear cells (PBMCs).

Results: Within 1 week of perfusion the epithelial cells formed selforganized and well-polarized villus-and crypt-like structures that resemble essential morphological characteristics of the human intestine. Dendritic cells were differentiated in the epithelial tissue that specifically respond to bacterial lipopolysaccharide (LPS) challenge. LPS is well-tolerated at the epithelial side corresponding to the gut lumen without signs of immune activation and a significant release of pro-inflammatory cytokines. In contrast, LPS stimulation at the endothelial side of the gut-on-chip model triggered release of the proinflammatory cytokines TNF, IL-1b, IL-6 and IL-8 by activation of macrophage residing in the endothelium. Perfusion of the endothelium with PBMCs led to an enhanced cytokine release. L. rhamnosus colonization of the model influenced cell signaling, however, the bacteria were tolerated by immune cells.

We developed a microfluidic gut-on-chip model to mimic a systemic infection with a dysregulated immune response under physiological conditions. The model facilitates the colonization of commensal bacteria and co-cultivation with facultative pathogenic microorganisms, which are tolerated by the host and contribute to cell signaling. The gut-on-chip model represents a promising tool to gain more detailed insight into the communication processes of the microbiome and its human host and moreover allows the co-cultivation with additional microorganisms to create an even more physiological environment. Introduction: Necroptosis, a rarely investigated form of programmed cell death, is employed in many cell types followed by injury and facilitates cell death in a controlled manner which simultaneously stimulates inflammation. This cell death mechanism is characterized by swelling of the cell followed by rupturing of the plasma membrane. Phosphorylation of the receptor-interacting serine/threonine kinase 1 (RIPK1) and hyperphosphorylation of receptor-interacting serine/threonine kinase 3 (RIPK3) forms the necrosome and activates mixed lineage kinase domain-like protein (MLKL) which leads to pore formation and cell death. Monitoring of necroptosis regulating proteins in response to therapeutic interventions would help to develop a novel treatment strategy to patient under several disease entity following impaired liver function e.g. cholestasis or inflammation. However, the role of RIPK3 in the liver remains controversial.

Objectives: Here we investigated, in light of the fact that RIPK3 expression in the liver is controversially discussed, the impact of necroptosis in liver dysfunction. Methods: Protein expression of different mediators of necroptosis is examined in primary human hepatocytes (pHep) and HepG2 cells. Furthermore, HepG2 cells overexpressing human RIPK3 are stimulated with pro-inflammatory molecules (toxic bile acids, cytokines) and the activation of necroptosis and inflammation are determined. Slices from paraffin-embedded liver samples of patients that show characteristics of cholestasis or inflammation are also analyzed in relation to the expression of necroptosis mediators. Results: Our results depict that pHep and HepG2 cells are not able to undergo necroptosis under basal conditions due to the lack of RIPK3. The formation of the necrosome and the signal transduction following phosphorylation of MLKL is interrupted. Overexpression of RIPK3 in HepG2 cells leads to significant RIPK3 phosphorylation. Bile acid treatment of RIPK3-overexpressing HepG2 cells affects the RIPK3 and P-RIPK3 expression in dependence on the modification of the bile acids. Moreover, analysis of cytokines shows also a significant increase after overexpression of RIPK3 in HepG2 cells. In human liver, RIPK3 expression differed in dependence on inflammatory liver disease.

Conclusions: These results indicate that RIPK3 is increased during inflammatory liver injury and can lead to pro-inflammatory signaling and cell death. The inhibition of RIPK3 pathway is a potential target for anti-apoptotic strategies in liver diseases but requires further investigation in preclinical models of sepsis.

Infection 2019

Inhibition of phosphoinositide 3-kinase-c improves liver function in sepsis by preventing RhoA-mediated cholestasis

Organic and Macromolecular Chemistry (IOMC), Friedrich-Schiller University Jena, Humboldtstrasse 10, 07743 Jena, Germany

Introduction: Liver has been increasingly considered as a target and modulator of manifold infectious diseases, making it a key object for disease progression and prognosis. Excretory dysfunction of the liver is an early and frequent event in polymicrobial sepsis and is strongly associated with the survival of the host. Different pathogen-associated molecular patterns (cytokines, chemokines) and inflammatory cells affect hepatic integrity. PI3K-c is a promising target of membrane shaping processes, cytoskeletal remodeling, cellular polarization, metabolism, and many other essential cellular developments. Furthermore, it is associated with many G protein-coupled receptors, such as chemokine receptors in vivo, playing an important role in several immune processes. In hepatocytes, PI3K-c regulates the phase I and phase II biotransformation and the elimination of bile acids by affecting the activity of ATP-dependent canaliculi transporters. The latter regulates intracellular trafficking towards the membrane, significantly affecting the liver function. The activating interactions between small GTPases and PI3K isoforms are manifold, directly and indirectly mediating migration, cell growth, and survival. Objectives: The present study investigates for the first time the impact of the inhibition of PI3K-c via a RhoA pathway to prevent dysfunction.

Methods: In accordance with Thuringian animal welfare association guidelines, sepsis was induced by applying the model of peritoneal contamination and infection (PCI) while sham animals were injected intraperitoneally with sterile saline. All animals had supportive therapy. Different treatment regimens of AS605240 and respective vehicle groups were applied during the first 5 days of the infection, the phase in which liver failure determines the outcome. The underlying PI3K-c/RhoA molecular mechanism was tested in HepaRG cells, primary murine hepatocytes, and the liver tissue.

Results: The levels of cytokines and of bile acid conjugation revealed a conjugation inability and an increase in proinflammatory cytokines in PCI vehicle groups in comparison to AS605240 treated animals. RhoA activation was observed in CM-treated HepaRG cells, as well as in the PCI sepsis model in primary murine hepatocytes, both being resolved by pharmacological PI3K-c inhibition. The amount and aggregation of b-actin was elevated in the PCI groups while fluorescently stained MRP2 was retrieved to the membrane after treatment with AS605240 inhibitor indicating that PI3K-c inhibition attenuates RhoA activation, both in vitro and in vivo.

The results demonstrate a direct link between PI3K-c activity and selective RhoA activation leading to an aggregation of F-actin in hepatocytes. The pharmacological inhibition of PI3K-c by AS605240 interferes with the sepsis-induced RhoA activation and the subsequent development of cholestasis in vitro and in vivo, respectively.

Implantable wireless biometric animal telemetry system: a powerful tool to improve translational sepsis research Introduction: It is well established that sepsis often results in multiple organ failure (MOF). The affected organs are mainly the liver, lung, kidneys and heart and circulation [1, 2] . Since MOF is often the cause of death of the septic patient, detailed characterization of the underlying (patho-) physiological and molecular mechanisms and resulting treatment approaches can lead to an improvement in the course of sepsis. In order to identify or characterize these underlying mechanisms and to test appropriate therapies, animal models are often initially used. However, translation of preclinical findings into a clinical setting, thus far, has not been very successful. A contributing factor may be that in patients, vital parameters are continuously monitored in the intensive care unit (ICU), whereas in rodents such sustained monitoring is rare. In mice, sepsis is induced, for instance, by surgical cecum ligation and puncture (CLP) and experimental therapy is started at a predefined time point that is usually independent of vital parameters. This approach does not take into account either the innate differences in strength and duration of individual immune responses or the status of the cardiovascular and respiratory systems. In order to increase the translatability of the preclinical to the clinical situation, an implantable wireless biometric animal telemetry system can be used. This allows real-time monitoring, measurement and documentation of the vital parameters, blood pressure, electrocardiogram (ECG), heart rate, temperature and physical activity [3] [4] [5] [6] [7] . Such a system, which is very similar to the human situation in the ICU, offers the possibility in mice to start a therapy or to determine timing for organ/blood/serum sampling, based on their vital parameters. In addition, this much more precise monitoring of the mice provides detailed information on the course of sepsis and allows a more precise decision on whether the trial must be stopped prematurely in order to kill mice humanely [8] .

Objectives: The first aim of the study was to explore vital parameters such as heart rate, blood pressure, temperature and ECG in mice after induction of an experimental sepsis by CLP. These data provide an understanding of (patho-) physiological processes during the course of sepsis as a basis to develop new therapy approaches and to optimize existing therapies. The second aim was to investigate the impact of transmitter implantation per se by comparing implanted septic mice with non-implanted septic mice. Finally, the third aim was to determine whether insight into real-time vital parameters allows a more precise decision to be taken on termination criteria, thus assessing whether the use of a telemetry system introduces a refinement to the CLP model. Methods: We used the implantable wireless biometric animal Phys-ioTelTM HD-X11 telemetry system (Data Sciences International (DSI), St. Paul, USA) for in vivo measurement of blood pressure, ECG, heart rate, temperature and physical activity. Under the use of an animal application (authorization number V54-19 c 20/15-F152/ 1016), approved by the Institutional Animal Care and Use Committee of the State of Hesse, Germany, male C57BL/6N mice (Charles River Laboratories Germany GmbH, Göttingen, Germany) aged 10-12 weeks were utilized for all experiments. For the implantation of the blood pressure catheter under ketamine/xylazine (120/8 mg/kg) anesthesia, the left carotid artery was used. The transmitter device body was placed along the lateral flank between the fore limb and hind limb. The continuous data acquisition of all vital parameters began immediately after an animal emerged fully from the anesthesia. At the earliest 7 days subsequent to implantation of the telemetry transmitter, a 21-gauge, double puncture CLP with a cecal ligation of 2/3 was performed under isoflurane anesthesia. Sham animals underwent a laparotomy without CLP. A control group underwent only the CLP operation, as described above, without prior implantation of a telemetry transmitter. At the latest 48 h after CLP/sham operation, mice were euthanized and heparinized and whole blood samples were obtained by cardiac puncture using a sterile technique for detailed analysis of blood parameters. The peritoneal cavity was irrigated with sterile phosphate-buffered saline (PBS) solution for peritoneal lavage and samples were collected in a sterile fashion to determine the bacterial load as colony-forming units (CFUs) . Results: We studied murine physiology during the first 48 h after CLP/Sham operation. As expected, we observed marked heterogeneity in the physiological and thermoregulatory responses to sham operation. Nevertheless, blood parameters and CFUs in sham mice remained in the range of those in healthy control mice, which underwent neither implantation of the wireless biometric animal telemetry transmitter system nor a CLP/Sham operation. In contrast, mice subjected to CLP-induced sepsis showed distinct differences in physiological and thermoregulatory responses. Mice that died within the first 24 h after sepsis induction lost an average of nearly 30% of the core body temperature within this time. In parallel, during this period, the blood pressure of septic mice decreased by an average of almost 50%. In this case, the temperature was correlated with blood pressure, expressed as a ratio/score for sepsis progress/severity, as reflected in a signature in the progression curves. Along with this signature, a systematically decreased heart rate over a 10-h period before death was observed. The different trajectories in the physiological and thermoregulatory responses between septic mice that died and those that survived were also reflected in the very different blood parameters and CFUs. Mice that died showed a significantly higher bacterial load. In addition, blood parameters like aspartate transaminase (AST) and alanine transaminase (ALT), as well as the differential haematogram for neutrophils, monocytes, eosinophils, thrombocytes and leucocytes, were substantially different between mice that died and mice that survived. Additionally, we compared implanted with non-implanted CLP mice to determine the impact of the wireless biometric animal telemetry transmitter system on the severity and progression of sepsis. Fifty percent of non-implanted mice died within the first 24 h after sepsis induction. In contrast, only 37.5% of the implanted mice died within the same period following sepsis induction. This result demonstrates that prior implantation of the telemetry transmitter per se has an effect on survival after CLPinduced sepsis, possibly due to a pre-activation of the immune system, which helps to fight against infection caused by CLP-induced sepsis. The reasons for this are still unclear and must be characterized in detail in subsequent studies. This suggestion is supported by our initial data regarding different blood parameters. In non-implanted mice which died as a result of the CLP-induced sepsis, the levels of AST and ALT were clearly increased compared to implanted mice. In both groups, this also applied to surviving mice. Moreover, inspection of the differential haematogram reveals clear differences in the quantities of lymphocytes, monocytes, thrombocytes and leucocytes between implanted and non-implanted mice.

Conclusions: The combination of the murine polymicrobial CLP sepsis model with the implantable wireless biometric animal telemetry system is a powerful tool to better understand the (patho-) physiological processes during the course of sepsis by objective, realtime measurement of physiological and thermoregulatory parameters. Our data show that there are clear differences between septic mice that survived versus mice that did not survive, both in the vital parameters and in the ex vivo parameters. These data provide a basis for future testing of therapeutic interventions. In addition, the vital parameters allow us to detect and determine signatures/scores that define termination criteria. An objective decision can be made to kill animals humanely, thus increasing refinement and reducing animal suffering. Despite the many advantages and improvements that this system offers, our data also indicate that it is important to define new thresholds or baselines and to critically examine the data obtained, because an impact of the telemetry transmitter implantation per se on the course and severity of sepsis cannot be ruled out.

References: [1] Hotchkiss, R. S., Nicholson, D. W. (2006) Apoptosis and caspases regulate death and inflammation in sepsis. Nat Rev Immunol 6, 813-822. [2] Rittirsch, D., Flierl, M. A., Ward, P. A. (2008) Harmful molecular mechanisms in sepsis. Nature reviews. Immunol 8, [776] [777] [778] [779] [780] [781] [782] [783] [784] [785] [786] [787] [3] Lewis, A. J., Seymour, C. W., Rosengart, M. R. (2016) Introduction: Cardiac dysfunction in septic shock is, besides inflammation, related to metabolic dysfunction, i.e. impaired glucose utilization attributed to defective supply (1) . Glucocorticoid receptor (GR) signaling is essential for survival of several septic shock models (2) (3) (4) . GR is also important for normal heart function and development (5), correlated with PGC1alpha activity (6) an important mitochondrial biogenic factor and metabolic regulator. Results for GR expression in peripheral blood cells in sepsis are ambivalent (7, 8) . In the septic liver, a GR down-regulation is associated with organ dysfunction (9), however cardiac tissue expression is not reported. Oxytocin receptor (OTR) signaling is also critical for heart function and stimulates glucose uptake in cardiomyocytes (10), however little is known about its role in sepsis.

Objectives: Since both GR and OTR mediate anti-inflammatory effects as well as glucose metabolism, the aim of this post hoc study was to elucidate their expression in septic cardiomyopathy in a comorbid large animal model. Methods: Anesthetized and instrumented FBM (familial LDL-receptor-/-) pigs with high fat diet-induced atherosclerosis underwent S8 Abstracts poly-microbial septic shock (n = 8) induced by inoculation of autologous faeces into the abdominal cavity, or sham procedure (n = 5), and subsequently received intensive care therapy with fluid and noradrenaline administration for 24 h (11). Systemic troponin levels were determined as a marker of cardiac injury. Left-ventricular GR, PGC1alpha and OTR expression were quantified by immunohistochemistry of formalin-fixed paraffin sections. Results: Troponin was increased 37-fold in septic animals in contrast to sham (p \ 0.001). OTR (p = 0.004) and GR expression in septic hearts were reduced in comparison to sham animals, along with a down-regulation of PGC1alpha.

Conclusions: In this clinically relevant, co-morbid, resuscitated large animal model, we show a sepsis-induced loss of cardiac GR and OTR expression, which was associated with cardiac injury as indicated by the increased troponin levels. In the same model, we previously reported reduced renal cystathionine-gamma-lyase (CSE) expression, dysregulated glucose metabolism and attenuated PGC1alpha expression, contributing to acute kidney injury (12) . CSE, an endogenous hydrogen sulfide (H2S) producing enzyme, is a regulator of GCinduced gluconeogenesis via PGC1alpha (13). Reduced cardiac CSE expression is associated with septic cardiomyopathy (14). H2S in the heart also has a direct effect on OTR expression (15). Recently, the reperfusion injury salvage kinase pathway was suggested to upregulate OTR as a mechanism of CSE-mediated cardio-protection (16 Introduction: During sepsis, the immune system is confronted with a variety of factors, which are integrated within the individual cells and result in changes in their basal state of responsiveness (1) . Epigenetic mechanisms like posttranslational modifications of histone are known to participate in the control of cellular reactions (2) . Variation of these histone modifications within promoter regions is functionally correlated with the transcription of the associated genes in defined genomic regions (3). Moreover, chromatin variations have been linked to the binding of further higher-order regulatory elements, including the CCCTC-binding factor (CTCF) (4) . The 11 zinc finger domain-containing protein is known as a chromatin insulator with impact on the global organization of chromatin architecture and topology, thus capable of modulating gene transcription (5).

Objectives: To unravel inter-individual concordant pathophysiological alterations during sepsis by genome-wide analysis of histone modification, as well as the impact of CTCF binding on the expression of most affected genes in human CD14?? CD16-monocytes.

Methods: Blood was collected from patients with sepsis and healthy volunteers. Magnetic activation cells sorting of CD14?? CD16classical monocytes was followed by (i) gene expression analysis via quantitative PCR and (ii) chromatin immunoprecipitation and sequencing (ChIP-seq) to assess the genome-wide distribution of the chromatin modifications histone 3 lysine 4 (H3K4me3), 27 trimethylation (H3K27me3), lysine 9 acetylation (H3K9ac) and CTCF. In addition, flow cytometric analysis was used to identify sepsis-induced immune suppression and to measure cell purity. Inflammatory cytokine levels in blood were determined via ELISA. Approval of the ethics committees of the medical faculty of the Justus-Liebig-University of Giessen, approval number 155/12) and the medical faculty of the Heidelberg University approval number S-135/2016. Results: Differential analysis revealed a set of distinct promoters with significant enrichment or depletion of histone marks between patients with sepsis and healthy controls. Compared to healthy controls, 86 genes were found to exhibit a decreased coverage of the active marks H3K4me3 and H3K9Ac in their promoter regions, while the inactive mark H3K27me3 was strongly enriched (''silenced cluster''). Gene ontology (GO) analysis identified overrepresentation of genes involved in immune function, including those involved in antigen presentation, located within major histocompatibility (MHC) locus on chromosome 6. In addition, differential CTCF enrichment was detected in patients with sepsis within this locus concurrent with specific changes in gene expression, depending on the relative location to CTCF binding sites.

The genome-wide maps of histone modifications indicate that a defined set of immunologically relevant genes undergo specific alterations within their promotors in CD14?? CD16monocytes during human sepsis. These epigenetic alterations are accompanied by uncoupled MHC-II expression and a differential CTCF-binding inside this locus, suggesting a sepsis-induced enhancer blockade mediated by epigenetic variations. Vincentius-Clinic Karlsruhe gAG, Karlsruhe, Germany Introduction: The mucociliary clearance system of lower airways is an important defense mechanism to remove debris and infectious particles that can cause pneumonia. This clearance mechanism is driven by ciliary bearing cells, which is under control of many physiologic signal transduction cascades [1] . Further it can also be affected by different factors including fungal cell wall components like the polysaccharides Galactomannan and Zymosan and the pro-inflammatoric cytokine tumor necrosis factor-a (TNF-a) [2, 3] . The influence of ciliary activity by immunomodulators is still not sufficiently investigated.

Objectives: In the present study we investigated whether interleukin 1 (IL1) or interleukin 6 (IL6), can alter the mucociliary clearance of mice trachea. Both interleukins are known to play a pivotal role in early host response to infections in diverse organs. Thus, circulating IL1 and IL6 also reach concentrations in the lung and tracheal epithelial that ought to be high enough to change cell functions of ciliary bearing cells. As surrogate parameters for mucociliary clearance we measured the ciliary beat frequency (CBF) and the particle transport velocity (PTV). Methods: Tracheae from male C57BL/6J mice were explanted after death and the ciliary beat frequency of the epithelial cells measuring light-dark-transmissions. Functional transport velocity was detected analyzing recorded image sequences and calculation of particle tracks. PCR experiments were performed on isolated tracheal epithelium to identify receptor mRNA. Results: In the tracheal epithelium we found mRNA for IL1 and IL6 receptors, assuming, that both receptors are expressed in these organs. Both PTV and CBF are concentration dependent positively stimulated by IL-6 (100 ng/ml) via the epithelial expressed IL-6 receptor (n = 6). The increase in CBF is reversible using the serotonin antagonist methysergide (100 lM) (p \ 0.01) or the muscarinic receptor inhibitor (atropine (1 lM) (p \ 0.05). In addition, the PTV can be significantly decreased by methysergide (p \ 0.01) while atropine only shows a declining tent (p = 0.2476). Further the exposure to IL1 or IL6 induces the expression of their specific receptors, maybe as a positive feedback mechanism.

Conclusions: IL-6 as a pro-inflammatory cytokine has an increasing effect in ciliary beat frequency leading to a serotonin dependent increase in particle transport Introduction: Sepsis is a life-threatening pathological host response to an infection often leading to profound vascular leakage. The underlying molecular mechanisms remain incompletely understood and specially the role of the micro RNAs (MIR) has not been investigated in detail. We hypothesized that specific MIRs might be up-regulated in the septic endothelium thereby contributing to vascular barrier breakdown. Objectives: To identify regulated MIRs in the septic endothelium and to decipher their role in endothelial permeability. Methods: Regulated MIRs were screened by an unbiased MIR array approach from murine lung endothelial cells (CD146? magnetic isolation). In vivo, the effect of specific MIRs on permeability was assessed in a transgenic zebrafish (flk:mCherry) and a murine knockout (KO B6.Cg-Mir155tm1.1Rsky/J) model. Both Lipopolysaccharides (LPS) and cecal ligation and puncture (CLP) was used for the induction of systemic inflammation. Readouts were pulmonary permeability (Evans Blue), activity scoring and survival. Mechanistic studies were done in human umbilical vein endothelial cells (HUVECs) using standard molecular biology tools (RT-PCR, Immunoblots) and functional real-time permeability measurements (ECIS-TER). Additionally, immediate post mortem kidney biopsies (n = 16) were performed to evaluate the expression of MIRs in a septic human organism. Results: The array revealed that MIR155 was significantly upregulated (259). Transgenic overexpression of MIR155 in zebrafish-as a screening tool-led to a massive induction of vascular permeability (eye assay, p \ 0.01). The same was observed when MIR155 was experimentally overexpressed in human ECs in vitro and permeability was measured (p \ 0.04). Moreover, MIR155 inhibition protects against classical permeability mediators (i.e. thrombin, p \ 0.03). We identified Claudin-1 (a tight junction protein) both on protein and mRNA level as putative MIR155 target both by overexpression and inhibition. Interestingly, in vivo the heterozygote MIR155 knockout mouse showed a protected phenotype with regard to capillary leakage and survival (40% benefit, p \ 0.01). MIR155 was also increased in septic human renal tissue compared to kidney biopsies from nonseptic patients (p = 0.01). Conclusions: Using an unbiased MIR-array approach, we found that MIR155 is upregulated in septic endothelium in mouse and men and might contribute to the pathophysiology of vascular leakage in a Claudin-1 dependent manner. Future studies have to clarify if MIR155 could be a potential therapeutic target.

Cholesterol supplementation protects hepatocytes against the cholesterol-dependent pore-forming toxin pneumolysin and impairment of innate immunity. Remotely, pneumococcal CAP triggers a hepatic activation of the sterol biosynthesis through a yet unknown mechanism that depends on PLY. Objectives: The direct impact of PLY on hepatocytes is determined. Supplementation of cholesterol species is evaluated as candidate for adjuvant therapy to counteract pore formation. The underlying principle of a protective effect of cholesterol supplementation is investigated. Methods: Membrane integrity and pore formation is analyzed by LDH-release assay and PI staining using dynamic live cell imaging techniques. Stress and sterol signaling adaption of HepG2 cells towards PLY challenge is examined by Western blot and quantitative RT-PCR. Cellular cholesterol of HepG2 cells is quantified by mass spectrometry and Filipin III staining. Membrane viscosity is characterized by applying live-cell two-photon fluorescence lifetime imaging of a bimodal dye sensing the cholesterol environment. In a clinical trial at the Jena University Hospital, a link between patient plasma cholesterol level and CAP score was assessed. Results: Supplementation of cholesterol resulted in amelioration of PLY toxicity, increased membrane integrity and a delay of pore formation. PLY directly activates key regulators of the sterol homeostasis on the protein level whereas gene expression is downregulated globally in HepG2 cells. Intriguingly, cells respond to PLY by increasing their cholesterol content despite cholesterol being the toxin's receptor for cell membrane recognition. By supplementation of cholesterol the membrane is shifted to a more rigid phase, hindering pore formation. In the clinic, CAP patients with elevated plasma cholesterol are significantly less prone to the progression of CAP. Conclusions: We conclude that PLY pore formation preferentially targets cholesterol in a highly flexible membrane resulting in shedding and cell death. Together, we highlight the role of a hepatic induction of cholesterol biosynthesis following PLY stress and outline the biophysical reasons for a protective effect of cholesterol supplementation against a cholesterol-dependent pore forming toxin.

Hemoadsorption with CytoSorb, a new treatment for Sepsis Ferrara G (1), Giuliano L (2), Gatta G (1), Gorgoglione G (2), Melchionda G (2), Aucella F (1) (1) Department of Nephrology, (2) Intensive Care Unit Introduction: Sepsis is the most common cause of death in medical intensive care units (ICU). If sepsis progresses to refractory septic shock, mortality may reach 90-100% despite optimum current therapy. One of the hallmarks of sepsis is the excessive release of cytokines and other inflammatory mediators causing refractory hypotension, tissue damage, metabolic acidosis and ultimately multiple organ failure. Objectives: Cytokine reduction by hemoadsorption represents a new concept for blood purification, developed to attenuate the systemic levels of pro-inflammatory and anti-inflammatory mediators released in the early phase of sepsis. Methods: We evaluated the impact of a new hemoadsorption device (CytoSorb), used as adjunctive therapy, on hemodynamics and clinically relevant outcome parameters in 24 critically ill patients with septic shock and in need of renal replacement therapy (RRT) in Intensive Care Unit. Mean levels of MAP, procalcitonin, noradrenalin need and SOFA score were evaluated. RRT of acute renal failure was performed either as continuous venovenous hemofiltration (CVVH) or continuous venovenous haemodialysis (CVVHD) at the discretion of the attending physician. Flow rates were set to achieve a dialysis dose of 25 ml/kg/h, blood flow rate was set accordingly. Hemoperfusion was started after refractory shock was diagnosed. The adsorber [total volume 300 ml, priming volume 120 ml, filled with sterile normal saline (NaCl 0.9%)] was connected in a PRAE-filter position into the RRT circuit. The first exchange was performed within 24 h without interruption. Further adsorber exchanges were at the discretion of the physicians. Results: After Cytosorb treatment procalcitonin, C-reactive protein and white cells count all decreased vs basal levels (Table 1) . This feature was associated with hemodynamic stabilization and a reduction of noradrenaline infusion. SOFA score improved in 11/24 patients, overall mortality was 63%. Treatment using the CytoSorb device was safe and well-tolerated with no device-related adverse events during or after the treatment sessions. Conclusions: In severe septic shock unresponsive to standard treatment, haemodynamic stabilization and inflammatory parameters improved using cytokine adsorption therapy. These effects seem to be more pronounced in patients in whom therapy started within 24 h of sepsis diagnosis, whereas a delay in the start of therapy was associated with a poor response to therapy in terms of reduction of catecholamine demand and survival. Detailed studies and registry reports may better define the potential benefits of this new treatment option. Introduction: One possible long-term consequence of sepsis is hematogenous osteomyelitis, i.e. the spread of bacteria from the blood stream to the bone. The clinical picture of this disease is swelling and deformation of the bone, pain, redness, fever and weakness. The most common underlying pathogen is the Gram positive Staphylococcus aureus, which is one of the most frequently found pathogen in hospital. These bacteria can invade cells, adopt an intracellular life cycle and persist in the bone for years before causing symptoms. Their intracellular appearance is especially difficult to treat with antibiotics because of a reduced metabolic rate and the challenge to target the drugs into the host cells.

Objectives: In this study we aim to define the localization of bacteria within the bone in an established mouse model [1] during the acute and chronic state of hematogenous osteomyelitis. Methods: Mice were infected via a lateral tail vein with S. aureus and pathological changes in behavior and by X-ray were recorded. After 1 week (acute phase) or 6 weeks (chronic phase) of infection mice were sacrificed, bones were isolated and fixed in 4% paraformaldehyde. After decalcification and cryosectioning, the slices were immunolabelled using fluorescent secondary antibodies to visualize S. aureus and osteocalcin, a marker for bone formation. Cellular structures were counterstained using DAPI (nuclei) and I555-phalloidin (actin fibres). For histopathological comparison hematoxilin & eosin (HE) staining was applied to some slices. Images were taken using confocal or two photon microscopy (Zeiss CLSM 780 Meta).

Results: An activation of the bone marrow was detected by HE staining and residing pathogens could be identified in the immunolabelled slices from both the acute and the chronic phase of hematogenous osteomyelitis. Pathogen load was high but not restricted to sites of bone deformation identified by X-ray in the chronic osteomyelitis model. In the acute phase no deformations were detectable by X-ray, still we found numerous bacteria in these bones.

Remarkably, sites of bacteria detection were quite diverse ranging from the hard bone, bone marrow to associated connective tissue and attached muscle fibres in both the acute and the chronic phase. Intracellular localization could be proven for both the acute and the chronic phase. Colocalization with osteocalcin, a marker of bone formation, which is produced by osteoblasts and secreted into the extracellular matrix of bone, was detected for some of the bacteria, especially in the acute phase of osteomyelitis. Conclusions: S. aureus can be found intracellularly and residing at multiple different locations in bone, both in the acute and in the chronic phase of osteomyelitis. The infection leads to an activation of the bone marrow and S. aureus can be found partly in areas of bone formation. Treatment strategies should therefore have a broad drug distribution and cover the different sites of bacterial residence. Introduction: The sphingolipid sphingosine 1-phosphate (S1P) is involved in many physiological and pathophysiological processes and also plays an important role in the function of the immune system. As a part of the inflammatory response, S1P mediates distinct signaling pathways that protect against sepsis. In a recent study we showed that S1P signaling could mimic the positive effects of genotoxic stress achieved in vivo in experimental sepsis experiments in mice and in vitro in cell culture upon stimulation of the lung epithelial cell line H1975 with lipopolysaccharides (LPS). These studies revealed that particularly inhibition of the S1P degrading enzyme S1P lyase and activation of the S1P receptor type 3 (S1PR3) are involved in genotoxic stress-induced S1P signaling, leading to tissue tolerance and protection against systemic inflammation. Now we focus on S1P and its receptors as potential candidates for sepsis treatment.

Objectives: The aim of the project is to investigate the influence of pharmacological modulators of the S1P metabolism and signaling pathways on the course of sepsis. Focusing on the positive effects of S1P signaling and metabolism under inflammatory conditions, a S1P lyase inhibitor and a S1PR3 agonist are tested for their therapeutic value. Methods: In in vitro studies, inflammatory conditions are achieved upon the stimulation of cells with LPS and cytokines. Additionally, these cells are treated with pharmacological modulators of S1P signaling and metabolism to inhibit the S1P lyase or activate the S1PR3. Focusing on the production of pro-inflammatory cytokines, enzymes, and metabolites of the sphingolipid metabolism, analyses are carried out by ELISA, flow cytometry, Western blot, and mass spectrometry. Furthermore the effects of pharmacological modulators will be examined under more complex conditions in a mouse model of experimental sepsis. To this end the model of polymicrobial contamination and infection (PCI) will be applied. Septic mice will be treated with a S1P lyase inhibitor and a S1PR3 agonist and afterwards the effects of those pharmacological modulators on cellular signal transduction, immune cell functions and tissue tolerance, as well as genetic regulation mechanisms will be investigated. Results: Currently, the role of sphingolipid signaling as well as the long-term efficiency of these signaling events are investigated in vitro, and the treatment of cells with pharmacological modulators of S1P signaling and metabolism showed positive effects regarding the production of pro-inflammatory cytokines. Following these initial results, the effects of pharmacological modulators of S1P metabolism and signaling will be examined in vivo using a mouse model of experimental sepsis. Conclusions: Carrying out these analyses, the role of S1P metabolism and signaling under inflammatory conditions will be characterized to reveal pathways resulting in protection against sepsis and to identify possibilities of sepsis intervention.

Infection 2019

In vitro synergism and anti-biofilm activity of dalbavancin antibiotic combinations against vancomycin-susceptible and vancomycin-resistant Enterococcus faecium Introduction: As a common sense, systemic inflammatory reactions as specially sepsis are an acute life-threatening condition with a high rate of death and very intensive treatment. Improving existing therapies and developing new approaches are therefore highly desirable. A contribution to this could come from the field of nanomedicine.

Objectives: Magnetic as especially superparamagnetic particles can be used for different medical applications. These particles can, for example, be navigated in the body with the use of magnetic fields or be deployed for several in vitro diagnostics. In our working group, we have successfully developed various particle systems to date. Examples are formulations for drug delivery [1] , MR imaging [2] , regenerative medicine [3] and targeted immunotherapy [4] . In addition to extensive physical-chemical characterization, we have established a systematic assay cascade [5] to test biocompatibility and efficacy in vitro and in vivo. Only particles with proven safety profile will be utilized for further biomedical applications. A new approach is to coat nanoparticles in such a way that they are able to selectively bind pathophysiologically relevant microbial products and thus remove it from body fluids. In human saliva there is an agglutinating saliva protein that is able to bind various pathogens. Interestingly, even a small consensus sequence of the macromolecule has similar binding properties, which facilitates orthogonal binding strategies to nanoparticles.

Methods: In this case we have developed promising candidates like APTES (3-Aminopropyltriethoxysilan) as a coating. Using APTES as a coating has several advantages. First it is already in use for the coating of implants and second it has amino groups on bound to the silica which make it easy to find linkers that can be connected to a peptide. In recent works, we synthesized superparamagnetic iron oxide nanoparticles with stable aminoalkylsilane layer that have later been functionalized with heterobifunctional linkers like N-succinimidyl bromoacetate (SBA). These linker were further chemoselectively reacted with the thiol group of singularly present cysteines of selected peptides.

Results It was possible to see that we could extract a model toxin from a solution which did not work with the control peptide [6]. Currently we are improving the synthesis to get particles that are stronger attachable by a magnet and to gain a more efficient coating as well as a higher linkage of peptides. Furthermore new linkers are under development as well as new assays to prove the binding of further toxins and simultaneously we are about to bind different bacteria, using our particles. Beyond that, we attempt to create a system that can extract pathogens from not only blood but also other fluids where a separation of toxins or bacteria is needed for diagnostic and therapy.

Conclusions: Proof of principle of a new particle based method to lower the pathogen load in sepsis patients Acknowledgement: Introduction: In SHV-b-lactamases single amino acids changes cause a change of substrate spectrum from narrow-spectrum-b-lactamases to extended-spectrum-b-lactamse (ESBL) or inhibitorresistant-b-lactamse (IRBL). At present time 147 SHV variants are listed in the NCBI database but only in 48% the phenotype is known. The focus of clinical testing is in terms of ESBL at CTX-M. But often SHV is also carried by the pathogens. With increasingly use of blactamase-inhibitors (BLIs) the prevalence of inhibitor-resistant SHV may rise and call for updated test panels. A well understood genotype-phenotype correlation is the prerequisite for molecular testing.

Objectives: The aim of this study was to identify phenotype relevant amino acid substitutions of SHVs by a targeted mutagenesis and genotype-phenotype correlation. Methods: We used mathematical models to evaluate phenotype-relevant amino acid substitutions based on the validated SHV variants stored at the NCBI data base. The SHV-1 gene cloned into pBTvector was altered with primer mediated site directed mutagenesis. 30 single mutations as well as three double and one triple mutation were generated. Antibiotic susceptibility of the mutants was tested towards a wide substrate spectrum of penicillins, cephalosporins, b-lactams/ BLIs combinations and carbapenems using broth microdilution and E-test.

Results: With the mathematical algorithms we identified 18 positions within the SHV proteins being related to the ESBL phenotype and 6 to the BL-inhibitor resistant phenotype in silico. The phenotypic testing of mutations at this positions revealed only substitutions G238S/A (Ambler position) being related to the ESBL phenotype. To refer resistance to BL-inhibitors mutations at two positions were identified: M69L and E240K/R. Many substitutions led to an increased MIC against ceftaroline, a 5th generation cephalosporin.

Conclusions: This is the widest substrate and substitution analysis for SHV alleles so far. The genotype-phenotype correlation of distinct amino acid changes showed only a few amino acids accounting for extension of the antibiotic and b-lactamase-inhibitor spectrum of shv. The increased resistance to ceftaroline, indicate that beforehand manifested random substitution may already confer resistance to very recently introduced beta-lactams. Thus, susceptibility testing against ceftaroline should be considered also for non-ESBLs.

Infection 2019

The role of Ezrin in hepatocytes in the onset of excretory liver failure Introduction: Sepsis is a clinical syndrome that is associated with systematic inflammation and metabolic changes. Liver failure is a serious complication in up to 10% of sepsis patients and linked to high mortality rates. Disease tolerance is a host protective mechanism in which cells undergo metabolic, molecular changes without directly targeting pathogen. Liver has a high tolerance capacity and ability to withstand assaults but this may be critically affected in sepsis and inflammation.

Objectives: Objective of this study is to establish an in vitro hepatic model in order to understand effect and changes in hepatocytes and how different signaling pathways are involved in disease tolerance in response to septic insult in hepatocytes. Methods: Mouse primary hepatocytes isolation protocol is optimized in order to maintain cells in culture for long term experiments. Coculture settings composed of AML12 and HepG2 hepatic cell lines and HMEC endothelial cells are also tested. Hepatocytes and endothelial cells are co-cultured in a 3:1 ratio and stimulated with cytokines (TNFa, IFNc, IL1, IL6) and PAMPS (LPS) for different time points to induce inflammation. In order to induce hypoxia, cells are incubated in hypoxic chamber and activation of HIF1 a is studied by western blot and immunocytochemistry. Activation of autophagy and development of stress granules are also checked by western blot and immunocytochemistry.

Results: Initial experiments are done using the mouse hepatic cell lines AML12 co-cultured with HMEC human endothelial cells. Primary hepatocytes co-cultured with endothelial cells maintain their morphology longer than hepatocytes cultured alone. Activation of HIF1 a is observed by immunocytochemistry in hepatocytes treated with cytokines and LPS but, it is negligible compared to hypoxia in both single AML12 culture and co-culture systems. Further experiments to check metabolic master pathways, mTOR and AMPK with/ without hypoxia are in process. Experiments to check stress response at the level of autophagy and stress granule production are still running and results will be presented at the conference. Conclusions: Hepatocytes co-culture with endothelial cells can be used to investigate stress pathways in the context of disease tolerance to inflammation. Inflammation induces HIF1a but undergoing disease tolerance mechanisms still have to be studied in more detail. Experiments to check whether autophagy and stress granule are also involve in hepatocyte disease tolerance mechanisms or not, are also in progress. Introduction: The role of the host immune response is highly controversial under septic conditions. In addition to a hyperinflammatory response, recent studies indicate that immunosuppressive pathways are triggered concomitantly. The Spred protein family (Sprouty-related EVH1 domain-containing protein) is involved in the regulation of the Ras/MAPK pathway, which is activated in sepsis in numerous immune cells. Strikingly, knockout of SPRED2 was shown to promote proinflammatory events resulting in radically opposed outcomes in mice subjected to different sepsis triggers: Thus, whereas deficiency of Spred2 exacerbated LPS (lipopolysaccharide)-induced acute lung inflammation, it exerted protective effects in septic peritonitis. These findings indicate that Spred's regulation of the Ras/MAPK pathway can have drastically diverse consequences on sepsis outcome in distinct clinical scenarios. Despite these appreciations, the precise mode of action of Spred is still unsettled.

Objectives: In this study, we investigate the mechanisms underlying the modulation of the Ras/MAPK pathway by Spred1. Understanding the molecular basis of the signal transduction regulation by Spred may reveal new opportunities for immunomodulatory approaches in sepsis therapy. Methods: After stimulation with EGF (epidermal growth factor), HeLa cells overexpressing Spred1 were lysed and exposed to glutathione-Sepharose for a Ras-GTP pull-down. Ras activation was analyzed by Western blotting. HEK293 cells overexpressing Spred1 were fixed and immunostained using fluorophor-labelled secondary antibodies prior to analysis via fluorescence microscopy. Using the proximity biotinylation assay followed by liquid chromatographymass spectrometry-based quantitative proteomics, we intend to identify Spred1 interactors and proximate proteins that team up with Spred1 for Ras regulation. The biotinylated target proteins were visualized by Western blotting with Avidin. Results: Overexpression of Spred1 reduced EGF-stimulated formation of active Ras-GTP, showing that Spred1 inhibits the pathway at the level of Ras. Immunocytochemistry analysis further revealed that Spred1 overexpression resulted in formation of giant early endosomes. This abnormal phenomenon affected the steady-state distribution of Ras causing an accumulation of Ras in the giant endosomes. Initial screening results revealed a huge amount of Spred1 interaction partners playing a role in endocytotic processes which is consistent with the observed effect of Spred1 on endosome formation. Conclusions: Our findings indicate that Spred1 is involved in the regulation of early endosome trafficking and may attenuate Ras activity indirectly by affecting its subcellular distribution in terms of altered endosome dynamics. This might represent a molecular mechanism for targeting the immune imbalance in sepsis at its roots. Introduction: Hepatocytes express transporting proteins like the multidrug-resistance associated polypeptide 2 (MRP2) on their apical pole to eliminate bile salts as well as endo-and xenobiotics into the bile. To secure its eliminating function, MRP2 has anchored into the canalicular membrane via the ezrin-radixin-moesin (ERM) family proteins that link it to the scaffolding fibrillary actin (f-actin). In the course of sepsis, local cytokine release will stimulate numerous signaling cascades. Among many others, the production of reactive oxygen species (ROS) and the upregulation of Protein Kinase C (PKC) family enzymes has been notified. In preliminary cell culture experiments, PKC activation as well as ROS inducement has led to the loss of MRP2 from the canalicular membrane and therefore to excretory dysfunction in hepatocytes (cholestasis). Objectives: To understand the mechanisms behind hepatocyte scaffolding and MRP2 internalization that lead to excretory dysfunction, we investigated the link to ROS release and PKC upregulation in small rodent liver tissue. Methods: We established a method of liver perfusion in mice with physiological buffer solution. In the treatment groups, ROS inducer tert-Butyl hydroperoxide (tBHP) or the PKC activator phorbol 12-myristate 13-acetate (PMA) were added to the buffer. Perfused liver tissue was stained for MRP2, ERM-proteins, and f-actin. The quantity, as well as the localization of stained proteins in hepatocytes, were analyzed via confocal imaging. To assess and quantify the impact of ROS and PKC treatment on liver excretory function, Indocyanine Green (ICG)-excretion rate of hepatocytes was quantified by in vivo microscopy of perfusion experiments.

Results: TBHP and PMA treatment lead to an internalization of MRP2 from the canalicular membrane. Also, f-actin is diminished in width and overall signal intensity at the hepatocyte membrane which correlates with a loss of ERM proteins in the treatment groups. Furthermore, the ICG-excretion rates show longer storage times and decreased MRP2 activity in liver tissue of PMA or ROS treated mice compared to the control group.

Conclusions: In clinical diagnostics, ICG is used to determine hepatic clearance rate. Our data depict, that treatment of mice with PKC activator or ROS inducer affects the excretion rate of ICG in liver tissue of small rodent. To investigate the mechanisms behind this loss of functionality, we analyzed morphological changes in our treatment groups. The diminishment of f-actin scaffolding and ERM proteins are consistent with the internalization of MRP2 into the cytosol of hepatocytes in treated liver tissue. These findings show, that PKC upregulation and ROS are involved in signaling cascades that lead to morphological rearrangement and ultimatively cholestasis in hepatocytes.

Infection 2019

Redox regulation of PPARc in polarized macrophages Introduction: Sepsis is characterized by a hyper-and a hypo-inflammatory response, occurring in parallel. As key players of the innate immune system, macrophages (MU) are involved in these responses and can shape specific phenotypes according to their task. Thereby, MU can be classical activated by lipopolysaccharide (LPS)/ interferon-c (IFNc) resulting in a pro-inflammatory expression profile. Contrarily, an alternative activation of the MU is triggered e.g. by interleukin-4 (IL4), leading to an anti-inflammatory expression pattern. These distinct expression patterns of polarized MU account for variable intracellular redox conditions, which may induce redox modifications, affecting protein function, and perhaps altering sepsis outcome.

Objectives: We determined how the MU redox status affects the nuclear receptor PPARc, which has been shown to be mandatory during alternative MU activation. Because additional to its function as a transcription factor, the protein is also known to interact DNA unbound with pro-inflammatory transcription factors such as NF-AT or NF-jB, transrepressing target genes. Therefore, we hypothesized a role of the cellular redox status in regulating these two functions of PPARc.

Methods: To prove our assumption, we first determined the redox status of MU following alternative and classical activation, by employing the redox sensitive marker protein roGFP2, coupled to Grx1 or PPARc to analyze cytosolic and nuclear redox stress. The redox status of PPARc was identified by mass spectrometry (MS). PPARc function was followed by reporter gene assays. Its cellular localization was examined by Western blot analyses. Co-IP studies were used to identify specific redox stress-based protein-protein interactions of PPARc.

Results: Using the redox sensitive GFP2 (roGFP2), we validated oxidizing and reducing conditions following classical and alternative MU activation. MS analysis of PPARc revealed that cysteine residues located in the zinc finger regions, reflected by the MS analyzed fragments aa 90-115, aa 116-130, and aa 160-167 of PPARc, were highly oxidized in response to LPS/IFNc, accompanied by phosphorylation of serine 82. IL4-stimulation provoked minor serine 82 phosphorylation and less cysteine oxidation, referring to a reductive milieu. Mutating these cysteines to alanine to mimic PPARc oxidation reduced its reporter gene transactivation, indicating lower transactivating ability of PPARc associated with classically activated MU. Moreover, classically compared to alternatively MU polarization alters PPARc expression and localization, giving rise to vimentin binding of PPARc in classically activated MU only, suggesting an altered protein-protein interaction profile of PPARc accompanying the MU phenotype shift. Conclusions: Our data support the notion that altering the redox status in MU will alter protein function as exemplified for PPARc. Therefore, it will be interesting to follow up this approach in an in vivo setting of polymicrobial sepsis in the mouse by testing whether altering the redox profile will shift MU phenotypes and thus, improve sepsis outcome.

Introduction: Streptococcus pyogenes is a highly versatile, grampositive human pathogen causing a broad range of infectious diseases. These vary from common mild infections such as pharyngitis to severe, invasive infections such as necrotizing fasciitis. Although the mechanisms by which innate immune cells detect S. pyogenes have increasingly been understood, the pathogen-specific innate immune cell adaptations evoked by S. pyogenes encounter have not yet been investigated.

Objectives: We aimed to analyze the metabolic, epigenetic and functional adaptations of murine bone marrow-derived macrophages (BMDMs) upon S. pyogenes infection compared to E. coli infection in vitro.

Methods: For analysis of cellular metabolism, BMDMs from wild type (WT) mice were infected with S. pyogenes or E. coli, respectively, and real-time analysis of extracellular acidification rate and oxygen consumption rate using Seahorse technology was performed.

To evaluate an S. pyogenes-induced functional reprogramming, infected BMDMs were washed 22 h post infection, exposed to a secondary stimulus, and inflammatory cytokine levels were measured by ELISA. To assess epigenetic rewiring, chromatin immunoprecipitation followed by high-throughput sequencing was performed.

Results: While oxidative phosphorylation was completely disrupted in WT BMDMs upon both S. pyogenes and E. coli infection, glycolysis was upregulated solely in S. pyogenes infected cells. Moreover, only BMDMs initially infected with S. pyogenes displayed enhanced responsiveness to TLR4 restimulation via LPS, with markedly increased inflammatory cytokine levels observed in S. pyogenes primed cells compared to E. coli infected or non-treated controls. Strikingly, both S. pyogenes and E. coli infection induced a profound, but distinctive, epigenetic reprogramming of BMDMs within the promotor regions of immune-relevant genes, mediated by the trimethylation of lysine 4 of histone 3. Conclusions: S. pyogenes launches a profound and pathogen-specific functional reprogramming in BMDMs, leading to an increased release of inflammatory cytokines in vitro upon restimulation. Those functional changes are corroborated by a major metabolic rewiring with a complete shutdown of mitochondrial respiration and distinctive changes in the epigenome. Recent studies using in vitro and in vivo animal models suggest that host factors and contamination of the CVCs with skin-associated and other bacteria are important for C. albicans biofilm formation, but the data has not been validated in CLABSI patients.

Objectives: We hypothesize that CVC-associated host proteins and bacterial colonization contribute to C. albicans biofilm formation in clinical settings. Therefore the aim of this project is to establish a protocol for the formation of C. albicans and E. faecalis dual species biofilms to further test the influence of single human blood proteins. Methods: Different incubation conditions like growth media, inoculation density and adherence times have been tested in 24-well plates. Biofilm metabolic activity and biomass were quantified using colorimetric assays (resazurin, crystal violet). To test the influence of host factors, the wells were coated with human serum before inoculation. To visualize fungal and bacterial behavior in biofilms, life/ dead and fungal-specific staining were applied and analyzed by fluorescence microscopy.

Results: The E. faecalis strain ATCC 29212 adheres better to uncoated plastic surfaces compared to OG1RF strain. Solid 24 hbiofilms were formed after an initial adherence phase of E. faecalis (4.5 h, 7 9 10 7 cells/ml) in tryptic soy broth, supplemented with 1% glucose and a second adherence phase of C. albicans in RPMI (1.5 h, 5 9 10 6 cells/ml) or vice versa. Initial adherence of C. albicans to the plastic surfaces produced mixed biofilms with higher biomass and metabolic activity compared to biofilms where E. faecalis adhered first. In addition, E. faecalis ATCC 29212 strain allowed for more robust biofilm formation. Serum-coated wells lead to further improvement of biofilm biomass and metabolic activity. Different fluorescence dyes allowed optical evaluation of C. albicans hyphal morphogenesis and for evaluation of biofilm viability.

Conclusions: The experimental conditions for in vitro mixed species biofilm formation of C. albicans and E. faecalis were determined. The fungus seems to serve as a scaffold for mixed biofilm formation. Next, we aim to dissect the influence of single abundant blood proteins on single and mixed biofilm formation. Further, we will now transfer these results to an in vitro and ex vivo central venous catheter model and to a flow model using a BioFluxTM device.

Infection 2019

Lactate as metabolic checkpoint of innate immunity (1), Uhle F (1) (1) Department of Anesthesiology, Heidelberg University Hospital, 69120 Heidelberg, Germany, (2) Metabolomics Core Technology Platform, University of Heidelberg, 69120 Heidelberg, Germany

Introduction: Patients suffering from sepsis frequently show elevated blood lactate levels. As the universal end product of glycolysis, the systemic occurrence of lactate is considered to be a result of tissue hypoperfusion and cellular oxygen shortage. Nevertheless, also activated immune cells like monocytes undergo metabolic reprogramming and increase glycolysis, while simultaneously shutting down mitochondrial respiration despite sufficient oxygen tension. Objectives: Linking these two aspects with each other, we hypothesized that apart from its role as a biomarker, lactate might exert long-distance effects on immune cells altering their response upon activation. Methods: The monocytic MonoMac6 cell line was stimulated with various toll-like-receptor agonists (e.g., LPS and advanced glycation endproducts) after priming with increasing concentrations of Nalactate (1-10 mM). As readout, IL-6 production was measured using ELISA or the expression of the corresponding gene by qPCR. Mass spectrometry was used to assess intracellular metabolite concentrations. Real-time assessment of intracellular energy pathways was done using Seahorse technology.

Results: Irrespective the stimulus, short Na-lactate-priming (1 h) strongly reduced IL-6 cytokine production, accompanied by a reduced expression of the corresponding gene. Notably, this effect was also observable when Na-lactate was added simultaneously or even after the immunogenic stimulus. Lactate as well as sorbitol was found to accumulate intracellularly. The latter indicates a shunt of glucose into the branching polyol pathway, hinting towards a shutdown of glycolysis. This was further substantiated by a decreased glycolytic flux in Seahorse assay. Opposingly, long-term priming (24 h) with Nalactate induced cellular adaption and abolishment of the lactate effect.

Our results indicate that, apart from its passive role as a clinical biomarker, exogenous lactate acts as a metabolic checkpoint, shaping monocyte function by indirectly blocking intracellular glycolysis. Therefore, a transient elevation of systemic lactate might be considered as an evolutionary mechanism of the organism counteracting excessive inflammation, also raising the question of potential therapeutic applicability.

Infection 2019

The critical role of SPNS2 in regulating endothelial barrier function under healthy and sepsis condition (S1PRs), a class of G-protein coupled receptors on endothelial cells. Signaling via S1PRs is mediated by sphingosine 1-phosphate (S1P). S1P at low nanomolar levels are sufficient to activate S1PRs, while high concentrations lead to internalisation and desensitisation of S1PRs, particularly S1PR1. Studies show that barrier function is maintained via S1PR1 however, the mechanism behind EC barrier stabilisation at high S1P levels in plasma is unclear.

Objectives: The principal aim of this study was to investigate the mechanism behind EC barrier stabilisation at high plasma S1P levels under physiological and pathophysiological conditions by using human umbilical vein endothelial cells (HUVEC) and the endothelial cell line EA.hy926 as endothelial models.

Methods: Electric cell-substrate impedance sensing (ECIS) measurements under the static condition and organ-on-a-chip under dynamic condition along with fluorescein isothiocyanate (FITC)dextran assay were used to demonstrate EC barrier function upon stimulation with different pharmacological compounds. Further, liquid chromatography coupled to triple-quadruple mass spectrometry (LC-MS/MS) was used to analyse the production and excretion of lipid metabolites in cell and medium samples. Besides, quantitative polymerase chain reaction (qPCR), Western blot and immunofluorescence microscopy were used to investigate the expression and alteration of VE-cadherin at gene and protein levels under healthy as well as inflammatory conditions. Results: In HUVEC ECIS measurements demonstrated a change in cell monolayer resistance upon stimulation at 6 kHz. Blocking of S1PR1 with specific antagonist led to barrier destabilisation. Also, LC-MS/MS revealed S1P traces in the medium. Scavenging S1P by S1P antibody also led to barrier destabilisation. While EA.hy926 did not respond to S1PR1 antagonist or S1P antibody treatment, LC-MS/ MS showed that EA.hy926 did not excrete S1P. This could be due to the lack of S1P transporter, spinster homolog 2 (SPNS2) in EA.hy926 but not in HUVEC, which was supported by media exchange experiments. Interestingly, lack of exogenous S1P leads to decrease in VE-cadherin association at the cell junctions in EA.hy926 and HUVEC. Furthermore, organ-on-a-chip analyses in combination with FITC-dextran permeability assays revealed that the fluorescence intensity was reduced at high concentration of S1P-perfused cell monolayers compared to vehicle, implying barrier maintenance at high S1P level. Based on these findings, it was speculated that autonomously produced S1P is most likely transported via SPNS2.

Transported S1P can then act on S1PR1, which is expressed on the tissue facing side of the endothelial cells, in an autocrine or paracrine manner, illustrating the autonomous mode of action. This serves as the mechanistic model explaining the regulation of S1PR1 activity in the presence of high plasma S1P concentrations. Also, the effects of cytokine mix together with lipopolysaccharide treatment (mimicking inflammation) on the production and transportation of S1P was examined. Interestingly, S1P secretion was reduced, and this was likely due to the increase in S1P-lyase (SGPL1), lipid phosphate phosphatase 3 (LPP3) and decrease in SPNS2 expression. Besides, VE-cadherin production was reduced after cytokine treatment due to alteration in S1P production and excretion.

Conclusions: To sum up, S1P release is steadily controlled by SPNS2 and, it is crucial for the maintenance of barrier function. Lack of SPNS2 expression can lead to loss of basal barrier integrity. Based on these findings, it is concluded that under high plasma S1P, EC barrier function is maintained by S1P, which is produced and excreted by endothelial cells in the nanomolar range. This low concentration of S1P is sufficient to mediate signaling via S1PR1, which is expressed on the endothelium and faces the tissue side of the vascular system. Also, under inflammatory conditions, expression of SPNS2 along with SGPL1 and LPP3 are altered thus, the autonomous mode of action is hindered and leads to loss of barrier integrity. Additionally, an auxiliary supply of S1PR1 agonists or production of S1P from tissues may, thus, support the recovery of EC barrier function mediated by S1PR1 expressed on the plasma or tissue facing side as they are unaffected during sepsis condition.

Infection 2019

Influence of the sphingosine 1-phosphate (S1P) carrier molecules serum albumin (SA) and high-density lipoproteins (HDL) on the signal transduction of S1P and its receptors Introduction: S1P, a bioactive phospholipid, is a ligand for five G-protein-coupled cell surface receptors designated S1PR1-5. It regulates pathophysiological processes involved in sepsis progression, including endothelial permeability, cell migration, cytokine release and vascular tone. S1P acts to promote endothelial cell (EC) barrier function. In plasma, S1P is carried by the ApoM ? sub-fraction of HDL (* 65%) and SA (* 35%). In patients with sepsis, serum-S1P levels are dramatically decreased as well as HDL-and SA-levels. This may contribute to increased vascular permeability during sepsis progression.

Objectives: In this study we investigate the different effects and the underlying molecular mechanisms of both carriers to the signal transduction of S1P.

Methods: In order to study the influence of S1P carriers with regard to EC barrier stabilization, electric cell substrate impedance sensing (ECIS) measurements were performed. The endothelial cell line EA.hy926 as well as primary human umbilical vein endothelial cells (HUVEC) served as endothelial model systems. Transwell migration assays with primary mouse lymphocytes (PML) followed by flow cytometry were used to study the impact of the S1P carriers on cell migration.

Results: ECIS results with EA.hy926 and HUVEC pointed to different physiological effects of both S1P carriers. Stimulations with 300 nM S1P and 50 mg/ml human SA enhanced the EC barrier significantly better compared to S1P with 0.5 mg/ml HDL or S1P alone. Initial experiments with PML demonstrated increased cell migration with 10 nM S1P and HDL compared to S1P and SA or S1P alone. Conclusions: These results show that HDL and SA are not only S1P carriers, but also impart different effects on S1P signaling. SA enhanced S1P-mediated EC barrier stabilization better than HDL, whereas HDL supported lymphocyte migration more than SA. Furthermore, the molecular basis of S1P signaling underlying these physiological effects is currently investigated. Based on our current results, SA could be a valuable target for pharmacological interventions in the treatment of sepsis to maintain a stable EC barrier.

Infection 2019

The effects of serum albumin on degradation and metabolism of sphingosine-1-phosphate

Xin H, Gräler MH Introduction: Sepsis is the main cause of death in intensive care units worldwide. Sepsis is defined as a life-threatening organ dysfunction owing to an abnormal host response to infection. The endothelial function, which is able to be regulated and influenced by sphingosine-1-phosphate (S1P), plays a crucial role in the organ failure caused by sepsis. While blood-borne S1P is a critical requirement for a functional vascular endothelial barrier, the regulatory pathways underlying its maintenance are largely unknown. The gradient of S1P concentration in plasma [ lymph [ tissue is essential for the maintenance of endothelial barrier function and other S1P-dependent processes such as leukocyte trafficking. Therefore, modulation of S1P degradation is also an important event for endothelial barrier function. The function of serum albumin in sepsis patients is not fully understood. A low concentration of serum albumin is associated with poor outcome in septic patients. S1P in blood is mainly associated with high-density lipoprotein (HDL) and serum albumin. More than 50% of plasma S1P is bound to HDL and about 40% is associated with albumin. Current data indicate that S1P is not physically bound with serum albumin at physiological concentrations. Our data indicate that serum albumin may protect S1P from degradation by a yet unknown mechanism.

Objectives: The effects of bovine serum albumin (BSA) and human serum albumin (HSA) on degradation and metabolism of S1P were investigated. Methods: Flow cytometry, cell culture experiments, liquid chromatography coupled to triple-quadrupole mass spectrometry (LC/MS/ MS), and SDS-PAGE electrophoresis were used to investigate the effects of BSA and HSA on degradation and metabolism of S1P. Results: After overnight incubation with rat hepatoma HTC4 and human endothelial EA.hy926 cells, S1P was protected from metabolism by BSA and HSA. Pepsinized BSA and ovalbumin didn't have this effect. After overnight incubation with HTC4 cells, cell media with S1P and BSA induced more S1P1 receptor internalization of S1P1-hemagglutinin epitope-tagged HTC4 cells, compared with cell media only with S1P.

Conclusions: Based on our current data, serum albumin may be able to protect S1P from degradation. To determine the molecular mechanism, incubations with different S1P-metabolizing enzymes and different spectrometric analyses for structural analyses and determinations of S1P-serum albumin-interactions are currently investigated.

Infection 2019

Co-infection with Staphylococcus aureus after primary influenza virus infection leads to damage of epithelial and endothelial cell layers Introduction: The seasonal influenza virus (IV)-associated bronchopneumonia is one of the infectious diseases with the highest population-based mortality. Likewise, respiratory tract infections represent the most common cause of sepsis. Beyond the virulence of the virus itself, epidemiological data suggest that bacterial co-infections are the major cause of increased mortality. In this context, Staphylococcus aureus (S. aureus) represents a frequent causative bacterial pathogen in secondary pneumonia.

Objectives: The aim of the present work was to determine the crosstalk between IV and secondary bacterial infection regarding the pathogen load and the inflammatory response of epithelial and endothelial cell layers. For this, a human in vitro alveolus model system composed of vascular and epithelial cell structures with cocultured macrophages resembling the human alveolus architecture and functions will be established. Methods: Singular and complex co-infection between IV and S. aureus of different epithelial and endothelial cell lines as well as infection of the human-alveolus-on-a-chip were performed in vitro. The pathogen load was determined via the standard plaque assays and serial dilution on agar plates. Cell death was determined via caspase cleavage by western blot analysis. In addition, LDH assays, FACS analysis, and immunofluorescence assays were applied. Results: During viral and bacterial co-infection, we measured an enhanced epithelial and endothelial cell damage compared to single infections. Furthermore, an accelerated bacterial release could be detected for the co-infection scenario. Here, we found significant endothelial cell damage associated with loss of barrier function. Endothelial cell damage was associated by the rapid spread of bacteria from the epithelial to the endothelial cell layer 2.5 h post co-infection.

Conclusions: Taken together, our results demonstrate the close interaction between host cells during infection. While epithelial cells within the alveolus are important for the first step of infection, endothelial cells become infected via the epithelial cells in a second step. This process is also influenced by various immune cells. Finally, the combined cell activation results in a robust inflammatory response of immune cells, which contributes to the lung damage.

Infection 2019

The non-canonical NF-jB signalling pathway is involved in the pathogenesis of Shiga-toxin-induced haemolytic-uraemic syndrome in mice . Microthrombotic haemolytic anaemia, thrombocytopenia and acute kidney failure are the typical clinical symptoms in HUS. However, the neurological or cardiovascular system can also be severely affected. It has been shown previously that proinflammatory cytokines that are associated with the induction of the classical NF-jB signalling pathway are increased in HUS patients. Stx is the most important virulence factor of STEC and contributes to this host response. The role of the non-canonical NF-jB signalling pathway, in which the heterodimeric transcription factor NF-jB2/RelB is involved, has not been yet investigated in HUS.

Objectives: The aim of this study was to elucidate the impact of the non-canonical NF-jB signalling pathway in the pathogenesis of STEC-HUS in mice.

Methods: HUS was induced by Stx administration in C57BL/6J mice as described previously [1] . Seven days after the induction of disease, kidney biopsies were taken from sham-and Stx-challenged mice. The activation of the non-canonical NF-jB signalling pathway was analysed comparatively in these kidney samples by investigating the gene and/or protein expression level of the key proteins involved in the pathway, NF-jB2, RelB, NIK, IKKa, TRAF2 and TRAF3, and of the pathway-activating receptors Fn14 and CD40 by performing western blot and real-time quantitative PCR analysis. The degree of the translocation of NF-jB2/RelB into the nucleus, and thus its functional activation, was verified on protein level by cytosolic and nuclear fractionation. The statistical analysis was performed using Mann-Whitney U test.

Results: Compared to sham mice, Stx-challenged mice developed severe clinical symptoms, including acute kidney injury, as described previously for this model. In kidneys of Stx-challenged mice we observed a significantly increased protein expression of the heterodimeric transcription factor NF-jB2/RelB and a significantly elevated translocation of these signalling proteins into the nucleus. Furthermore, Fn14 protein expression and CD40 gene expression-both upstream activators of the non-canonical NF-jB signalling pathwaywere significantly increased. These results indicate an activation of the non-canonical NF-jB signalling pathway in the kidneys of mice with HUS. Conclusions: For the first time, we provide evidence for the involvement of the non-canonical NF-jB signalling pathway in the pathogenesis of STEC-HUS. 

Infection 2019

Fuss J (1), Voloboyeva (2) (1) Department of surgery, Regional Central hospital in Pustomyty, Ukraine, (2) Department of Intensive Care, Hospital No. 8, Lviv, Ukraine

Introduction: Despite the current advances in surgery, the actual clinical problem in medicine is the treatment of large wound defects with the presence of infection [1, 2, 3] . One of the effective methods used in the treatment of both acute and chronic wounds is VAC [4] .

Objectives: Explore using VAC-therapy for treatment for soft tissue wounds of different genesis. Methods: Clinical observation was performed in 46 patients with wound defects of soft tissues of various genesis. The patients were divided into two groups: study group-32 patients, control-14 patients. The bacterial landscape of the wounds was determined by a qualitative and quantitative method of analysis of the microflora of soft tissue biopsy tissues obtained during dressing from the area of the bottom and edges of the wound (Table 1 ). In the first group, additional use was made of NPWT (HEACO). Results: Study showed that the use of the vacuum therapy method in first group significantly reduces the treatment period compared with the use of conservative techniques. In first group, the average terms of treatment were (19.3 ± 13.9) days, in second-(40.0 ± 28.2) days. After treatment, the wound area in the first group of patients was (68.1 ± 45.3) cm 2 , in the second-(74.4 ± 46.2) cm 2 (p \ 0.05). The study showed that the level of bacterial insemination on the 4th day of treatment against the background of bacterial therapy in the first group was, on average, 10 3 -10 4 microbial cells per 1 g tissue, in the second 10 5 -10 6 . Operative recovery was performed in 46 patients ( (5), Genz B (6), Abshagen K (6), Pützer BM (7), Sha LK (1), Weigert A (1), Syed SN (1), Schulz M (1), Shah AM (8), Ernst A (2,3), Putyrski M (2), Finkelmeier F (9), Pesic M (9), Greten F (9), Hoghardt M (10), Kempf VAJ (10), Gunne S (2), Parnham MJ (2), Introduction: Multi-organ failure (MOF) is an important characteristic in sepsis progression, often associated with the patients death. Therefore, understanding mechanisms leading to MOF, which allow new treatment strategies, are mandatory to improve sepsis survival. Objectives: One of the affected organs is the liver. Considering the established role of cytotoxic T lymphocytes (CTL) in murine sepsis, we focused on targets known to induce CTL tolerance and activation, thus contributing to CTL-dependent liver damage. Methods: We used the Hepa1-6 cell line in vitro and a mouse model of polymicrobial sepsis, following cecal-ligation and puncture (CLP) in wild type, myeloid-specific NADPH oxidase (NOX)2, global NOX2 and NOX4 knockout-mice for in vivo experiments. As readouts, we determined mouse survival, the release of alanine-and aspartate amino-transaminases as well as liver mRNA and protein expression. We restored hepatic expression of PD-L1 by adenoviral transfer and a transposon-based setting applying hydrodynamic injection (HDI). As a pharmacological approach, we utilized recombinant PD-L1-Fc to induce PD-1 signaling. N-Acetylcysteine (NAC) was employed in vitro to scavenge reactive oxygen species (ROS).

Results: Expression of PD-L1 was downregulated on hepatocytes following CLP induction. Restoring hepatic PD-L1 expression by adenoviral transfer or HDI significantly improved mouse survival. Interestingly, adenoviral transfer itself reduced CLP-dependent liver damage, whereas HDI itself did not alter the release of liver damage markers following CLP operation. With a pharmacological approach, applying recombinant PD-L1-Fc directly following CLP operation, liver damage was also reduced. Because we mechanistically found that NAC inhibited PD-L1 downregulation on Hepa1-6 cells stimulated with LPS, we determined the role of ROS in vivo. We found an increase of hepatic PD-L1 expression in sham-operated NOX4 knockout mice. Its expression in myeloid specific NOX2 knockout as well as global NOX2 knockout mice was comparable to that in wild type animals. Following CLP, PD-L1 expression stayed only high in global NOX2 knockout mice, associated with a significant reduction of liver damage.

Conclusions: We suggest that contrary to the common assumption, PD-L1 expression on hepatocytes is important to maintain CTL tolerance. Therefore, administering recombinant PD-L1 or scavenging ROS formation might be a new therapeutic strategy in sepsis.

Infection 2019

Mitochondrial Dysfunction as a predictor for Sepsis Severity and Outcome? Introduction: Sepsis is associated with mitochondrial dysfunction and exercise capacity influences sepsis outcome. However, exercise capacity consists of a genetic and an acquired component which cannot be assessed separately in humans. High exercise capacity has been suggested associated with improved mitochondrial function and mitochondrial function may predict mortality. However, the effect of sepsis on cardiac and skeletal muscle mitochondrial function may depend on intrinsic exercise capacity.

Objectives: The objective of this study was to evaluate the influence of sepsis on mitochondrial function in both rats with high and low intrinsic exercise capacity. Methods: Sepsis was induced by intraperitoneal injection of human feces (peritoneal contamination and infection model-PCI) in rats with high (HCR) or low (LCR) intrinsic exercise capacity. Sepsis severity was assessed after 6 and 24 h. Cardiac, skeletal muscle and hepatic mitochondria were isolated after 24 h of sepsis.

Results: PCI resulted in severe sepsis in both rats with high and low exercise capacity. In HCR, sepsis severity score as well as body temperature was higher at 6 h post induction. Cardiac as well as hepatic citrate synthase activity was comparable in HCR and LCR with or without sepsis. In gastrocnemius, HCR presented with higher citrate synthase activity. This was not affected by sepsis. Maximal respiratory capacity was higher in cardiac interfibrillar mitochondria (100) (IFM) in HCR using glutamate as a substrate. Cardiac subsarcolemmal mitochondria (SSM) were not different between HCR and LCR.

No difference between HCR and LCR was found in hepatic as well as skeletal muscle mitochondrial function. Sepsis reduced respiratory capacity in cardiac IFM but had no effect on hepatic and skeletal muscle mitochondrial function. Furthermore cardiac SSM remained unaffected by sepsis as well. Mitochondrial ATP/O ratio as a measure of efficacy was not different between HCR and LCR and not affected by sepsis.

Our results indicate that isolated mitochondrial function and sepsis severity do not seem to be related in our model of high and low intrinsic exercise capacity. The results question a potential relationship between mitochondrial function and sepsis severity.

Infection 2019

Protecting the endothelium with hemoperfusion during sepsis (2), Büttner S (1), Spilok J (1), Ward R (2), Geiger H (1) (1) Medical Clinic III, Goethe University Hospital, Frankfurt/Main, Germany, (2) Exthera Medical, Martinez, CA 94553, USA Introduction: Sepsis is the systemic response to an uncontrolled infection. Sepsis can also be described as a ''severe endothelial dysfunction syndrome in response to intravascular and extravascular infections'' (NIH/NHLBI Sepsis Panel 2010), leading to multiple organ failure by disrupting the nurturing microcirculation. There are many mediators that contribute to endothelial dysfunction, so targeting only a single one is unlikely to improve sepsis outcomes. Three potent sepsis mediators significantly elevated in septic shock and believed to be significant contributors to endothelial dysfunction are Heparin-binding protein (HBP), Histone H4 and lipopolysaccharidebinding protein (LBP). HBP is a mediator with positive local, but disadvantageous systemic effects. After contact with the endothelium HBP is released by activated neutrophils, functioning inter alia as a chemoattractant and inducer of endothelial leakage for neutrophil extravasation. However, in sepsis patients HBP is an early indicator of developing circulatory failure and death. Histones are intracellular proteins that compact chromosomal DNA in the nuclei of eukaryotic cells. They are released upon cellular damage and contribute to initiation, amplification, and propagation of DIC. High histone levels can be found in sepsis, trauma, and pancreatitis. Histones are highly inflammatory and induce the release of several pro-inflammatory cytokines. They contribute to DIC by activating platelets and promoting thrombin generation. LBP is an acute phase protein that mediates the inflammatory effects of lipopolysaccharides (LPS) by Toll-like receptor 4 activation. LBP is necessary for inducing inflammation on human glomerular endothelial cells, and reduction of plasma LBP levels has been shown to prevent endothelial and tubular dysfunction in animal models (Castellano et al. 2014 Results: Results showed a very rapid and significant reduction of HBP, Histone H4 and LBP by the heparinized beads. For HBP, reductions of 99.9%, 89.9%, and 50% for heparinized beads, hydrophilic control beads, and cationic control beads, respectively. For Histone H4, reductions of 98% and 17% were observed for heparinized and hydrophilic control beads, respectively. LBP was cleared at 100% (heparinized beads) and 0% (untreated beads). The hydrophilic control beads are slightly electronegative, which may explain the binding to this surface, however the hydrophilic beads are not sufficiently blood compatible, whereas the heparin-functional beads are antithrombogenic.

The endothelial function is primarily regulated through the glycocalyx as it mediates adhesion of platelets and leukocytes, hemostasis, and vascular barrier functions. Therefore, shedding of glycocalyx is the first step of endothelial disfunction, and is caused by many mediators. Important examples include HBP, histone H4 and LBP; they are considered to be therapeutic targets. In this study, it was found that the heparin-functional adsorption media used in Seraph filters demonstrated high affinity for HBP, Histone H4 and LBP with rapid binding kinetics. Previous studies with a broad spectrum of viral and bacterial pathogens, which includes animal and in vitro results, have shown the ability of Seraph 100 to rapidly reduce the pathogen load from the bloodstream. The ability to rapidly reduce both sepsis mediators and pathogens should provide a much-needed tool to prevent and/or treat sepsis.

Infection 2019

The role of mTOR1-dependent metabolic adaption for macrophage function Introduction: The liver plays a central role in the host immune response towards microbial infections. Accordingly, this organ is highly prone to sepsis-induced inflammation and liver dysfunction. The liver harbors high macrophage counts, comprising 80% of the body's macrophages. In order to prevent liver damage as a result of an exaggerated inflammatory response, the activation and transformation of monocytes to macrophages underlies a strict regulatory mechanism. Upon infection, monocytes/macrophages undergo a metabolic reprogramming regulated by one of the most important metabolic regulators, mTORC1 (mechanistic target of rapamycin complex 1). The serine/threonine protein kinase mTOR is a crucial regulator of cellular metabolism, growth and survival in response to hormones, growth factors and nutrients. Nevertheless, the underlying mechanism of mTOR signaling in regulating macrophage activation is not fully understood.

Objectives: This study focuses on mTORC1-induced metabolic reprogramming of macrophage activity during inflammation and liver failure as well as the effect of an overactive mTORC1 signaling in the same context. Methods: Modified monocytes exhibiting a high mTORC1 activity will be analyzed for their beneficial capacity in a liver organoid fluidic chip model. For this purpose, HepaRG and HUVEC cells were seeded on a membrane that functions as a cell culture substrate. Monocytes were isolated from a human donor and subsequently electroporated for a siRNA-mediated knockdown of the vital negative regulator of mTOR, TSC1/TSC2 (Tuberous Sclerosis Complex). The mutated monocytes were seeded on the chip and cultured for 10 days. The cells on the membrane of the chips were fixed and immunostained for microscopic analyses. Furthermore the cytokine profile as well as clinical parameters were examined from cell culture medium supernatants. Additionally, pharmacological approaches were performed by using inhibitors (Rapamycin and Torin 1) and an activator (MHY145) of mTOR in the chip model. Results: Pharmacological inhibition of mTOR with Rapamycin and Torin 1 resulted in damage to the vascular layer in the liver chip organoid under LPS (lipopolysaccharide) challenge. Expression of the vascular endothelial cell marker VE-cadherin was not detectable 72 h after stimulation with LPS. Inhibition of mTOR further caused elevated levels of proinflammatory interleukines (IL-1b, IL-18 and IL-6). In case of the anti-inflammatory cytokine IL-10, a significant decrease was detected after measuring the cytokine expression from the cell supernatant. Upon stimulation with the mTOR activator MHY1485, no damage of the vascular layer was detected and VEcadherin was present even after LPS stimulation. Moreover, improvement of the hepatic function, as assessed by expression of the transporter protein MRP2 (Multidrug resistance-associated protein 2), was detected.

Conclusions: Inhibition and activation of mTOR vastly influenced the integrity of the vascular and hepatic layer in the organoid model. Expression of proinflammatory cytokines was highly increased whereas secretion of anti-inflammatory cytokines was reduced following mTOR inhibition with Rapamycin/Torin 1. This indicates that mTOR signaling crucially affects the monocyte/macrophages-dependent inflammatory response, which might represent an interesting target for re-calibrating the dysregulated immune response in sepsis. Introduction: Organ damage and mortality in sepsis have been attributed to deleterious and inappropriate host response during infection. Over the course of sepsis a systemic hyper inflammatory response as well as innate immunosuppression may occur. Activation of the innate immune system is a key host defense strategy for clearance of the bacteria. The innate immune system recognizes pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs) through a set of germline-encoded proteins called pattern recognition receptors (PRR). Apart from surface PRRs, a wide array of microbial structures like lipids and nucleic acids also activate the cytosolic inflammasome signaling cascade. Depending on the ligand, inflammasome activation can occur through either the canonical and non-canonical inflammasome activation pathways. Gram-positive bacteria such as Staphylococcus aureus (SA) and Group B Streptococcus (GBS) are the main causal organisms of adult and neonatal sepsis. Molecular mechanisms which activate inflammasome pathways during Gram-positive bacterial infection and sepsis is poorly understood. Objectives: In the pathogenesis of sepsis, how Gram-positive bacterial PAMPs activate the immune system is a major question. Having recently discovered outer membrane vesicles as mediators of inflammasome activation, we are currently analyzing pathways activated by extracellular vesicles derived from SA and GBS. Methods: Re-addressing the formation and activation of the inflammasome pathway induced by bacterial extracellular vesicles. We used an array of interdisciplinary techniques including electron microscopy combined with state-of-the-art CRISPR/Cas genome editing technique and label free vibrational spectroscopic methods to study Grampositive bacterial recognition by human macrophages. Results: We carried out a comprehensive structural and biochemical analysis of extracellular vesicles isolated from Gram-positive bacteria. We found that a subset of extracellular vesicles enriched with lipids encoded by a pathogenicity island were potent inducers of canonical inflammasome pathway. Further characterization of the different biomolecules of these extracellular vesicles has allowed us to decipher a novel pathway by which SA and GBS stimulate human macrophages. (1), Bohm P (7), Godmann M (7), Grinenko T (4), Beretta M (1), Stunnenberg HG (2), Joosten LAB (2), Netea MG (2), Bauer M (1,6) and Weis S (1, 6, 8) Signalling pathway analysis was performed by the use of specific inhibitors and Western Blot analysis. In addition, in vivo analysis with mice were performed assessing disease severity, survival as well as immune-cell and hematopoietic stem cells composition by flow cytometer.

Results: We provide experimental evidence derived from primary murine and human myeloid cells that specific but not all DAMPs are potent inducers of trained immunity. The histone and RNA analysis revealed that, when compared to b-glucan, a strong inducer of trained immunity, there is a common set of genes induced by both stimuli. Potentially, these genes, mostly involved in metabolism, lysosomes and phagocytosis, are necessary for trained immunity. In vivo DAMP training leads to an increase in the myeloid progenitor cells, on the basis of the self-renewing hematopoietic stem cells as well as on the direct myeloid progenitor cells in the bone marrow. In response to LPS shock DAMP trained mice have a pronounced disease severity as assessed by the clinical severity score, a greater loss of temperature and a higher mortality rate compared to untreated animals. This goes along with increased amounts of inflammatory monocytes and neutrophils in the peritoneum. Mice lacking mature cells of the adaptive immune system still show the same effect.

Conclusions: In conclusion, we provide evidence that specific DAMPs can induce trained immunity in human and murine primary macrophages. In vivo DAMP induced trained immunity is deleterious upon secondary LPS application. We reveal distinct pathways and molecular mechanisms underlying trained immunity. This phenomenon is a long lasting phenomenon as it primes hematopoietic stem cells towards myeloid cells in vivo.

References: Larsen, R., Gouveia, Z., Soares, M.P., and Gozzelino, R. Introduction: Metabolic adaptation has been described as the ability to prevent metabolic dysfunction and damage imposed to tissues in face of excessive inflammation during infections. Adaptation to hosts imposed stress can be achieved through conserved metabolic mechanisms (1) (2) (3) (4) . Systemic infections, concur with host maladaptive stress and damage responses, resulting in insufficient adaptation and failure to restore tissue homeostasis, ultimately leading to disease (3, 4) . Expression of the stress-inducible Heme oxygenase (HO)-1, encoded by the HMOX1 gene, has been shown to lessen disease severity in various inflammatory disease including sepsis (5-8) which has been attributed to the removal of the pro-oxidant labile heme preventing cellular damage. Moreover, in humans, polymorphisms in the promoter of HMOX1 could be related to disease severity and outcome in bacterial sepsis and malaria (8, 9) . Previous work using a constitutive Hmox1 knock out animal model, as shown that HO-1 affords disease tolerance against a polymicrobial infection (7). However, embryonic deficiency in HO-1 is partially lethal, which leads to a reduced amount of successful Hmox1 constitutively deleted mice pups (9, 10). Successfully deleted Hmox1 animals, were reported to show progressive chronic inflammatory disease characterized by enlarged spleens and hepatic inflammatory lesions, making them an unsuitable model to study metabolic adaptation (10). To bypass this obstacle, a novel animal model ROSA26Cre-ERT2 Hmox1lox/lox, was developed by Miguel Soares, IGC, Portugal. Changing the deletion type from constitutive to conditional, allowed to avoid the lack of HO-1, during embryonic development. Deleting Hmox1 after birth prevented the progressive chronic inflammation observed in the Hmox1 constitutive knock-out model. Objectives: We here assessed the hypothesis that conditional deletion of Hmox1 impairs tissue damage control to pathogen and danger associated molecular patterns and affects the outcome in S. aureus bacteremia.

Methods: Hmox1 conditional knock-out animals were infected with the hemolytic 6850 wt S. aureus strain, treated with different dosages of lipopolysaccharides (LPS), and pathophysiological relevant levels of heme to simulate a highly hemolytic intravascular milieu. Targeted metabolomics were assessed in kidney and liver.

Results: Here, we provide evidence that expression of Hmox1 regulates metabolic adaptation, and stress resistance in response to inflammatory stress induced by heme. Hmox1 conditional knock-out animals exposed to heme, rapidly succumbed due to multiorganfailure (predominantly kidney) that was associated with hypoglycemia, hyperkaliemia and disruption of thermoregulation. Targeted metabolomics analyzes for energy metabolism intermediates, of liver and kidneys, revealed a distinct metabolic response from ROSA26Cre-ERT2 Hmox1D/D, after 20 h of heme treatment. Noteworthy, serologic markers shown an evident increase in kidney damage markers, accompanied with elevated levels of potassium. These results reveal a maladaptive stress response in conditional delete Hmox1 mice after 20 h of heme treatment. Labile heme is a known strong damage inducer mainly through redox reactions. However, administration of N-acetylcysteine a potent antioxidant, did not affect the survival rate. Notwithstanding these results, absence of HO-1 was proven not detrimental during LPS induced endotoxic shock, nor with acute and chronic S. aureus infection. Introduction: We and others have recently provided experimental evidence that host defense mechanisms in sepsis rely on an appropriate metabolic adaptation of parenchymal tissues. While the state and activity of immune system can routinely be assessed during infection, measuring metabolic adaptation in parenchymal tissue is hardly possible due to the inaccessibility of the compartment. Therefore, the identification of surrogate parameters reflecting metabolic function are needed to allow to determine host defense mechanisms in sepsis allowing to monitor the disease course disease and ultimately to adapt treatments. Stable isotopologues of exhaled carbon dioxide (CO 2 ) such as 13 C have been shown to be differentially metabolized in tissues under stress, including sepsis in animal models. Its applicability in humans and its association with the host defense response are currently unknown. Objectives: We assessed whether the CO 2 isotopologues in exhaled breath are affected by experimental human endotoxemia and can be used as a surrogate parameter for the host inflammatory response in humans.

Methods: In order to assess feasibility to assess breath-exhaled CO 2 isotopologues during systemic inflammation, we used the established experimental human endotoxemia model. Breath samples were collected from volunteers who were intravenously challenged with 1 ng/ kg LPS twice, with an interval of 1 week. Exhaled breath was collected at different time points before and after LPS administration. The VTT breath isotope analyzer was used to determine the 13 C/ 12 C and 18 O/ 16 O in exhaled CO 2 .

Results: LPS administration induced a robust inflammatory response, reflected by the development of fever, flu-like symptoms, and neutrophilia as well as pronounced increases in plasma levels of pro and anti-inflammatory cytokines (peak TNFa, IL-6, and IL-10 concentrations of 571 ± 76, 243 ± 43, and 329 ± 117 pg/mL, respectively). Breath analysis of exhaled CO 2 isotopologues was successful in all volunteers at all time points. The breath signal was stably detected in all measurements. Rations of 13 C/ 12 C doubled after the first LPS challenge compared to control levels. The inflammatory response elicited by the 2nd LPS challenge was significantly attenuated, for instance exemplified by a 35 ± 11%, 45 ± 9%, and 56 ± 11% decrease in plasma concentrations of TNFa, IL-6, and IL-10 respectively, indicative of endotoxin tolerance. However, a significant higher 13 C/ 12 C ratio was induced by the 2nd LPS challenge compared to the first challenge. Conclusions: We provide evidence that the ratio of 13 C/ 12 C of CO 2 isotopologues can be determined in human volunteers subjected to experimental endotoxemia and that systemic inflammation induced by a bacterial PAMP alters isotopologue composition. This might provide an important measurement tool via which we can assess inflammatory responses in sepsis patients non-invasively. Further studies in animals and humans are currently being performed to substantiate these findings.

Infection 2019

The role of sphingosine in host defense against fungal pathogens Methods: Sphingolipid levels of frequently and non-frequently exposed tissues of C57BL/6 wild type mice were determined using LC-MS/MS. The effect of Sph on yeast cell growth and hyphal formation of the Candida albicans (C. albicans) clinical isolate SC5314 was tested in vitro. Candida albicans SC5314 yeast cells were incubated in RPMI media at 37°C for 2-6 h or in YPD medium at 30°C for 21 h with Sph concentrations of 3-5 lM. Sphingosine uptake and metabolism was determined using LC-MS/MS. To check for a potential target of hyphal formation inhibition a PepTag nonradioactive PKC assay was used. An activity inhibition of C. albicans Pkc1 by Sph was determined using yeast protein extracts. An intestinal epithelial cell C. albicans infection model was used to check for beneficial effects of Sph treatment. SC5314 yeast cells were preincubated with 5 lM Sph or 3 lM FTY720 (Sph synthetic analog). Differentiated polarized C2BBe1 cells were then infected with preincubated or non-treated SC5314 yeast cells. Membrane integrity was determined after 4 to 22 h using TEER measurement and Western blotting on junctional proteins. LC-MS/MS was used to determine Sph and FTY720 levels in supernatants to exclude an S1P mediated effect on membrane barrier. Results: On the host site, in C57BL/6 mice, highest Sph concentrations were found in common pathogen exposed tissues like lung or kidney. On the fungal site, we found in vitro that Sph and and its synthetic analog FTY720 inhibit hyphal formation in Candida albicans in a concentration depend manner, while yeast cell growth was not affected. External Sph was taken up by yeast cells and phosphorylated to S1P. Preliminary results showed reduced activity of a fungal protein extract incubated with increasing concentrations of Sph or FTY720. Infection of polarized C2BBe1 intestinal epithelial cells with Candida albicans cells that were preincubated with Sph or FTY720 led to postponed and decreased barrier breakdown determined by transepithelial electrical resistance measurement and junctional protein expression levels.

Conclusions: Sphingosine can be used to inhibit hyphal formation in C. albicans, on of its most important virulence factors. We hypothesize that the underlying mechanism of filamentation inhibition is mediated by the protein kinase C-like 1 (Pkc1). Preliminary results support the hypothesis. Pkc1 is involved in cellular integrity and drug resistance and plays a crucial role in C. albicans morphogenesis under serum conditions involving the GTPase-activating protein Lrg1 and the small GTPase Rho1. In mammals, Sph inhibits PKC. Our principal aim is to investigate the interaction of Pkc1 and Sph in C. albicans and to find new strategies to treat life-threatening fungal infections by manipulating the SL metabolism in host cells and by identifying new target molecules for sphingolipid intervention in fungi.

Infection 2019

Regulation of non-canonical inflammasome during sepsis induced innate immunosuppression

Ghait M (1), Duduskar SN (1) Introduction: Cellular adaptations to innate immune signaling may have implications during sepsis-related organ damage. In some patients with sepsis, a state of organ-damage-associated immunosuppression occurs. Immunosuppression is an altered immune response to a subsequent pathogen challenge and hence that characterized by reduced inflammatory cytokine production, secondary infection, an increased risk of organ failure and mortality. Lipopolysaccharide tolerance is a form of innate immunosuppression, which recapitulates several key features in some sepsis patients.

Recently, we demonstrated regulatory miRNAs namely miR-221 and miR-222, that are upregulated during innate immunosuppression and were positively correlated with organ dysfunction. miR-221 and miR-222 regulate innate immunosuppression by transcriptional silencing of the chromatin remodeling transcription factor known as BRG1 and thereby down-regulates a subset of pro-inflammatory genes. Although during innate immunosuppression cell surface signaling is attenuated, the host cell may remain equipped with distinct cytosolic receptors that defend against invading pathogens. Inflammasomes are cytosolic multi-protein complexes, which assemble in response to cytosolic PAMPs or endogenous DAMPs. The non-canonical inflammasome, which comprises inflammatory caspase-11 in mice and caspase-4 and caspase-5 in human, is activated in response to bacterial infection. Activation of non-canonical inflammatory caspases is essential for initiating pyroptotic cell death through activation of GSDMD and ultimately leads to release of intracellular cytokines. Regulation of inflammatory caspases and their overall function as cytosolic receptors during sepsis-associated immunosuppressive state remains poorly studied.

Objectives: We employed our recently discovered miR-222 expression profile as a surrogate to stratify patients with signs of innate immunosuppression and organ dysfunction. In this study we aim to investigate the mechanism of non-canonical inflammasome regulation during innate immunosuppression. Methods: PBMCs (cohort I n = 30 patients) and CD14? monocytes (cohort II n = 10) from patients with decompensation of cirrhosis and suspected bacterial infection were analyzed for miRNA-221 and miR-222 expression. Patients were stratified according to the presence of organ failure (ACLF). Correlation analysis of miRNA-222 expression with clinical scores including MELD score, Creatinine, CRP and WBCs counts was performed. For regulation and activation of noncanonical inflammasome genes CASP4, CASP5 and GSDMD, PBMCs (cohort I) and serum from septic patients without cirrhosis (cohort III n = 19) patients were subjected to analysis using qPCR, immunoblotting and ELISA. SOFA (-CLIF) scores were used for grading of organ dysfunction. miRNA-222 expression was used as a surrogate to stratify patients with sepsis-related immunosuppression. Results: Sepsis patients undergoing innate immune suppression (cohort I and II) showed elevated expression of miRNA-222. In these groups of patients we further studied regulation of non-canonical inflammasome activation and its association with organ damage. Noncanonical inflammasome genes including CASP4 and CASP5 were differentially regulated during sepsis related immunosuppression (cohort I). Moreover proteolytic activation of GSDMD (cohort III) underlines occurrence of inflammasome singling during sepsis organ damage. Our data provide important human specific information for non-canonical inflammasome.

Conclusions: Our data demonstrate highly regulated activation of the non-canonical inflammasomes in sepsis and hence further analysis of these molecular events may provide important insights into how noncanonical inflammasome is regulated in sepsis associated innate immunosuppression. Considering severity of the condition of septic and pre-septic patients, comprehensive examination of the functional state of the respiratory system is not adequate.

Objectives: The aim of the study is to find disorders of the functional state of the respiratory system in patients with sepsis. Methods: All the patients received the lessons of respiratory exercises in the complex of post-operative rehabilitation before the study. Seven patients with sepsis (I group), nine patients after planned surgery on uncomplicated hernias of the anterior abdominal wall or varicose veins on the lower limbs (II group) have been examined. The control group (III group) included 12 healthy and practically healthy volunteers. Anamnesis of all the patients was not complicated by bronchial-pulmonary pathology. Parameters were detected on 2-3 day after operation enabling reduction a direct influence of surgery on the functional state of the respiratory system. Examination and estimation of the respiratory function (RF) conducted with standard respiratory maneuvers on a portable computed spirograph.

Results: The vital capacity index sharply reduced in the II group of patients, but in sepsis it was triple as low as compared to the control group hardly reaching 38.2% out of the calculated standard index. FVC was twice less in group I compared to group II. The muscular apparatus tonus participating in respiration was sharply reduced, lung excursion decreased, permeability of the bronchial tree deteriorated on all the levels. pO 2 index at rest prevailed in the III group and II group as compared to the patients of the I group (p \ 0.05). With the dynamics of pCO 2 at rest a reliable difference was found in the group I (p \ 0.05) and III group (p \ 0.05) as compared to the biggest value in the II group. After oxygen inspiration the following pO 2 dynamics was observed: the result was higher in the III group as compared to the I one (p \ 0.05), without substantial differences in the II group. pCO 2 variation after oxygen is characterized by reliable differences in the II group concerning the I one (p 0.05). According to the level of pO 2 and pCO 2 gases in the arterial blood reliable arterial hypoxemia against mild hypocapnia occurs (adaptive respiratory alkalosis with metabolic acidosis due to alveolar hyperventilation against the ground of pronounced tachypnea). Dynamics of pO 2 and pCO 2 indices after hyperventilation test and respiration with 100% oxygen during 5 min are most likely indicative of prevailed disorders of ventilation-perfusion and metabolic correlations in genesis of hypoxemia, and respiratory alkalosis was not characterized by the tendency to normalization.

Conclusions: The found disorders of the functional state of the respiratory system in patients with sepsis are indicative of the necessity to correct medical treatment of such patients considering functional changes of the respiratory function and oxygen therapy inclusion into the therapeutic protocol of such patients as an early compulsory component.

Infection 2019 Impact of source of infection on frequency and severity of organ dysfunction in patients with severe sepsis and septic shock

Walter L (1), Kuhn SO (1), Vollmer M (2), Gibb S (1), Gründling M (1), Scheer C (1) (1) Department of Anesthesiology, University Medicine Greifswald, ) . In contrast, we observed significantly between-group-differences in SOFA-subscores and organ dysfunction at sepsis onset. Patients with pulmonary sepsis had higher respiratory scores and more respiratory organ dysfunction than other infection sites. Abdominal sepsis was associated with the highest cardiovascular and hepatic subscores. Patients with sepsis due to urogenital tract infection had higher renal scores in contrast to those in the abdominal and pulmonary group. Furthermore, renal dysfunction was most frequent in the urogenital group and rarest in the pulmonary group.

Conclusions: In patients who met severe sepsis or septic shock due to different sources of infection we observed comparable 28-day mortality. In contrast, frequency and severity of the single respiratory, cardiovascular, hepatic and renal organ dysfunction varied significantly between sepsis induced by different sources of infection.

Infection 2019

The role of aromatic microbial metabolites, inflammatory and autoimmune biomarkers in the development of neurological disorders in chronic critical ill patients in serum, which are associated with the severity of bacterial infection in critically ill patients [1] . It is also known that neurological diseases are accompanied by neuroinflammatory reactions in the brain and an increase in immune markers in the blood (2) . Objectives: The aim of our study was to identify AMM, inflammatory and autoimmune markers, biomarkers and to evaluate correlations of these markers in chronic critical ill (CCI) patients with neurological disorders. Methods: The study included 60 serum samples taken from 37 chronic critical ill (CCI) patients with neurological disorders (stroke, traumatic brain injury, neurosurgical intervention for brain tumors). Serum samples of 20 healthy donors were used as a control. The level of AMM was measured using GC-MS (Thermo Scientific), biomarkers (procalcitonin (PCT), S100) were measured by Elecsys immunoassay. The activity of enzymatic activity of leukocyte elastase (LE) and functional activity of a1-proteinase inhibitor (a1-PI) was determined by spectrophotometry, and the levels of autoantibodies to neuroantigens S100b and myelin basic protein (MBP) by ELISA.

Results: The median level of the sum of 8 aromatic metabolites (benzoic, phenylpropionic, phenyllactic, p-hydroxybenzoic (p-HBA), p-hydroxyphenilacetic (p-HPhAA), p-hydroxyphenylpropionic (p-HPhPA), homovanilic (HVA) and p-hydroxyphenillactic (p-HPhLA) acid) were 4.5 lM (significantly higher than in control (p \ 0.05)) against the background of reference values of high sensitive PCT that may indicate of chronic dysregulation of host-microbe interactions. This was accompanied by inflammatory and autoimmune markers abnormalities. The indirect Spearman's correlation between the level of PCT and LE (-0.48, p \ 0.05) was revealed. The correlations between other immune biomarkers and some microbial metabolites were also revealed ( Table 1) . Conclusions: Connection between aromatic microbial metabolites and examined biomarkers indicates the potential involvement of gut microbiota in the pathogenesis of neurological dysfunction in chronic critical ill (CCI) patients.

References : Introduction: Despite of antifungal prophylaxis liver transplant recipients are at increased risk of opportunistic invasive fungal infections (IFI) in the early course following liver transplantation (LTX). Early and reliable diagnosis is a prerequisite for successful treatment of IFIs and thereby for the outcome of these patients. However, culture-based routine diagnostics of IFIs are associated with relevant weaknesses, especially since they are time consuming and a relevant number of infections get missed. Therefore, plasmatic biomarkers for early and reliable IFI diagnosis following LTX would be desirable. In this context, Intercellular Adhesion Molecule (ICAM-1)-1 was shown to be suitable in septic patients, whereas Midregional-proAdrenomedullin (MR-proADM) proved to be a suitable tool for IFI diagnosis in septic and LTX patients as well.

Objectives: The aim of this study was therefore to evaluate the value of ICAM-1 (in combination with and without MR-proADM) for IFI diagnosis in patients following LTX. Methods: In an observational, prospective clinical study, 93 patients following LTX were screened for the emergence of IFIs by the use of culture-based as well as imaging diagnostics. In brief, Candida spp. in the respiratory tract or in fluids from drainages were classified as colonization. Positive results in blood cultures, intraoperative swabs and Aspergillus spp. in deep respiratory tract specimens with accompanying pulmonary infiltrates were classified as infection. In parallel, plasma samples were collected at seven consecutive time points within a 28-day observation period after LTX. Plasma concentrations of ICAM-1 and MR-proADM were measured using immunoassay-based methods as well as mass spectrometry.

Results: Compared to patients with fungal colonization or without any fungal findings, patients suffering from an IFI revealed significantly elevated plasma levels of ICAM-1 within the 28-day observation period following LTX. Accordingly, ICAM-1 proved to be a suitable tool for the identification of IFIs in patients following LTX (receiver-operating-characteristic (ROC)-analysis: e. Introduction: Progranulin (PGRN) is a pleiotropic growth factor and a multifaceted immune-regulatory molecule involved in the regulation of signaling pathways of host-defense during infection and inflammation. It is critical in innate immunity against bacteria and is known to target TLR4 which recognizes LPS (1-3). PGRN deficiency in experimental animals leads to increased vulnerability to LPS-induced septic shock and high mortality (1). Increased PGRN plasma levels have been described in in patients with sepsis (4). Objectives: To quantify the performance characteristics of PGRN plasma levels as a biomarker for sepsis in comparison to the established marker procalcitonin (PCT) and others, and to delineate molecular networks involving upregulated PGRN in sepsis. Methods: The diagnostic performance of PGRN was assessed by calculating Area Under Curve (AUC) and Receiver Operating Curves (ROC) for PGRN alone, as well as in comparison to PCT in 114 consecutively recruited patients with sepsis, who served as an initial exploratory sample. A further 127 independently recruited patients were included in a second confirmatory cohort (all fulfilling Sepsis-3 criteria). One hundred-eighty-two ICU patients with a sterile inflammatory reaction (SIRS) after cardiac surgery were studied to quantify the ability of PGRN to discriminate between SIRS and sepsis. In order to establish logic validity of the novel biomarker, we isolated miRNAs and mRNAs samples from blood cells of septic shock patients (n = 7) and healthy volunteers (n = 7) for deep sequencing (NGS). The resulting FASTQ files were processed by QIAGEN Bioinformatics software to obtain the differentiation profile Abstracts S29

after DESEq2 analysis (OmicSoft ArrayStudio) and for biological interpretation using Ingenuity Pathway Analysis (IPA). The NGS data were then used to identify the canonical gene network (targeted miRNA-mRNA network) involved in the early antimicrobial response of PGRN followed by RT-qPCR confirmation in an independent and larger sample (n = 39 for septic shock and n = 23 for controls). Introduction: Patients with a sepsis-induced Acute Respiratory Distress Syndrome (ARDS) are hallmarked by high mortality rates. However, a detailed analysis of patients pulmonary microbiome as well as its influence on the course of the disease has not been performed yet.

Objectives: The objectives of this study were therefore to examine changes in the pulmonary microbiome in ARDS patients and their influence on patients' outcome by the use of a Next Generation Sequencing (NGS)-based approach. Methods: In total, 30 patients in two groups were enrolled in the presented study: (1) patients with sepsis-associated ARDS (n = 15) and (2) patients undergoing esophageal resection (n = 15) representing the control group. In the ARDS group, blood samples were collected at ARDS onset as well as 5 days and 10 days afterwards. At the same timepoints, bronchoalveolar lavages (BAL) were performed in order to collect epithelial lining fluid for culture-based as well as NGS-based longitudinal analyses of the pulmonary microbiome. In the control group, epithelial lining fluids and blood samples were collected only once, immediately prior to the surgical procedure. Results: ARDS patients showed a significantly decreased a-diversity (p = 0.003) and an increased dominance (p = 0.005) of their pulmonary microbiome. The a-diversity negatively correlated with the length of ICU stay and the need for mechanical ventilation. In 42.9% of the ARDS patients, culture-based results were not concordant with the NGS-based results. Moreover, 5 culture-based analyses were even negative, although the NGS showed a bacterial colonization. Conclusions: As assessed by a NGS-based diagnostic approach, patients with a sepsis-associated ARDS reveal a significant dysbiosis of the pulmonary microbiome. Moreover, these changes correlate well with the clinical course of the disease. Therefore, a NGS-based diagnostic approach might be a promising tool for pathogen identification as well as therapy guidance in ARDS patients. Introduction: Sepsis is frequently associated with critical ill patients. Assessment of disease severity at the time of initial presentation could be helpful in the patient management. Procalcitonin (PCT) is commonly used in the diagnosis of sepsis. Presepsin (PSEP) has been shown to provide powerful prognostication in sepsis. Objectives: We thought to evaluate the validity of PCT and PSEP for the diagnosis of sepsis and the assessment of disease severity and outcome prediction in the setting of a cardio-vascular intensive care unit (ICU). Methods: 71 patients admitted to a cardio-vascular ICU were included. PCT and PSEP were determined at the time of initial presentation by using Elecsys BRAHMS PCT (Roche, Switzerland) and PATHFAST Presepsin (LSIM, Japan). Samples were obtained at the day of admission to ICU. The primary study endpoint was death during hospitalization. Results: 16 patients obtained a transfemoral implantation of a prostethic aortic valve (TAVI), who recovered rather quickly without complication and served as control group. Of the remaining group 27, 23 and 5 patients were assigned to sepsis, resuscitation after sudden cardiac death and pneumonia requiring assisted ventilation, respectively. The PCT values of the control group were found to be below the cutoff for bacterial infection of 0,5 pg/L (max 0.29 pg/L), whereas the corresponding PSEP values failed to comply. In 9 patients PSEP values were below the cutoff of 350 ng/L (max 346 ng/ L) but in 7 patients PSEP exceeded the cutoff (min 634 ng/L). 24 patients of the sepsis group exhibited PCT values above the established cutoff of 2 pg/L but 3 patients had values \ 2 pg/L (max 0.645 pg/L) although these patients were assigned to septic shock. PSEP values in all patients of the sepsis group exceeded the cutoff of 350 ng/L (min 1030 ng/L). 23 (41.8%) patients died and 29 (52.7%) needed dialysis. The majority of non-survivors occurred in the sepsis group 17 (63.0%) whereas only 6 (13.6%) where belonging to patients without sepsis. ROC analysis for sepsis revealed AUC values for PCT and PSEP of 0.806 and 0.923. AUC values for death in the sepsis group revealed AUC values of 0.706 (sens 76.5%, spec 70%, crit. 2337 ng/L) and 0.506 (sens 88.2%, spec 30%, crit. 3.06 pg/L) for PSEP and PCT, respectively. For prediction of need of dialysis for PCT and PSEP the ROC analysis revealed AUC values of 0.798 and 0.874. These findings showed that PCT and PSEP could complement each other in the diagnosis of sepsis and risk stratification in patients admitted to a cardio-vascular ICU. PCT could be used for the identification of sepsis at admission with high diagnostic validity, whereas PSEP is superior in prognostication and prediction of outcome in sepsis. Conclusions: Combination of PCT and PSEP provided a higher validity in identification and risk stratification of septic patients admitted to a cardio-vascular ICU. PSEP demonstrated strong relationship with disease severity and outcome. The PATHFAST system allows early determination of PSEP from whole blood in the ICU in addition to PCT and may improve the management.

Simultaneous assessment of D-dimer, presepsin and qSOFA in patients admitted with early sepsis to the emergency department Spanuth E (1), Engesser P (2) Introduction: Coagulation activation during sepsis can initiate multiple organ failure. D-dimer as a highly sensitive marker of coagulation activation may early indicate the presence of sepsis induced organ failure. Presepsin (PSEP) provide early prognostication in sepsis. The qSOFA score defined by the Third International Consensus Definitions for Sepsis and Septic Shock can be assessed at patient admission without time delay by laboratory tests. The qSOFA score has been shown to be associated with an increased probability of mortality and can be used for prognostication. Objectives: To compare qSOFA and the POC tests D-dimer and presepsin (PSEP) at admission for assessment of organ dysfunction and differentiation of complicated clinical causes or mortality risk. Methods: 109 Patients admitted with signs of sepsis were included. The qSOFA score was calculated from respiratory rate, GCS score and systolic blood pressure using the recommended thresholds. The DIC score was established according to the ISTH (International Society of Thrombosis and Haemostasis) guidelines. D-dimer, PSEP and NT-proBNP were determined using the PATHFAST TM platform from LSI Medience Corporation. Creatinine, CRP and lactate were measured using laboratory methods. Results: Discrimination between patients with uncomplicated sepsis (N = 49) and septic shock (N = 69) revealed AUC values of 0.657, 0.626, 0.613, 0,553, 0.762 and 0.884, 0.858 for CRP, lactate, NT-proBNP, qSOFA, PSEP and D-dimer, and 0.978 for the simultaneous assessment of qSOFA ? D-dimer ? PSEP, respectively. 19 patients died during hospitalization. AUC values of mortality prediction were 0.517, 0.605, 0.665, 0.708, 0748, 0.842 and 0.848, 0.876 for CRP, D-dimer, NT-proBNP, lactate, PSEP, qSOFA, and the simultaneous assessment of qSOFA ? D-dimer and qSOFA ? D-dimer ? PSEP, respectively. Comparison of the DIC score and D-dimer revealed higher discriminatory validity of D-dimer for prediction of death and septic shock. The AUC-values for DIC score and D-Dimer for prediction of death were 0.514 vs. 0.605 and for discrimination between sepsis and severe sepsis or septic shock 0.526 vs. 0.639, respectively. Using the threshold C 2 of qSOFA, C 0,5 mg/L of D-dimer and [ 500 ng/L of PSEP, qSOFA detected 18 non-survivors (95%) and 41 patients of the high-risk group (82%), D-dimer detected 17 non-survivors (89%) and 33 (66%) patients of the high-risk group (n = 50) and PSEP detected 18 non-survivors (95%) and 45 patients of the high-risk group (89%). qSOFA score C 2 and B 1 was estimated in 34 (31.2%) and 75 (68.8%) patients, from whom 27 (79.4%) and 35 (46.6%) were assigned for prediction of septic shock by using an algorithm of PSEP [ 1000 ng/L and D-dimer [ 0,5 mg/L, revealing finally 79.4% of patients with qSOFA C 2 for septic shock and 53.4% of patients with qSOFA B 1 for uncomplicated sepsis. Conclusions: The simultaneous assessment of qSOFA ? D-dimer or qSOFA ? D-dimer ? PSEP provided added value to assess the severity and early mortality risk in patients admitted with sepsis to the emergency department. Combining qSOFA and D-dimer or PSEP improved the validity significantly. These parameters could be determined already at admission without time delay as D-dimer and PSEP could be measured as POC assays in parallel in anticoagulated whole blood samples using the PATHFAST TM system within 17 min. Introduction: Influenza constitutes a major public health problem, occurring as seasonal epidemics and sporadic pandemics which result in significant morbidity and mortality worldwide. Influenza virus infection itself as well as the secondary bacterial infection-a common complication during influenza infection-often lead to severe pneumonia, organ failure, and sepsis. Typical bacterial species isolated from patients with secondary infections are common colonizers of the nasopharynx, such as Staphylococcus aureus and Streptococcus pneumoniae. However, primary influenza virus-mediated sepsis might occur as well. Nosocomial acquisition of seasonal influenza constitutes a serious risk among patients with underlying illnesses, the elderly and neonates. In infected adults, the highest viral replication and probable spread occur within the first 3-5 days of illness. Thus, the Robert Koch Institute recommends an isolation period for influenza patients of 7 days. However, immunocompromised persons and young children show longer periods of viral spread, exceeding the recommended isolation period. So far, diagnostic tests available for detection of influenza viruses in respiratory specimens include molecular assays, such as RT-qPCR and other nucleic acid amplification tests that are fast and reliable but provide limited information on the infectivity of the patient, as they do not measure the ability of the virus to replicate. Antigen detection assays often showed poor performance and are rarely used in clinical practice. In consequence, a rapid system to identify infectious virus particles is required since the existing tests for infectivity are time and cost intensive. Objectives: The influenza A virus genome consists of segmented single-stranded RNA (vRNA) with negative orientation. The eight segments are not only transcribed into messenger RNA (mRNA) for protein synthesis but also used to generate a copy RNA (cRNA) which serves as a template for the viral reproduction. In consequence, three different virus-related RNAs are present within an infected cell. Based on strandspecific real-time RT-qPCR we aim to develop a rapid method to detect infectious influenza virus particles by the analysis of the three types of influenza viral RNA (vRNA, cRNA and mRNA). Methods: To achieve this goal, several approaches are performed using laboratory strains as well as patient samples which have been collected from influenza A positively diagnosed patients at day one, two, four and six upon hospitalisation. We analyse the levels of viral RNA via RT-qPCR as well as the number of infected cells by FACSanalysis upon infection of MDCK-cells. Furthermore, we aim to directly measure levels of the different viral RNAs (vRNA, cRNA and mRNA) by strand-specific RT-qPCR in patient samples. The results of these experiments are then correlated with the viral titres determined by a standard plaque assay. Results: Our findings indicate that we were able to detect different viral RNAs from laboratory strains and patient samples upon propagation in cell culture. Furthermore, we were able to monitor infected cells by FACS analysis. Different viral RNAs, as well as the number of infected cells, correlate to viral titres that were determined by standard plaque assay. While the detection of viral nucleic acid from patient samples by RT-PCR is successful, specific measurement of vRNA, cRNA and mRNA has to be improved. Conclusions: Both FACS-analysis and RT-qPCR showed promising results after infection of MDCK-cells independent of the samples used. Nonetheless, further tests are needed to optimise infection procedures and determination of detection limits. For direct analysis of vRNA, cRNA and mRNA derived from patient isolates further optimisation is required. Acknowledgement: Hagel, Giebeler and Deinhardt-Emmer contributed equally, Center for Sepsis Control and Care.

Microarray gene expression analysis using artificial intelligence: a valuable approach for precise sepsis diagnosis Schaack D (1), Brenner T (1), Weigand MA (1), Uhle F (1) Department of Anesthesiology, Heidelberg University Hospital, Im Neuenheimer Feld 110, Heidelberg, Germany Introduction: Early and reliable diagnosis of sepsis remains a major challenge for the successful treatment of the highly complex syndrome. The blood of ICU patients is an essential source of vital diagnostic information and is used for both general and biomarkerbased laboratory analyses to support the process of clinical decisionmaking. The second domain of assessable information in blood samples is contained within the transcriptomic data of comprising immune cells. Measurement and evaluation of global gene expression is usually achieved by differential analysis of DNA microarrays. Instead of conventional statistical solutions, we here propose a new approach based on artificial intelligence which offers new perspectives in the field of sepsis diagnosis. Objectives: A pilot study for large-scale assessment of publicly available sepsis microarray data was performed using deep-learning artificial neural networks to prove their utility and superiority for precise sepsis diagnosis. Methods: NCBI GEO and EBML-EBI Array Express databases were searched for distributed data series of patients with sepsis (n = 1354), trauma patients (n = 478), and healthy controls (n = 383) and were combined in an encompassing meta-dataset. Only data series using microarray technology to analyze whole blood transcriptome data on samples drawn within 24 h after ICU admission were included. After data preprocessing, 5932 common genes were identified between all data series and selected for subsequent analyses. Available samples were randomly split into training (65%), validation (20%) and testing (15%) subsets. Samples from training and validation subsets were used to iteratively adapt deep-learning neural networks to the objective of binary classification either as ''Sepsis'' or ''Non-sepsis'' (trauma patients and healthy controls). After the training was finished, the classification accuracy for the reserved testing samples was assessed. To test the resilience, stratified ShuffleSplit cross-validation was implemented effectively, repeating the steps of random data sampling, network adaptation and binary classification for 250 times. Conclusions: Our pilot study demonstrates the superior performance of artificial intelligence algorithms applied to high-dimensional gene expression data in a use case of sepsis diagnosis. Even when information is largely reduced, the analytical performance remains robust, thereby underlining the possibility of application within emergency departments and ICU in the future, aiming to overcome the limitations of current biomarkers.

Introduction: The role of macrolide/beta-lactam combination therapy in community-acquired pneumonia (CAP) of moderate severity is a matter of debate. Macrolides expand the coverage to atypical pathogens and attenuate pulmonary inflammation, but have been associated with cardiovascular toxicity and drug interactions. Objectives: We developed a decision tree based on etiological and clinical parameters, which are available ex ante to support a personalized decision pro or con macrolides for the best clinical outcome of the individual patient. Methods: We employed machine learning in a cross-validation scheme based on a well balanced selection of 5646 patients after propensity score matching to data available on admission of 6440 hospitalized patients with moderate severity (non-ICU patients) from the observational, prospective, multinational CAPNETZ study. We aimed to improve the primary outcome of 180 days survival. Results: We found a simple decision tree of patient characteristics comprising chronic cardiovascular and chronic respiratory co-morbidities as well as leukocyte counts in the respiratory secretion at enrolment. This decision tree reduced the death rate considerably (OR = 1.82, CI [1.49, 2.23] , P \ 0.0001) of patients treated in compliance with our treatment suggestion compared to the observed standard of care. Conclusions: Stratifying macrolide treatment in patients following a simple treatment rule may lead to considerably reduced mortality in community-acquired pneumonia. A future randomized controlled trial confirming our result is necessary before implementing this rule into the clinical routine.

Rapid antimicrobial susceptibility testing (AST) using Raman spectroscopy combined with a multiwell-dielectrophoresis chip Kirchhoff J (1, 2, 3, 4) , Grohs R (1) Introduction: Over the past years multi-resistant pathogens have become an acute healthcare problem worldwide, which also critically affects sepsis patients. In order to initiate appropriate antibiotic therapy, the pathogen must be identified and its antibiotic sensitivities characterized. Due to several cultivation steps classical microbiological diagnosis takes up to several days from patient sample to result. Faster antimicrobial susceptibility testing (AST) is urgently needed to avoid both, inappropriate undertreatment resulting in increased mortality in sepsis patients and the unnecessary use of broad-spectrum antibiotics fostering further spread of multi-resistant bacteria. We develop a rapid cultivation-free procedure for isolation of the pathogen directly from patient's body fluid, followed by spectroscopic identification and subsequent AST. The whole procedure from urine sample to AST result takes as little as 3.5 h [1] . Here, we present a simple and fast on-chip spectroscopic technique to identify antibiotic susceptibilities and furthermore, to determine the minimal inhibitory concentration (MIC) of bacterial isolates within 2 h. Objectives: Our aim is the development of a parallelized and automated procedure based on Raman spectroscopy in combination with a multiwell-dielectrophoresis (DEP) chip for identification and AST of sepsis pathogens within a few hours. Methods: Raman spectroscopy is a nondestructive and label-free method for analysis of chemical composition, enabling measurement of biological samples in aqueous environment. A laser is irradiated on the specimen and the inelastically scattered light yields its vibrational fingerprint, which allows identification of the pathogen and comprises information of its phenotypic behavior [1, 2] . To concentrate the bacteria from patient's bodily fluids, e.g. urine, DEP is used. In an alternating inhomogeneous electric field, dielectric forces capture particles along the field lines. Utilizing a quadrupole electrode design, bacteria can be accumulated in the middle of the electrodes for direct spectroscopic characterization [3] . The novel multiwell-DEP-chip allows parallelized analysis of 20 individual conditions of bacteria-drug combinations. The test panel consists of four clinically relevant antibiotics with four concentrations permitting quantitative AST results within 2 h [4] . Results: When treated with antibiotics, spectral changes in susceptible bacteria can be observed, whereas resistant bacteria keep on growing and generate normal spectral growth signatures. The comparison of sample spectra to a database yields species identification. Principal component analysis defined spectral marker bands indicating effective antibiotic treatment. The spectroscopic approach was adapted to determine the MIC after only 90 min interaction time. The AST method using the novel multiwell-DEP-chip is demonstrated with gram-negative urosepsis pathogens, namely Klebsiella pneumoniae, Proteus mirabilis and Escherichia coli. Conclusions: We show how Raman spectroscopy can be utilized to visualize the effect of antibiotics with different mechanisms of action on sepsis pathogens with various antibiotic susceptibility patterns. Only minimal sample preparation is required and short-time cultivation of 90 min is sufficient. The combination of DEP for concentrating bacteria and Raman-spectroscopy for fast and non-destructive characterization has the potential to become a fast and reliable diagnostic tool. References: [1] Cell cycle biomarkers and soluble urokinase-type plasminogen activator receptor for the prediction of septic acute kidney injury courses requiring renal replacement therapy Nusshag C (1), Rupp C (2), Schmitt FCF (2), Krautkrämer E (1), Speer C (1) (1). In the absence of kidney-specific therapeutic alternatives, renal replacement therapy (RRT) is often the final therapeutic option. Whether and when to start RRT remains an ongoing controversy (2) . A major issue that persists is the early differentiation of patients with progressive AKI and need for RRT from those with autonomous renal recovery (3, 4) . Surrogate parameters of glomerular filtration, however, are only vague indicators of the true damage to the kidneys and thus inefficient for renal outcome prediction (2) . Objectives: We hypothesized, that the product of the two cell cycle arrest and tubular injury biomarkers tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 ( . If adjunctive therapy in this time was applied, application of hydrocortisone and/or a second catecholamine/vasopressor was analyzed as well. All data were documented in a matrix (Fig. 1) .

Results: Of the 425 patients analysed, 167 received additional CytoSorb treatment and 259 received standard therapy. Both groups were comparable in regard to age, sex, weight, and initial SAPS and APACHE II score. Demographics, clinically relevant variables and statistics are depicted in Table 1 . Analysis revealed that septic shock was typically represented by a maximum score of 5 points, while 6 or more points indicated a situation refractory to standard therapy with the worst outcome in patients [ 8 points. The differences in mortality between score groups were significant (Fig. 2) . Analysis however showed a significant survival advantage in those patients where CytoSorb therapy was started early ( Table 2 , Fig. 3 ). Only in Cyto-Sorb treated patients with a score below 6 and a late start of cytokine adsorption (mean 52.6 h), mortality was higher than initially expected, explained by a significantly higher rate of acute renal failure (100% vs. 13.3% in control group) (Fig. 2) . Among the overall patient population, a total of 184 patients (43.3%) had pneumonia with 66 (35.9%) of those receiving CytoSorb treatment (Table 3 , Fig. 4) .

Interestingly, comparing the subgroups with 6-8 points (Table 4) , those patients with an early start of CytoSorb therapy (n = 30) showed a clear benefit in hospital survival compared to patients receiving standard therapy (n = 63) (56.7% vs. 41%) (Fig. 5) .

Conclusions: In this study we aimed to validate a scoring system that allowed the identification of patients with early refractory septic shock who benefit most from adjuvant CytoSorb hemoadsorption therapy. Applying this approach using four clinical and one laboratory parameter, we were able to detect three populations with significant S38 Abstracts distinct mortality characterized by different scores. Particularly pneumonia patients with early start of therapy showed a clear advantage when treated with cytokine adsorption. This easy-to-apply scoring system enables to track the early dynamic evolution of septic shock and might be a useful tool in the decision to indicate CytoSorb hemoadsorption regardless of kidney failure (1, 4, 9) , Bocklitz T (2,3), Popp J (1, 2, 3, 8, 9) , Neugebauer U (1, 2, 3, 8, 9) Introduction: Effective sepsis treatment relies on early diagnosis. As yet there is a lack of reliable and fast detection methods for sepsis aiding the treating physician to differentiate non-infectious and infectious stimuli as origin for inflammation and shock. Host specific immune response could be helpful for identifying infection as cause for organ dysfunction. Raman spectroscopy is an emerging method in the field of biomedical research with diagnostic potential. This technique provides information on the biochemical features of the cells in a non-destructive and label-free manner, which in turn enables discrimination of different cell types as well as their functional states. In Raman spectroscopy laser light gets scattered upon interacting with the biomolecules and the inelastically scattered light contains information about the molecular vibrations that are very specific to the molecule. Hence, complex information about the cell and its activation status is captured by the Raman spectra. Previously, Raman spectroscopy has been applied for leukocyte phenotyping [2, 3] and it has been demonstrated in an ex vivo study of splenocytes isolated from endotoxemic mice possibility to follow dynamic changes in the T-lymphocytes [4] . In the current work we have applied Raman spectroscopy as a new diagnostic platform for leukocytes screening to obtain infection-specific information and for sepsis detection.

Objectives:

The study aims at validating Raman spectroscopy signature of isolated blood leukocytes for fast, reliable and precise identification of sepsis patients and to stratify patients having infection or non-infectious shock.

Methods: In this observational study (HemoSpec, DRKS-ID: DRKS00006265) 54 patients, with pre-defined criteria of (a) inflammation: patients with no proven infection, undergone elective surgery, (b) infection: patients with clinically or microbiologically proven infection, (c) sepsis: patients with organ dysfunction in accordance to sepsis-3 definition were recruited. Peripheral EDTA blood (4.9 ml) was collected for routine clinical diagnostic (Blood hemogram), biomarkers (IL6, procalcitonin [PCT], C-reactive protein [CRP], suPAR) and for Raman spectroscopy. Blood leukocytes were isolated from 500 ll blood by removing erythrocytes by lysis method followed by chemical fixation with 4% formaldehyde. The cells were coated on CaF2 and measured immediately. Raman spectral data analysis was performed with aid of chemometric analysis methods: canonical powered partial least square (CPPLS) analysis and logit regression analysis to build Raman spectral based model for patient stratification.

Results: Raman spectroscopy allowed extracting phenotype information of leukocyte subtypes. The logit regressions of the Raman spectroscopic data shows good separation between patients having infection (AUC = 0.83) and patients with sepsis (AUC = 0.82). The major spectral differences were observed in the vibrational peak of proteins and nucleic acid. To calculate the added value of Raman spectroscopy, advanced statistical analysis was performed by employing CPPLS, which allows including demographic data of the patients as additional responses. All the models were validated by leave-one-patient-out cross validation, that allows robust evaluation of models, similar to testing on an independent data set. The CPPLS model for Raman spectroscopic data allowed discrimination of sepsis patients from the two other groups with a mean sensitivity of 72% (AUC = 0.82) being equivalent to the mean sensitivity obtained by using the biomarker panel consisting of IL6, PCT, CRP and suPAR. The mean sensitivity for sepsis detection was increased to 91% (AUC = 0.99) by combining Raman responses and the biomarkers. There was no change in the sensitivity for sepsis detection by including the qSOFA values. Similarly, we also show presence of infection can be identified with mean sensitivity of 80% (AUC = 0.86) by using only Raman spectroscopic data and when combined with biomarkers, the sensitivity was increased to 99% (AUC = 0.97).

In this study we demonstrate diagnostic capability of the Raman spectroscopy and show the added value of Raman spectroscopy for fast, accurate and reliable detection of presence of infection and sepsis.

Conclusions: With the aid of chemometric methods, the Raman spectroscopy model allows identifying patients with reactive leukocytes leading to adverse immune responses manifesting as inappropriate responses, i.e. sepsis. Further, we show Raman spectroscopy brings in added value for qSOFA and biomarkers by increasing sensitivity for sepsis patient detection. Raman spectroscopy has high potential especially in a decentralized health care system for patients screening with suspected infection. Introduction: Sepsis, defined as a organ dysfunction due to severe infection (1), is one of the leading causes of mortality worldwide (2) . Early treatment, correct and rapid identification of offending microorganism and timely administration of appropriate antibiotics is of crucial importance in reducing mortality of sepsis. (3, 4) Blood culture is considered gold standard in invading microorganism identification in sepsis diagnosis. Nevertheless, the culture takes time and is not suitable for targeted antibiotic treatment in the early stages of management of septic patient. Molecular -biological methods based on polymerase chain reaction (PCR) testing could be a useful tool in improvement of appropriate and early microbiological work-up in septic patients, but they have certain limitations, namely costs and laboratory equipment. (5) Furthermore among many other septic biomarkers PCT is well established diagnostic tool to confirm or rule out suspected bacterial sepsis. But its interpretation needs to be put in the context with results of other tests, investigations and specific clinical situation. (6, 7) Objectives: The aim of ongoing study is to compare blood cultures with PCR assay for presence of microorganisms. Secondary aim is to evaluate levels of PCT at the time of clinical diagnosis of sepsis and 24 h after the first sampling considering results of blood cultures and PCR. Methods: In time of suspected or documented sepsis blood cultures were drawn. Simultaneously other blood samples were frozen for later PCR detection of infectious agent on the cooperating laboratory. PCT levels at the same time (PCT1), respectively after 24 h (PCT2) were examined. Results of blood cultures and PCR were combined and divided into 4 groups: AP (infectious agent positive), AN (infectious agent negative), AP G? (only grampositive infectious agent), AP G-(only gramnegative infectious agent). Mixed flora and fungal infection were not further evaluated. Basic statistic methods were applied to describe PCT levels in individual groups. Non parametric (Wilcoxon test and Mann-Whitney U test) statistic methods were used to assess difference between groups and cases in time.

Results: Out of 65 blood samples were 26 (39%) blood culture positive and 31 (47.7%) PCR positive. By combination of both methods we found 41 (63.1%) agent positive results. In 20 (30.8%) cases grampositive and 12 (18.5%) gramnegative were detected. Mixed flora or fungal pathogen was found in 9 (13.8%)subjects. In 34 (52.3%) cases corresponded the results of bloodcultures with PCR detection. In 1 (1.5%) sample partial congruence was documented. 15 (23.1%) samples was PCR positive and blood culture negative. 10 (15.4%) microbial agents was found in blood culture and not detected in PCR. One of them was fungi, which is undetectable by used PCR assay. 5 (7.7%) results was nonconcondart in infectious agent between both methods. PCT levels in individual groups are shown in Fig. 1 . The difference in procalcitonin levels in individual groups or in time was not statistically significant. Conclusions: PCR showed better detection ability than conventional blood cultures. In part of examined samples was proven infectious agent only in blood cultures. In some cases was nonconcordant positive results between both methods. We observed higher prokalcitonin levels at the time of suspected or documented sepsis and also 24 h after first sampling in group with detected gramnegative microorganism in blood stream. This difference was statistically not significant. Collection of new data continues. Objectives: Authors present a rare case of sepsis-like inflammatory response to accidental intravenous administration of the Hylak Forte germfree eubiotic preparation with evaluation of the kinetics of several biomarkers including myristic acid.

Methods: A 30-year-old woman with no significant medical history was admitted to the department of gynaecology with suspected infection from uterine myoma necrosis. She was treated by intravenous antibiotics. On admission her laboratory tests were unremarkable except for C reactive protein (CRP) of 300 mg/l. She had stable vital signs with no symptoms of systemic inflammatory response. On the second day the patient accidentally self-administered 2 ml of the Hylak Forte eubiotic preparation (Merckle GmbH) in her peripheral intravenous cannula. The patient developed septic/SIRS reaction shortly afterwards, she became febrile (39.6°C) with tachycardia (125 beats/min) and tachypnoea (22 breaths/min), her blood pressure remained normal. The treatment consisted of fluid resuscitation and corticosteroids, intravenous antibiotic therapy was continued. Remission of the clinical symptoms occurred within 12 h after the event. The patient had no signs of organ dysfunction and was discharged on day 5. During a period of 96 h following the accidental injection of Hylak, serum levels of CRP, PCT, interleukin 6 (IL-6), presepsin, copeptin and free myristic acid were evaluated. The myristic acid levels were determined by the gas chromatography/mass spectrometry (GC/MS) method.

Results: 23 min after the insult, we recorded peak level of myristic acid reaching a 10 times higher value (230.9 lmol/L) than the reference range, with gradual decrease in the 96 h to 26.6 lmol/L. The reference values were determined as the median of myristic acid levels measured in healthy subjects (18.9 lmol/L min 7.8 max 27.7 n = 66). Myristic acid was not detected in the eubiotic itself. All the biomarkers under evaluation showed dynamics typical of septic inflammatory response. Dynamics of all examined biomarker levels are shown in Fig. 1 .

We conclude that myristic acid may be a promising biomarker in the early identification of septic patients. It is currently being systematically evaluated (NCT03314831). Introduction: Hemoadsorption devices are used to treat septic shock by adsorbing inflammatory cytokines and as yet incompletely defined danger and pathogen associated molecular patterns. In an ideal case, hemoadsorption results in immediate recovery of microvascular endothelial cell function and rapid recovery from catecholamine-dependency [1] . Objectives: One reason for the functional deterioration of vital organs in septic shock is the loss of barrier function by endothelial cells. Out of a panel of innovative therapeutic options, blood filtrations are promising strategies. To verify their efficacy, bioassays using endothelial-cell integrity as a read-out parameter comprise a valuable experimental approach. Methods: Patients were treated with CytoSorb Ò according to the hemoadsorption SOP of our intensive care unit (ICU). The microvascular endothelial cell (mEC) bioassay was performed by a rat brain derived cell line, co-incubated with protein fractions recruited from hemadsorption devices used for treatment of septic shock. Impaired endothelial growth as well as impaired viability of mEC was documented by long-term video microscopy using Incu-CyteZOOMTM (sartorius.com). Protein fractions resulting in significantly impaired mEC viability were subjected to mass spectrometric analysis (Orbitrap, thermofisher.com).

Results: Protein fractions with mass spectroscopy and sorting of ligands relevant in a context of DANGER ligand induced inflammation, cell migration, and antimicrobial potential in patients suffering from septic shock. Fifteen proteins turned out to be central with either increased or decreased concentrations after CytoSorb Ò treatment. Out of those the protein diminished most frequently was Serum amyloid A 1 (SAA1), followed by Alpha-1-acid glycoprotein 1 (ORM1), adrenomodulin (ADM), selectively involved in the pathogenesis in septic shock leading to vasodilatation [2] . ADM plays a major role as a biomarker predicting outcome [3, 4] and guiding novel treatment approaches [5] . In addition to ADM, other antimicrobial peptides [6] (AMPs) such as cathelecidin derived LL-37 played a role in a subgroup of patients by its significant downregulation after treatment.

Conclusions: Mass spectroscopy is a valid approach to identify individual response patterns of hemoadsorption therapy in patients within septic shock. Introduction: Sepsis is a killer. While the majority of international research focus on the biomedical mechanisms of sepsis, with significant breakthroughs in recent years, epidemiological studies investigate population patterns that highlight import areas to include to the research agenda, such as high-risk groups, high risk diseases and high risk clinical settings. The actual functioning (or non-functioning) of health systems, however, remain unclear. Yet health system conditions (such as staffing, communication, information flow, etc.) and their influencing factors have a significant impact on earlier detection of sepsis and could thus contribute to the reduction of morbidity and mortality of sepsis worldwide.

Objectives: A pilot project within the CSCC aims to identify conditions and influencing factors for earlier sepsis detection and to develop interventions that could speed up the detection of sepsis.

Methods: The pilot applies an innovative social laboratory approach and uses systematically pooled group intelligence and group generated insights, using an incubator workshop methodology, to disrupt current thinking and create interventions for earlier detection.

Results: This presentation reports from the first of three workshops within this EARLIER detection project. Early indications point to the role of information sharing, multi-disciplinary communication and coordination at various levels of the health system. Conclusions: Participants of the first workshop developed two discrete proposals to accelerate primary and secondary care sepsis detection.

Infection 2019

Towards a blood culture-independent diagnostic tool for blood stream infections using Raman spectroscopy Introduction: Clinical routine identification of blood stream infections is still problematic due to low colony forming unit numbers per ml of blood or difficult to meet culture conditions of certain pathogens [1] . Thus, progress in this traditionally microbiological area is necessary. Advances in immunology research have shown that Raman spectroscopy is a powerful tool to detect immune cell activation [2] . Objectives: In this study we used a blood culture-and pathogen isolation-independent diagnostic approach to identify bacterial and fungal infection from non-infected neutrophils. Methods: Freshly isolated neutrophils were co-incubated with Staphylococcus aureus as representative for Gram-positive bacteria, Escherichia coli for Gram-negative bacteria or Candida albicans for fungi. By using a High-Throughput-Screening-Raman Spectroscopy (HTS-RS) system nearly 20,000 cells from three different donors were measured and the data was fed into a random forest classification model. Furthermore, 2D Raman scans were recorded using a commercial Raman imaging system. Results: Infected neutrophils were correctly distinguished from noninfected cells with 92% accuracy, while neutrophils challenged with bacteria or fungi were successfully predicted with 90% accuracy. Even bacteria species prediction, in S. aureus and E. coli, was 84% accurate. When the Raman scans were analyzed, phagocytized yeasts could be visualized in Raman false color images without the need for labeling or staining.

Conclusions: This proof-of-principle study shows that the Raman spectroscopic fingerprint of neutrophils carries the information about the activation state of the cell and furthermore, which pathogen species activated the cells.

Infection 2019

Organ deficiency development and early lethality in sepsis patients are linked to AQP5 promoter and intron polymorphisms

Pisarev VM (1), Chumachenko AG (1), Grigoryev EK (1,2,3), Karpun NA (2), Cherpakov R (4) Introduction: AQP5 belongs to the family of aquaporins (AQPs)membrane proteins responsible for selective transmembrane transport of water. Studies by Adamzik and colleagues have stressed the role the minor allele C of AQP5 1364A/C (rs3759129, mutation within the promoter region) in predicting increased 30-day survival in sepsis and acute respiratory distress syndrome (ARDS) patients and decreased leukocyte migration (1, 2) . In our previous studies, the role of another minor AQP5 mutation within the another region of gene, the intron of AQP5 gene (genotype AA, rs3736309) to mild course of ARDS has been established (3).

Objectives: The purpose of current study was to further clarify the possible contribution of both promoter and intron AQP5 mutations to heterogeneity of sepsis course and outcomes in Intensive Care Unit (ICU) patients. Methods: Sepsis and septic shock were defined in patients according to SEPSIS-3 (2016) consensus recommendations. Study groups (n = 231) included ICU patients with intra-abdominal sepsis (IAS, including pancreatitits, peritonitis, cholecystitis, appendicitis n = 72) and two ICU cohorts of sepsis patients with other sources of infections (n = 47 and n = 112). AQP5 polymorphisms were studied by analyzing PCR products in a 2% agarose gel using a AQP5 1364A/C and AQP5 2254A/G specific tetra primer sets for single nucleotide polymorphisms (SNP) rs3759129 and rs3736309, respectively. Data were analyzed by Kaplan-Meyer plot, Fisher test, Mann-Whitney test and Student t test when appropriate after determining the type distribution of variables odds ratios (OR) and P values were calculated using SigmaPlot 12.5 software (Jandel Scientific Software, CA, USA) and GraphPad InStat (GraphPad Software Inc., La Jolla, CA, USA). Results: Distribution of alleles A and G and genotypes AA, GA and GG of AQP5 2254A/G as well as alleles A and C and genotypes AA, CA and CC of AQP5 rs3736309 1364A/C in patients with sepsis versus control group of healthy volunteers did not differ. However, patients with non-intra-abdominal sepsis and AQP5 rs3736309 AG and GG genotypes (n = 112) exhibited enhanced maximum SOFA values and increased longevity of mechanical lung ventilation (MLV) during ICU stay compared to patients with alternative genotype AA (median values and 25-75 percentiles were: 13 (10.8-15) vs. 12 (7-14) for SOFA (P = 0.039), and 4 (1.8-11.8) vs. 2.3 (0.2-7.25) days for MLV(P = 0.024, Mann-Whitney test), respectively. In survived patients, the difference between AQP5 rs3736309 G gene carriers (GA ? GG) and AA homozygotic patients in maximum SOFA values remained significant: 9 (8.2-12.2) vs 4 (3-7.8), respectively (P = 0.002). Data demonstrate that allele G occurring with minor frequency in population compared to variant A associates with a more severe course of sepsis in non-ARDS and non-intraabdominal sepsis patients. No significant association of AQP5 intron mutation G with lethality was revealed in this study. Obtained data is complementary to our previous data demonstrating severe course of ARDS in AQP5 rs3736309 GA/GG patients (3, 4) . Further, our AQP5 rs3759129 promoter polymorphism study confirmed the trend for association of increased lethality and CC and CA genotypes of AQP5 1364A/C (rs3759129) variant. However, only patients with intra-abdominal sepsis and AQP5 CC or CA genotype, demonstrated significantly increased 30-day survival versus AA homozygotic patients (lethality: 20% versus 60%, respectively, n = 93, P0.05). Further studies revealed that the allelic variant C of polymorphic AQP5 1364A/C promoter site contributes to protection against increased SOFA: the median SOFA values were 10.5 (2.5-13) and 13 (3.7-16.2) for C? genotypes vs. AA genotype, respectively (n = 101 P = 0.039, Fisher test).

Conclusions: Polymorphism of intron region 2254A/G AQP5 rs3736309 affects development organ deficiency in sepsis whereas allelic variant C within promoter region of AQP5 1364A/C (rs3759129) protects against lethality The informative value of detecting the AQP5 CC or CA genotype for prognosis of 30-day survival versus AA homozygotic patients is increased only in abdominal sepsis patients.

Infection 2019

Gene-based signature predicts future severe course of disease in hospitalized patients with community acquired pneumonia . Mortality of CAP patients can be decreased, when high-risk patients are admitted to intensive care units. Conversely, late admission of CAP patients to ICU was associated with higher mortality rates (1, 2) . Recently, we could show that the Sequential Organ Failure Assessment score (SOFA) outperforms previously proposed clinical scores and laboratory parameters for discriminating currently present severe events (SEv), defined as a composite endpoint of either CAP-specific treatment on ICU or death within 28 days. Risk prediction tools which can be used to improve decision-making in treatment pathways (especially regarding admission to intensive care unit (ICU)) are urgently needed (3). Molecular predictors based on gene expression profiling from blood are a promising approach to address this. Current research successfully demonstrated the diagnostic value of transcriptome-based scores for discriminating between currently present CAP vs. non-CAP (4), CAP vs. hospital acquired pneumonia (5), or bacterial vs. viral infection (6). Objectives: We here aimed at identifying transcripts and pathways associated with present as well as with future SEv and to identify and characterize a transcript signature predicting future SEvs in patients with CAP. Methods: We assessed transcriptomic data from samples obtained within the PROGRESS cohort. Future SEv were defined as previously described (7). A total of 1247 samples was assayed. Raw data of 47,231 gene-expression probes were extracted by Illumina GenomeStudio without additional background correction. Data was further processed within R/Bioconductor. Expression values were log2-transformed and quantile-normalised. Batch effects of expression BeadChips were corrected using an empirical Bayes method (8) .

Finally analyzed samples comprised a total of 1189 gene expression measurements from 544 patients equally divided in a discovery and replication set.

Results: Using blood-based expression data from the PROGRESS study, we here identified transcripts and pathways associated with present as well as with future SEv. We find that after adjusting for current disease severity (i.e. present SOFA scores), effects of gene expression on present SEvs are not correlated with effects on future SEvs. We used our gene expression results to identify and successfully replicate a transcript signature for predicting future SEv. We observed good discrimination in the discovery cohort (AUCDiscovery = 0.82 (95% CI 0.71-0.91) and also in the replication cohort (AUCValidation = 0.81 (95% CI 0.70-0.90), superior to discrimination observed for other 14 clinical prognostic scores including the SOFA score. When combining the signature with the SOFA score, prediction could be significantly improved (p = 0.0002 and p = 0.03 for the discovery and validation, respectively). Conclusions: We suggest that clinical translation of this predictive signature has the potential to reduce CAP-mortality remarkably by improving clinical workflows (2), we tried to point out that such designs are no ''magic'' solutions for all the challenges. Objectives: We would like to initiate a more differentiated view of methodological, statistical and practical challenges related to adaptive designs.

Methods: Here, we consider four major components of adaptive designs: response adaptive randomisation (RAR), adaptive enrichment, seamless, and platform designs. For all components we display pros and cons. Results: Applying response adaptive randomisation (RAR), fewer patients are randomised to the trial arm with poorer observed outcomes (seemingly reduced efficiency). Thus, RAR is applied as a kind of futility monitoring which should be better addressed by planned interim monitoring without RAR. When applying RAR, one must define the decision rules for adaptation upfront of study initiation, monitor the randomisation weights to avoid instable estimates, account for time dependency of the outcome (if necessary) and has to rely on a short-term outcome. In particular, the latter have also been questioned in sepsis research. Adaptive enrichment designs are applied to identify patient (sub-) populations using biomarkers. One should distinguish between prognostic and predictive biomarkers even though some biomarkers are both prognostic and predictive (3). However, in clinical sepsis research very few biomarkers exist. Seamless designs allow moving from phase II to phase III without a recruitment stop while using all the information in a joint final analysis. In the application of seamless II/III designs, the optimal time point for transition and the adaptive changes (e.g. which arm(s) to drop) are crucial determinants of efficiency. In a platform trial, an IT infrastructure (overarching platform) is used to identify eligible patients across many trials in parallel to make trial conduct more efficient. For these designs, organisational and statistical issues introduced by the screening platform must be addressed already in the planning phase.

Conclusions: With respect to the major methodological challenges, costs and benefits must be carefully checked against bias that is introduced easily. Introduction: Early effective antimicrobial treatment is fundamental in sepsis therapy and should be initiated as soon as possible in order to reduce patient mortality. Yet, optimal drug exposure in sepsis and septic shock represents a key stumbling stone in the ICU due to a multitude of pathophysiological alterations that critically interfere with pharmacokinetic (PK) properties of b-lactams. PK-alterations and consecutively insufficient serum concentrations have been identified as a principle mechanism of treatment failure in patients receiving intermittent bolus applications of standard doses in the ICU. Customized drug-dosing regimen like the continuous infusion of (but not exclusively) b-lactams and the individual dose-application according to therapeutic drug monitoring (TDM) measures have been proposed as a reliable way to rapidly and effectively realize PK-target attainment in critically ill patients. Attainment of defined PK-targets has repeatedly been suggested to increase treatment success and reduce patient mortality.

Objectives: The present study focused PK-target attainment within 24 h of CI of PIP and the effect of consecutive TDM-guided doseadjustments on target-attainment. Secondary aims included evaluation of hospital and ICU-mortality depending on PK-targets. (Table 1) . Beyond the first 24 h, 449 TDMs were performed and accounted for major changes in target-attainment. While the fraction of low PIP serum concentration did not significantly change (\ 16 & 16-32 mg L -1 : 15.8%, n = 71/449), TDM-guided dose adjustment measures beyond day 1 managed to further increase the fraction of patients within the primary PK-target range by 62.4% (n = 280/449) with median PIP concentrations of 46 mg L -1 [16; 33-64]. Overall, TDM-guided dose adjustment accounted for an 82% increase of PIP target attainment compared to the first 24 h of treatment. At the same time, TDM-guided measures reduced moderately high PIP serum concentrations by 63.4% to 17.4% (n = 78/449). Furthermore, doseadjustments accounted for a major reduction of patients with potentially toxic concentrations C 100 mg L -1 ) by 85% to 4.5% (n = 20/ 449). Overall, TDM effectively enhanced target-attainment while reducing unreasonably high PIP serum concentrations at the same time.

Effect of target attainment on ICU-and hospital mortality (Fig. 1) . ICU (12.0% vs. 14.6, 17.1 & 30.8%; p = .0013 ) and hospital mortality (13.9% vs. 20.8, 21.1 and 36.3%; p = .0018) were significantly lower in patients with an expsoure to the PK-target concentration of 33-64 mg L -1 within the first 24 h of treatment (Fig. 1 ). Exposure to PIP concentrations C 100 mg L -1 resulted in significantly higher mortality (ICU: 30.8%, n = 45/146, p = .04; hospital: 36.3%, n = 53/146, p = .03) compared to the PK-target group and patients with moderately high (65-99 mg L -1 ). Conclusions: Our data strongly support TDM-guided CI of PIP as a safe strategy to significantly improve PK target-attainment in critically ill patients. CI generated sufficiently high PIP serum concentrations within the first hours of treatment while critically low concentrations rarely occurred. We further found evidence of both a reduction in ICU-and hospital mortality with achieving predefined target exposures of PIP. Validation through definite RCTs is, however, needed to confirm our findings. Introduction: Antimicrobials are amongst the very few drugs with perfect correlation of drug concentration and treatment effect. Optimal drug exposure aiming at predefined pharmacokinetic (PK)-targets has been recognized a key point in modern treatment of infections in critically ill patients, respectively those presenting with sepsis and septic shock [1] . While standard dosing of b-lactams in the mentioned setting is not recommended anymore [2] , customized drug-dosing of b-lactams including the principle elements of optimized infusion (continuous infusion, CI) according to substance-specific pharmacokinetics along with therapeutic drug monitoring (TDM)-guided dose-adjustments has evolved as a promising and beneficial alternative [3] [4] [5] . Data on CI/TDM are promising in terms of better patient outcome and higher treatment success [3]. However, most customized strategies start individual dosing after the initiation of a-usually fixed-loading dose (LD) and infusion of a more or less standardized dose for the first 24 h. A priori approximation of individual doses using PK-algorithms is a way to start customized drug-dosing from the early start of treatment.

Objectives: We hypothesized that the use of a dose-approximation algorithm prior to treatment initiation might substantially increase PK-target attainment compared to the standardized protocol (SP) used in the original study (fixed bolus and standardized PIP-dose for CI for the first 24 h of treatment). Methods: The present study is a secondary analysis of a dataset of 484 patients treated in the ICU of an academic teaching hospital for severe infections (23.3%, n = 113/484) and sepsis, severe sepsis and septic shock (76.7%, n = 371/484) respectively (manuscript submitted). We used the original patient data of the study and approximated individual PIP doses for each patient for the first 24 h of treatment using the Calculator to Approximate Drug-Dosing in Dialysis (CADDy). Hypothetical PIP serum concentrations were deducted by the patient's individual PIP-clearance and the calculated PIP-dose. Descriptive analysis was performed, and PK-target attainment compared to the standardized protocol used in the original study. PIP PKtarget was defined as a serum concentration of 33-64 mg L -1 , concentrations between 65 and 99 mg L -1 were regarded moderately high whereas concentrations C 100 mg L -1 exceedingly high.

Results: The a priori use of a PK dose-approximation algorithm to calculate individual doses other than administering a fixed regimen to all patients would have increased the number of patients attaining the predefined PK-target within the first 24 h of treatment by 53.6% (n = 255/484 vs. 166/484). This result is of major importance as the original study (along with previously published data) found that PKtarget attainment (33-64 mg L -1 ) within 24 h was associated significantly lower rates for both ICU and hospital mortality. At the same time, patients exceeding PIP serum concentrations of C 100 mg L -1 would have likely been reduced by 69.2%. The particular relevance of this finding is that there are data suggesting neuro-and nephrotoxicity with PIP serum concentrations C 100 mg L -1 .

Conclusions: Our data suggest that the use of dose-approximation algorithms and the application of individual PIP-doses within the first 24 h of CI significantly enhances PK-target attainment while preventing exceedingly high (probably toxic) serum concentrations at the same time. In our opinion, customized drug-dosing should be paired with dose-approximation in a ''right-dose, right now''-manner [1] . However, definite prospective studies are needed to confirm our findings. (1), Rubino G (2), Milano AD (2), Gesualdo L (1), Pertosa GB (1) (1) Department of Emergency and Organ Transplantation (DETO), Nephrology, Dialysis and Transplantation Unit, University of Bari, (2) Cardiac Surgery Unit, University of Bari Introduction: Multiple organ dysfunction syndrome (MODS) associated with septic shock is characterized by high mortality [1] . Current therapeutic guidelines on septic patient management do not support the use of extracorporeal intraoperative cytokine adsorber in patients undergoing cardiac surgery. Objectives: We report the clinical effects of an extracorporeal therapy performed with the CytoSorb Ò adsorber in a patient with acute kidney injury (AKI) and septic shock from infective endocarditis. Methods: A 45-years-old man with a ventriculoperitoneal (VP) shunt due to neonatal hydrocephalus, was hospitalized in Nephrology for septic shock. Blood tests showed a rapid deterioration of renal function (sCr 5 mg/dl, BUN 151 mg/dl), oliguria, anemia, neutrophil leukocytosis and elevated inflammatory markers [C-reactive protein (CRP) and procalcitonin (PCT) were 350 mg/l and PCT 7 ng/ml, respectively]. Endocarditis on mitral and aortic valve was diagnosed (multiple endocarditic vegetations, probable fistulized abscess with pseudo-aneurysm formation and dissection of the mitro-aortic junction). The patient was transferred to Cardiac Surgery Unit to undergo surgery. Due to the presence of persistent arterial hypotension not responsive to fluid therapy and vasopressors, continuous haemodialysis (CRRT: CVVHDF with citrate) was started in association with a 12-hour CytoSorb Ò hemoperfusion treatment. The extracorporeal cytokine hemoadsorption CytoSorb Ò is a novel non-pharmacologic technology, able to remove medium-size molecules such as pro-and anti-inflammatory cytokines. CVVHDF has been set on the Fresenius Multifiltrate system. CytoSorb Ò (CytoSorbents TM ) was installed in series, downstream to the dialyzer on the same circuit. The patient underwent a double-replacement mitral-aortic valve replacement with a mechanical prosthesis. Subsequently, the patient continued haemodialysis treatment (CVVHDF) associated with a new Cyto-Sorb Ò cartridge for a further 24 h period. Results: The preoperative use of CytoSorb Ò resulted in a successful reversal of septic shock and a marked improvement in patient's hemodynamic stability in the first 48 h postoperatively. After the onset of Cytosorb Ò we observed significantly less vasopressor requirements with a stabilization of the mean values of arterial pressure (MAP). Furthermore, in the following 72 h a progressive improvement of renal function (sCr 2.89 mg/dl) has been observed, along with the recovery of diuresis and reduction of inflammatory markers (CRP 105 mg/l, PCT 3.05 ng/ml), as shown in Fig. 1 . The patient was discharged from the Cardiac Surgery Unit after 10 days from surgery with haemodynamic stability and without the need of dialysis therapy.

Conclusions: Patients with septic shock are at a remarkably higher risk for developing severe vasoplegia and multiorgan failure. Case reports and case series published recently demonstrated that CytoSorb Ò treatment in patients with septic shock results in improved hemodynamics, significant decrease in vasopressor dose and effective removal of proinflammatory cytokines after 24 h of application [2, 3, 4, 5, 6] . This case report also highlights the potential effects of CytoSorb Ò in controlling perioperative vasoplegia, likely modulating postoperative inflammatory response and clinical outcome. In addition, the early use of the Cyto-Sorb Ò may significantly reduce the need of postoperative renal replacement therapy, favoring a rapid recovery of renal function. Introduction: Sepsis-associated Purpura fulminans (SAPF) is a rare life-threatening condition emerging secondarily to severe bacterial and viral infections. It is characterized by disseminated thrombosis in dermal and systemic microcirculation, cutaneous hemorrhages with progressing necrosis and multiple organ failure. The underlying pathogenesis is based on the disruption of the intrinsic anticoagulation cascade, with protein C deficiency being considered the leading factor in this process.

Objectives: Epidemiological data on sepsis-associated purpura fulminans (SAPF) are scarce. Evaluations of the efficacy of different therapeutic approaches in randomized clinical trials are lacking. The causal role of individual microbial pathogens requires comprehensive evaluation. The goal of the registry is comprehensive collection and evaluation of information on epidemiology, etiology, clinical course, biomarkers, and treatment of SAPF. Methods: The multicenter registry was established in 2015 on the international level. All patients diagnosed with sepsis-associated Purpura fulminans can be included except for premature neonates. Primary outcome is in-hospital mortality. A catalogue of all outcome data is provided at ClinicalTrials.gov ( https://clinicaltrials.gov/ct2/show/NCT02238795). Participating centers enter data via a web application on the servers of the Centre for Clinical Studies (ZKS) at the University Hospital Jena. The study management software ''OpenClinica'' fully complies with regulatory requirements (GCP, 21 CFR Part 11). Results: 15 patients have been included until the end of 2018, of whom 6 were female and 9 were male. The mean age of 42 is a result of the inclusion of two children and two adolescents, the mean age of adult patients was 54 years. Most patients were not vaccinated against meningococcei nor pneumococcei. The dominant source of infection causing sepsis and SAPF were community acquired. The spectrum of positive bacterial cultures included S. aureus and N. meningitis, Streptococcus species but also E. coli, Klebsiella species and Pseudomonas aeruginosa. In-hospital mortality was 35%. Conclusions: The continuous monitoring of SAPF can provide knowledge for a better understanding of this very rare condition and will help to improve quality standards of care.

Supportive therapy of acute pancreatitis with CytoSorb Ò Adsorber. Case report Lévai T (1), Marjanek Zs (1), Brenner D (1) Jávorszky Ö dön Hospital, Vác, ICU Introduction: Extracorporeal removal of inflammatory mediators (CytoSorb Ò ) is a novel treatment modality used for immune modulation in septic shock. The method is based on molecular weight and concentration dependent adsorption of cytokines, toxins and inflammatory mediators to biocompatible polymer particles with an overall surface area of more than 40,000 m 2 . Objectives: Our aim was to examine and insert additional methodes to supportive therapy in the treatment of our patient suffering from severe acute pancreatitis. Methods: A 42 year old male patient was admitted to the intensive care unit with severe acute pancreatitis [APACHEII: 13 Ranson score 4 (24 h) and 8 (48 h)] as a result of food abuse and hypertriglyceridemia (71 mmol/l). Along with supportive treatment (haemodynamic and respiratory support, renal replacement therapy) we applied plasma exchange on two consecutive days. Despite normalizing serum triglyceride levels the patient had deteriorating multi organ failure. Due to abdominal compartment syndrome surgical decompression was performed (open abdomen with vacuum dressing). To attenuate the ,,cytokine storm'' and remove pancreatic catabolic enzymes we used pre-filter CytoSorb Ò with continuous veno-venous renal replacement therapy (with regional citrate anticoagulation) for 4 9 24 h. Results: The patient was successfully stabilized and the recovery from multi organ failure was accompanied by improving respiratory, kidney and liver function and decreasing oedema. Bilirubin and procalcitonin was excessively removed by CytoSorb Ò . We experienced no clotting in the extracorporeal system and only mild and transient pH disturbances (caused by citrate/calcium dosing) during the treatment. The abdomen was completely closed by day 35, the patient was discharged from the ICU on day 41 and returned home on day 45. Conclusions: In addition to standard treatment of severe acute necrotizing pancreatitis extracorporeal removal of inflammatory mediators using CytoSorb Ò contributed to the recovery of our patient. Given the complex pathophysiology, individualized approach and further research is needed to examine the efficacy and safety of extracorporeal cytokine removal.

Infection 2019

Blood purification with Cytosorb Ò in abdominal septic shock: a case report Zuccari S (1),Vannicola S (1), Scorcella C (1), Domizi R (1), Damiani E (1), Giaccaglia P (1), D'Arezzo M (2), Donati A (1) (1) Clinica di rianimazione generale, respiratoria e del trauma, Direttore Prof. P. Pelaia, Dipartimento di scienze biomediche e sanità pubblica, Azienda Ospedaliera Ospedali Riuniti UNIVPM, Ancona, Italia, (2) SOD Nefrologia e Dialisi, Dipartimento Gastroenterologia e dei trapianti, Azienda Ospedaliera Ospedali Riuniti UNIVPM, Ancona, Italia Introduction: Cytokines reduction by hemoadsorption represents a new concept for blood purification in septic shock. Objectives: In the present case report, we evaluated the impact of Cytosorb Ò hemoadsorption device, used as a weapon in patient-tailored therapy, on macro and micro haemodynamics. Methods: A 70-years-old man was admitted to emergency department with peritonitis, severe lactic acidosis (serum lactate: 6.8 mmol/L), and imaging was consistent with ureteral rupture after operative ureteroscopy with peritoneal purulence. He underwent a surgical ureteral repair and debridement of the peritoneal cavity. This patient was admitted to ICU with the diagnose of septic shock, SOFA score was 14, and underwent an adequate fluid resuscitation. Vasopressors were needed to treat hemodynamic instability. Microbiological samples were promptly taken and a broad spectrum antibiotic therapy was started. Procalcitonine levels were elevated: 330 lg/L. We implemented invasive hemodynamic monitoring using Picco2 Ò that showed an adequate cardiac output (Ci 4.69 L/min/m2), but a reduced systemic resistance (SVRI 903 dyness/cm-5/m 2 ). We started microcirculation analysis in sublingual mucosa with a non invasive videomicroscopy technique using Cytocam Ò . A perfused vessel density (PVD)of 15,17 was detected and mean flow index (MFI) was 2.58. Terlipressin was needed to treat vasoplegic shock that was unresponsive to high dosage of norephinephrine: 2 lg/kg/min The patient developed an acute kidney failure with anuria that was unresponsive to massive diuretic therapy and continuous veno-venous heamodialysis (CVVHD) was started. Admission blood essays shown severe lactic acidosis accompanied by high levels of cytochines: IL1 5 pg/ml, IL6 361 pg/ml, IL8 214 pg/ml, IL10 28 pg/ml, TNF-a 126 pg/ ml. We started a combined treatment of hemoadsorption using a Cytosorb Ò hemoadsorber and CVVHD.

Results: After 6 h of treatment we noticed a reduction in plasmatic cytochines levels: IL1 5 pg/ml, IL6 118 pg/ml, IL8 145 pg/ml, IL10 27.8 pg/ml, TNF-a 61.1 pg/ml. After 24 h of treatment we described an improved quality of blood flow: MFI 2.75 and increased PVD 16.08. Cytochines levels were reduced IL1 5 pg/ml, IL6 178 pg/ml, IL 8 67 pg/ml, IL10 34.8 pg/ml, TNF-a 38.1 pg/ml. SOFA score was 11. We extended Cytosorb Ò treatment for 72 h using 3 cartridges with a restoration of macrohaemodynamic parameters. Conclusions: We present a case of septic shock with peritonitis leading to multi organ failure. The early treatment with a combination of CVVHD and Cytosorb Ò led to a rapid hemodynamic and metabolic stabilization, suggesting that hemoadsorption might be an option in patient-tailored therapy of septic shock. Introduction: Sepsis is a serious complication of infection and an influential cause of morbidity and mortality worldwide. The use of intravenous IgM-enriched immunoglobulin (Pentaglobin Ò ) as complementary therapy in sepsis patients appears promising, but data from clinical studies alone are not sufficient to define quality standards for therapeutic use. Objectives: Aim of the registry is to assess the effects of Pentaglobin Ò use on clinically relevant outcome parameters and its side effects under real-life conditions based on a prospective, high-Quality data documentation. Methods: Data collection in adult patients with severe infections began in January 2018. The participating centers collect the data via a web application on the servers of the Center for Clinical Studies (ZKS) at the University Hospital Jena. The study management software ''OpenClinica'' fully complies with the regulatory requirements (GCP, 21 CFR Part 11). Results: By March 2019, 17 patients had been included. Of these, 13 patients have already reported data on treatment and post-treatment visits. The average age of patients was 53 years. 8 patients were male, 9 were female. The majority of patients were admitted for the nonsurgical emergency. The mortality rate was 7 out of 13 patients treated. In 4 cases sepsis was identified as a direct cause of death (multiorgan failure). Conclusions: Early data show the potential of the registry, although the results must be interpreted with caution as the number of patients included is small. A future extension of the data set could help to improve clinical management of sepsis. Introduction: APRV is a widely used and highly protective modality of mechanical ventilation in severe hypoxaemic respiratory failure. The old-new tool for the PEEP setting strategy is esophageal manometry derived transpulmonary pressure. Although it has a plenty of the strategies, how to set up the time ratio and PEEP level, there is a lack of recommendation for time and pressure settings based on transpulmonary pressure. Our aim is to prove the transpulmonary pressure relevance under APRV ventilation in comparison of classic targets of setting (as time constant, flow analysis and volumetric method). Objectives: Inclusion criteria: We included moderate to severe ARDS cases, in adults, which are potentially have benefit from higher pressures, according to static PV curve analysis. Methods: Methods: We compared 4 different methods to determine expiration time. Flow curve analysis (targeting 70% of PEF), volumetric method (targeting 6 ml tidal volume/IBW), expiratory time constant (Tlow = 1 9 time constant). Goals: We aimed to prove the transpulmonary based method superiority over the other three methods. And analyse the effect on oxygenation (Horowitz score, Oxigenation index, volumen, intrinsic PEEP). Results: Results: We found, that all the methods improved oxygenation. The PEEP was significantly higher in the time constant guided group. The transpulmonary pressure guided group allowed lower PEEP, under the threshold of significance. But resulted better CO2 elimination, and much more physiologic pH. Conclusions: Conclusion: The transpulmonary pressure measurement has a potential benefit in APRV set up. The low number of involved patient has a limitation of the estimation of it's significance Introduction: Mortality due to septic shock is very high, particularly in patients with heart failure or septic myocardiopathy. Objectives: In this case VA-ECMO could be useful to prevent irreversible organ damage, however, lack of control of a dysregulated immune reaction may compromise survival. Association of VA-ECMO and cytokine hemoadsorption could be effective in this context. Methods: A 59-year-old male patient, admitted 20 days after inferolateral AMI complicated by cardiac arrest, underwent off-pump myocardial revascularization. Ejection fraction was 35%, therefore IABP and Swan-Ganz catheter were placed. IABP was removed on third POD, and weaning from dobutamine and mechanical ventilation were started. Hyperthermia and arterial hypotension with no modification of cardiac index (CI) showed at that moment. Nor-adrenaline and adrenaline i.v. infusions were initiated after fluid challenge (T0). Despite CI increase, systemic hypotension worsened and lactate raised (T0 ? 6 h). After 12 h the mean arterial pressure(MAP) was 50 mmHg although high catecholamine dose, and lactate reached 15 mmol/l. Renal, hepatic and coagulation dysfunctions became evident, and SOFA II score was 15 with 80% expected mortality. PCT, PCR and leucocytes raised too (T0 ? 12 h). Peripheral VA-ECMO was established to increase CI and oxygen availability (DO2I), while CVVHDF was started to replace renal function, and CytoSorbTM was installed into the CRRT circuit to prevent cytokinemediated organ damage, and restore peripheral vasomotor responsiveness. Vancomycin and gentamicin were empirically added. Results: VA-ECMO increased CI, DO2I, and MAP. The patient's heart contributed to the overall CI (T0 ? 24 h). Hemoadsorption, delivered continuously for 3 days, favored rapid clearance of blood lactate, PCT, PCR, and leucocyte count normalization, preserved liver, kidney and coagulation system, and hastened vasoplegia resolution. Adrenaline and noradrenaline were reduced and stopped on 3rd and 6th day of treatment, respectively. CVVDHF was halted in 6th. VA-ECMO was removed on 8th day, when SOFA II score was 8 with 15-20% expected mortality. The patient was weaned from ventilation and dismissed from ICU 2 weeks later. Blood cultures were negative. Stafilococcus Aureus was isolated from broncoalveolar lavage.

Conclusions: Care of patients in septic shock need to be tailored. In this patient we considered the inability of the heart to increase cardiac output and match increased oxygen requirements, therefore, in front of a quick worsening of SOFA, we implemented VA-ECMO and cytokine hemoadsorption with CytoSorbTM. This allowed prompt reversal of the profound vasoplegia and quick removal of catecholamine, hastened clearance of lactate, PCT and PCR, and protected organs already made dysfunctional by sepsis. As matter of facts, SOFA score passed from 15 to 8, and the patient showed a complete recovery.

Effect of early therapeutic plasma exchange on microvascular coagulopathy in septic shock and an attenuation of anti-coagulant activity leading to microvascular stasis and consequent organ failure. No specific treatment approach exists to target this key pathophysiological process. Objectives: In this study, we explored the effects of early therapeutic plasma exchange (TPE) on microvascular coagulation dysbalance in septic shock. Methods: We conducted a prospective single center study enrolling 31 patients with early septic shock (onset \ 24 h) requiring high doses of norepinephrine (NE [ 0.4 lg/kg/min). Clinical and biochemical data, including measurement of protein C, ADAMTS13, and vWF:Ag were obtained before and after TPE. Results: Antithrombotic acting proteins such as antithrombin-III (ATIII) and protein C were markedly reduced in septic shock patients. After TPE, the activity of both proteins increased significantly [AT III: 51 (41-61) vs. 63 (48-70) %, p = 0.029 and protein C: 47 (38-60) vs. 62 (54-69) %, p = 0.029]. D-dimers were severely elevated in all patients and significantly declined following TPE [13.1 (2.7-17.5) vs. 4.7 (2.3-8.6 ) mg/l, p \ 0.001]. Median ADAMTS13 activity was markedly increased by TPE from 27 (21-42) before to 47 (38-62) % after TPE (p \ 0.001). In contrast, vWF:Ag was highly increased at study inclusion and could be reduced significantly by vs. 170 (117-232) iU/ml, p \ 0.001]. Thrombocytes were severely reduced in patients with ADAMTS13 \ 30% (39*1000/ll (26-63) and only slightly reduced in those with ADAMTS13 C 30% [193 9 1000/ll (86-281), p = 0.004]. Regression analysis yielded a significant correlation between ADAMTS13 activity and thrombocyte count (p = 0.001, R2 = 0.316). Conclusions: Severe septic shock was associated with activation of pro-coagulant pathways such as vWF:Ag and ultra-large-VWF-multimers and consumption of anti-coagulant factors such as antithrombin-III, protein C and ADAMTS13. TPE partially attenuated this dysbalance by removing pro-coagulant and by replacing anticoagulant factors. It has yet to be determined by an appropriately powered RCT that TPE improves outcome in septic patients.

Interim analysis from a randomized controlled trial on therapeutic plasma exchange in early and severe septic shock Introduction: Introduction: Sepsis is a life-threatening dysregulated host response to infection. Given the injurious role of (1) the overwhelming immune response and (2) the consumption of protective plasmatic factors (e.g. vWF cleaving proteases-ADAMTS13 etc.) we hypothesize that early therapeutic plasma exchange (TPE) in severely ill individuals might be beneficial. TPE combines two aspects in one procedure: (1) Removal of harmful circulating molecules and (2) replacement of protective plasma proteins. We have recently shown in an uncontrolled pilot study that TPE is safe and feasible in septic shock and that it might improve surrogate endpoints (1) . Objectives: To investigate the degree of organ failure in patients with early septic shock after a single initial TPE vs standard of care (SOC).

Key secondary outcome parameters were norepinephrine (NE) doses after 6 h and 28-day mortality. Methods: So far, we have included 20 septic shock patients (onset \ 24 h) requiring high doses of NE ([ 0.4 lg/kg/min) in this single center RCT. TPE was performed within 4 h after randomization (TPE n = 11, SOC n = 9). Clinical and chemical data (e.g. for SOFA calculations) were obtained at randomization, 6 h later and longitudinally over the following 9 days. Results: Mean NE doses at randomization were comparably high (SOC 0.69 ± 0.45 vs. TPE 0.78 ± 0.37 lg/kg/min) in both groups. While 6 h later the NE dose was significantly lower in the TPE group (0.26 ± 0.15 lg/kg/min, p = 0.002) it was unchanged in the SOC group (0.61 ± 0.51 lg/kg/min). The NE dose between randomization and 6 h was reduced by 64 ± 21% in the TPE group but only by 17.7 ± 29% in the SOC group (p [ 0.001). Mean total SOFA score d1-9 (SOC 16 ± 3 vs. TPE 14 ± 3, p = 0.15) and 28-day mortality was not significantly different (SOC 55.6 vs. TPE 45.5%, p = 0.34).

Conclusions: This interim analysis of a single center RCT supports our hypothesis that early TPE in highly unstable patients might be beneficial with regard to hemodynamic stabilization. Possibly due to the small sample size only trends were detected with regard to hard endpoints. A national multicenter RCT powered for mortality is planned and funding applications are currently under review. Introduction: Surgery for infective endocarditis (IE) is associated with a high incidence of multiple organ dysfunction syndrome (MODS) and mortality, which may be linked to increased release of inflammatory mediators during cardiopulmonary bypass (CPB). Objectives: We therefore assessed plasma cytokine and precursor hormone profiles in patients undergoing valve surgery with or without IE. Methods: We performed a prospective case-control pilot study comparing patients undergoing cardiac valve surgery with or without IE. Plasma profiles of inflammatory mediators were measured at 7 defined time points and reported as median (interquartile). The degree of MODS was measured using sequential organ failure assessment (SOFA) score. Results: Between May and December 2016 we included 40 patients (20 in each group). Both groups showed similar distribution of age and gender. Patients with IE had higher preoperative SOFA (6.9 2.6 vs 3. 8 (0.53) nmol/L, p = 0.003); MR-pro-atrial natriuretic peptide (MR-proANP) (479.49 (224.74) vs. 266.55 (308.26 ) pmol/l, p = 0.028). IL-1b and TNF-a were only detectable in IE patients and first after starting CPB. Plasma levels of IL-6, IL-18, MRproADM, and MRproANP during CPB were significantly lower in survivors than in those who died. Conclusions: The presence of infective endocarditis during cardiac valve surgery is associated with increased inflammatory response as evident by higher plasma cytokine levels and precursor hormone profiles. Actively reducing inflammatory response appears to be a plausible therapeutic concept. The aim of this registry is to record the use of CytoSorb adsorber device in critically ill patients in as many cases as possible.

The objectives of the registry ( http://www.cytosorb-registry.org/) are collection of data on a broad scale, centralized, structured and comprehensive documentation, and controlled data exchange. The registry records all relevant information in the course of product use, e.g. diagnosis, comorbidities, course of the condition, treatment, concomitant medication, clinical laboratory parameters and outcome (ClinicalTrials.gov Identifier: NCT02312024). Primary endpoint is in-hospital mortality as compared to the mortality predicted by the APACHE-II and SAPS-II-Score, respectively. Results: Data available from the start of the registry on May 18, 2015 to June 4, 2019 were analyzed. At this time point 247 centers from 31 countries participated in the registry, of whom 37 centers from 8 countries provided data for a total of 719 patients. Mean age was 59.5 ± 15.1 years, 464 (64.5%) were male. The majority of patients was admitted for non-surgical emergency, 363 (50.5%) patients, type of admission was surgical emergency for 218 (30.3%) of the patients and elective surgical for 138 (19.2%) of the patients. 663 (93.6%) had one to 5 Cytosorb adsorber applications. The mean (± standard deviation) duration of treatment was 50.0 ± 66.0 h for the sepsis group (N = 394), 7.9 ± 16.4 h for patients with preemptive use in cardiac surgery (N = 82), 52.1 ± 41.1 h for patients with postoperative use in cardiac surgery (N = 49) and 62.3 ± 61.7 h for patients with other indication (N = 183). Sepsis/septic shock was the most common indication for CytoSorb treatment (401 patients), followed by other indications (183 patients), incl. liver failure (76 patients), acute pancreatitis (29 patients), ARDS with ECMO (23 patients). 68 patients were treated intraoperatively while receiving open-heart surgery with the use of cardiopulmonary bypass. Mean APACHE-II score in the sepsis/septic shock group was 29.9 ± 8.7 [range 11-55]) with a predicted risk of death of 70.4%, whereas the observed mortality was 63.1%. This result for the sepsis group is supported by the high SAPS-II scores (67.9 ± 18.5 [20-111]), with a predicted mortality of around 72%. There were no significant decreases in the SOFA scores before and after treatment (15.7 ± 4.2 [3-24] vs. 15.3 ± 5.1 ). However, IL-6 levels were markedly reduced after treatment (median 5000 pg/ml before and 289 pg/ml after treatment, respectively). Conclusions: This 8th interim report demonstrates the feasibility of the registry with excellent data quality and completeness from 37 study centers. The disease severity is remarkably high and suggests that adsorber treatment might be used as an ultimate treatment in lifethreatening situations. There were no device-associated side effects Longterm Outcome 001 Infection 2019

Long-term mortality and outcome in survivors of septic shock, sepsis, and severe infections: the importance of aftercare Introduction: Patients with severe infections and especially sepsis have high in-hospital mortality. However, even hospital survivors may face long-term sequelae, decreased health-related quality of life and a greater, less appreciated risk of death. Specifically, data are lacking regarding long-term outcome comparing patients with former septic shock, sepsis, and severe infections without organ-dysfunction according to the Sepsis-3 definition. Objectives: To determine reliable estimates of long-term mortality, morbidity, and evaluate the importance of aftercare in hospital survivors with former septic shock, sepsis, and severe infections without organ dysfunction. Methods: 117,202 patients having suffered from severe infections within the years 2009-2016 were retrospectively analyzed to determine their in-hospital mortality, 3-month to 5-year mortality, and common health sequelae within the first year after hospital discharge. Cases were identified by clinical and pathogen-based ICD-10-GM codes for sepsis and infections, and stratified according to the recent Sepsis-3 definition.

Introduction: As a consequence of sepsis and intensive care, considerable proportions of patients but also of their spouses develop a post-traumatic stress disorder (PTSD). However, only a very small number receive psychotherapeutic treatment. Internet-based cognitive-behavioral writing therapy (IB-CBWT) has proven to be an effective treatment option for PTSD. It seems to fit the specific needs of this cohort and to overcome treatment barriers. Objectives: Aim of the REPAIR trial was to examine the efficacy, safety and applicability of IB-CBWT for PTSD in patients and their spouses after intensive care for sepsis. Methods: Participants were randomly assigned either to a treatment or a wait-list (WL) control group. The treatment group received therapist-guided IB-CBWT for PTSD comprising two written assignments per week over a 5 week period. Participants of the WL control group received treatment after a waiting period of 5 weeks. We present efficacy data for PTSD symptom severity assessed by the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5) at the end of treatment/waiting time. Results: Among enrolled 34 participants, 29 (18 patients/11 spouses, mean age 55 years, 55% female, ICU stay 29 days) completed the study. Five of 16 participants in IB-CBWT prematurely terminated treatment. At the end of treatment/waiting time, there was a significantly higher reduction in PTSD symptom severity in IB-CBWT compared to WL (standardized mean difference, d = 1.90, 95% CI [1.00, 2.79], p \ .001). Conclusions: IB-CBWT revealed large effects on PTSD symptom reduction in comparison to WL. Despite considerable efforts recruitment of participants was difficult raising the question of post-ICU accessibility of patients or spouses in need of treatment. Introduction: A large number of ICU patients and their spouses report considerable levels of posttraumatic stress after intensive care. However, current knowledge regarding the content and organization of traumatic ICU experiences is incomplete, and less is known about dyadic similarities in traumatic ICU experiences. Objectives: Aim of this mixed-method study was to explore the description of traumatic ICU memories by patients and spouses, to analyze the language used for expressing these memories, and to examine dyadic concordance in ICU experiences. Methods: ICU survivors and their respective spouses with severe posttraumatic stress symptoms were asked to give a detailed narrative of their traumatic ICU event. Trauma narratives were analyzed (1) with respect to the content using qualitative methods and (2) regarding verbal expression of emotions, cognitive processes, and other linguistic characteristics using the text analysis software program ''Linguistic Inquiry and Word Count'' (LIWC). Results: Eight couples were included. Patients (all men, mean age 52 years) were treated in intensive care for Mdn 28 days. They were married for Mdn 25 years with their respective spouses (7 women/1 men, mean age 51 years). Narratives were written by patients and their spouses Mdn 22 months after ICU discharge. Acute fear of dying (personally experienced by patients or witnessed by spouses) and strong feelings of helplessness were mentioned most frequently as worst traumatic experiences in the narratives. Conclusions: Examination of traumatic ICU experiences of patients and their spouses might contribute to a deeper understanding of ICU processes and traumatizing situations. Introduction: In January 2019 the Federal Joint Committee (Gemeinsamer Bundesausschuss-G-BA) requested Germany's central institute for quality assurance in healthcare, the so called ''Institute for Quality Assurance and Transparency in the Healthcare System'' (Institut für Qualitätssicherung und Transparenz im Gesundheitswesen-IQTIG), according to § 137a SGB V, to develop a report (,,Konzeptstudie'') on optimal care aspects and areas for improvement regarding ,,diagnostics, therapy and aftercare of sepsis''. The G-BA may use the developed evidence to take a decision whether to commission the development of a nation-wide quality assurance procedure for this specific care area. The developed quality assurance procedure would ultimately allow for the collection of health care data and implementation of quality indicators. Objectives: This report's objective is first, to identify sepsis-care related quality deficits and areas for improvement, second, to deduce relevant aspects of care, and finally to assess whether the identified and described quality of care issues would be effectively addressed by the available instruments that the nation-wide quality assurance procedure can offer. Methods: The methods are applied according to the institute's methods handbook (,,Methodische Grundlagen 1.1'') Results: The so called quality model (,,Qualitätsmodell'') which provides a framework for evaluating quality of sepsis care encompasses three stages of development. The first stage focuses on the qualitative and quantitative exploration of the topic area by researching literature, guidelines, norms and regulations, by conducting expert interviews and a quantitative analysis of health insurance data. According to the G-BA's directive, patients of all ages who receive treatment as out-or inpatients are included in the study. All care phases, before and during the hospital stay as well as post discharge are considered within the framework of the study. A care pathway is developed which reflects relevant care processes highlighting critical key points as well as intersections between health care sectors. Based on the researched and generated results from literature research, data analysis, the focus groups with patients, next-of-kin and healthcare professionals, relevant quality of care aspects are deduced. Conclusively, only those quality of care aspects will be selected and suggested which adhere to the scope of the G-BA's directive and which can be measured using the available instruments that the nation-wide external quality assurance has to offer (documentation by healthcare providers, data from health care insurances, patient interviews). The nation-wide external quality assurance procedure sets standards for quality of care to be fulfilled by providers. It is therefore critical to select care topics which can realistically be influenced by health care providers as well as suggest potential for improvement. Conclusions: The study's results will provide evidence for decision makers to decide in favour or against the development of a nationwide external quality assurance procedure according to § § 136 ff. SGB V. The final report will be published in December 2019 and give recommendations whether and if so, for which patient groups the development of an external quality assurance procedure is recommended. Introduction: The imagination of a safe place is a successful method in psychological therapy. Feeling safe is a requirement for any intervention and supports the positive effects of an intervention. Being a patient in the intensive care unit (ICU) represents an extreme situation which is usually accompanied by psychological distress. One of the key factors causing psychological distress is mechanical ventilation as breathing is externally controlled. Objectives: Aim of our study is to test the effects of safety suggestions in patients treated with non-invasive ventilation (NIV) during intensive care. Methods: In a pre-post study design we tested the effects of standardized safety suggestions provided in a face-to-face setting by trained therapists. Patients were asked to rate their subjective feelings of anxiety, valence and arousal before and after receiving suggestions of safety. We further recorded physiological parameters, e.g., heart rate, blood pressure, and breathing before, during and after the suggestions. Results: Our results show that suggestions of safety significantly improve subjective well-being of patients on NIV by reducing their anxiety and arousal ratings. Conclusions: Suggestions of safety are a successful method to reduce psychological distress in ventilated patients during intensive care. Our study supports the application of psychological interventions such as positive suggestions in the ICU. Long-term effects of safety suggestions on mental health after ICU discharge have to be examined in further studies. Introduction: Cognitive dysfunction is a major and debilitating problem in survivors of severe sepsis. Patients suffer from significant cognitive problems especially in the domains of attention and executive functions. So far, effective and feasible training strategies to improve cognitive deficits in those patients have not yet been evaluated. Objectives: In the present pilot study, we aim to better characterize the cognitive deficits in patients of the Mid German Sepsis Cohort and to evaluate if these deficits can be influenced by specific home-based neuropsychological training. Methods: A controlled pre-post-test-design with two arms is used. The specific training group performs a computerized, home-based attentional speed, capacity, concentration and selection training at increasingly complex levels of difficulty and speed (adaptive to the individual user). The active control group performs a computerized unspecific training, with equivalent training duration and intensity. Training effects will be assessed as changes in visual attention parameters in post-compared to pre-training sessions measured with a psychophysical whole report paradigm based on the theory of visual attention (TVA, Bundesen 1990) and a clinically established neuropsychological battery. Results: Primary outcome is parameter visual processing speed in items per second. We will assess whether the specific training group has a higher visual speed gain than the active control group. Besides, we will evaluate if the specific training group benefits in further TVA based parameters and neuropsychological test results, compared to the active control group. Conclusions: The results of this pilot study will provide first evidence on whether cognitive deficits in sepsis patients can be improved by targeted computerized training. Reference: Bundesen, C. (1990) . A theory of visual attention. Psychological review, 97(4), 523. Introduction: The University Hospital Dresden and the Bavaria Kreischa Hospital jointly founded the Comprehensive Sepsis Center Dresden-Kreischa in order to improve the quality of care for sepsis patients in the acute as well as in the rehabilitative phase. Objectives: By means of structured treatment algorithms and consented standard operating procedures along with joint boards and interdisciplinary discussion forums we aim at a sustainable quality improvement in the care of sepsis patient. Our primary goals are a reduction in sepsis related mortality and an increase in quality of life of sepsis survivors. We will compare the patients in the structured treatment plan with a matched cohort of patients not enrolled in this pathway in order to evaluate the benefits of this program. Methods: All patients with sepsis and septic shock admitted to the intensive care unit during the study period of 3 years will be included in the treatment pathway. It describes the cross-sectoral therapeutic concept by defining therapeutic goals in each treatment stage and determining assessment tools and therapeutic measures suitable for achieving these goals. Furthermore, parameters required to evaluate treatment success are defined. Informed consent will be obtained by the patient, a legal representative or an independent physician. We designed a database collecting over 800 variables during the whole process of care from the acute phase until discharge from the rehabilitation program. In addition, the patient's long-term outcome will be assessed in terms of independency in daily life, cognition, mental health, and quality of life. Furthermore, independent risk factors for relevant clinical endpoints will be identified as a basis for future prospective studies. Clinical endpoints are mortality, proportion of patients in need of in-patient care (re-hospitalization), mobility, cognition, mental health und quality of life after one/two/three years. In addition, parameters of process quality such as duration of ventilation, duration of tracheal cannula care, duration of renal replacement therapy, extent of wound care etc. are recorded, clinical endpoints will be analyzed by statistical tests. A follow up will be performed at fixed time points up to 3 years after discharge. Results: The clinical pathway as well as variables included in the database was consented among the participating institutions and an application for ethical approval as well as registration of this study has been filed. Conclusions: We developed an interdisciplinary, trans-sectoral treatment pathway for patients with sepsis in order to improve quality of care and outcomes. Our database will provide long-term data to identify potential risk factors necessary for clinical decisions making as well as planning of future prospective studies in this field.

Trends in sepsis and septic shock mortality between 2009 and 2019, a systematic review and meta-analysis Vogelmann T (1), Preissing F (2), Stiefel J (1), Adam Blanco D (2), (1) Linkcare GmbH, (2) CytoSorbents Europe GmbH Introduction: Previous research suggested that in-hospital mortality rates of septic shock and sepsis have been reduced at a 1% yearly rate, but numerous clinical trials have failed to show an improvement in mortality. Currently, no recent and systematic overview on mortality data at 30-days or 90-days, that take into account data from RCTs and observational studies and distinguish between sepsis and septic shock, is available. Objectives: We aimed to estimate the 30-and 90-days mortality rates for sepsis and septic shock in a systematic review and meta-analysis and assess, whether a change over the past decade can be observed. Methods: We performed a systematic review of English articles published between 2009 and 2019 in MEDLINE of interventional and observational studies. We extracted the following information for each study in a standardized way: study design, enrollment start and end date, country of the study, average age, SOFA score and APACHE II of patients, enrollment of patients with severe sepsis vs. septic shock, 28-days and 90-days mortality rate for septic shock and sepsis patients. A meta-analysis of pooled 30-and 90-days mortality separately for sepsis and septic shock was performed using a randomeffects model. Time trends were assessed using the joinpoint method. Results: 4,494 records were identified in the electronic database. After title/abstract screening, 783 articles were assessed in full text for eligibility and 166 studies were included in the quantitative analysis. 80 studies reported data on 30-days septic shock mortality, 37 on 90-days septic shock mortality, 72 on sepsis 30-days mortality and 27 on 90-days sepsis mortality. Average 30-days septic shock mortality was 34.82% (95% CI 32.70-36.94%) and 90-days septic shock mortality was 38.43% (95% CI 35.43-41.53%). Average 30-days sepsis mortality was 24.62% (95% CI 21.86%-27.40) and 90-days sepsis mortality was 32.36% (95% CI 27.36-37.35%). Estimated mortality from RCTs were slightly below prospective and retrospective cohort studies. Between 2009 and 2018, we found a trend in the reduction of 30 days mortality rate among sepsis patients but no reduction in mortality rate in septic shock patients at 30 or 90 days. Conclusions: Despite some evidence of a trend towards lower mortality rates in sepsis patients over the past 10 years, no reduction in mortality has been observed among septic shock patients, indicating a remaining high unmet need in those patients.

Counselling interviews for patients, relatives, and surviving relatives: updated experiences of the German Sepsis Aid Redlich U, Groenert H, Trumann A, Wegmann R, Herrmann V, Brunkhorst FM German Sepsis Aid, Germany Introduction: The German Sepsis-Aid is the world's first association of sepsis patients and their relatives. In 2007, the German Sepsis Aid was founded in Jena as a non-profit-organisation for and by people affected by sepsis. Before, there were no national or international contact persons in this field, nor was there a focal point for patients and their families. The German Sepsis Aid is financed exclusively by donations, membership dues and subsidies of health insurance companies. It waives funding from pharmaceutical companies or other entities that could create a conflict of interest. Objectives: The main objective is to provide a unique platform for sepsis survivors and their families to connect, to share experiences and to find support. The German Sepsis Aid pursues the target to raise awareness for the symptoms of sepsis and to fight the often longlasting sequelae of this disease. Women and men, who are aware of sepsis from their own experience, volunteer to give practical and psychosocial assistance and information, according to the principle of ,,sepsis patients help sepsis patients''. The German Sepsis Aid can be reached by telephone helpline (?49 700-737747-00) 12 h a day, website and email. Methods: 571 counselling interviews, requested between 01.01.2017 and 19.6.2019, were analysed. Multiple requests per enquirer were evaluated as one request. Results: 60% of the requests were made via the telephone helpline, 30% per email, 8% via the website and 8% of the enquirers used other communication ways. 49% of the requesting people were relatives/guardians or friends, former sepsis patients were 37%, and others 14%. The inquiries had a wide variety. They focused on questions about long-term consequences and rehabilitation in 49% of cases followed by questions about the acute disease in 38% cases. Other inquiries, such as questions about grief assistance or contact with affected persons, were made in 33% cases. After the consultation, the German Sepsis Aid provided additional printed free information material for 30% of the help seekers. Frequently, the support is not finished after a call or an answered email, and further conversations occurred in the course of the time and the total number of contacts was 801 during the reported time frame. Conclusions: On the part of patients affected from sepsis and their relatives there is a strong need to receive information with regards to the acute disease, therapy, possibility of prognosis and rehabilitation as well as to request for support and contact with other concerned people. Acknowledgement: Supported by the German Sepsis Society (GSS).

Infection 2019

Microbiota alleviates host resistance and causes septic complications in diabetic foot syndrome Sydorchuk RI (1), Sydorchuk LP (1), Khorshani Bilel (1), Hrushko OI (2), Plehutsa OM (2), Sydorchuk I (1), Plehutsa IM (2), Vakarchuk AV (2), Sydorchuk AR (1), Ilaschuk II (2), Plehutsa N (2) (1) Bukovinian State Medical University, Chernivtsi, Ukraine, (2) A&E Hospital, Chernivtsi, Ukraine Introduction: Limb sepsis in diabetic foot syndrome (DFS) patients is associated with significant morbidity and mortality. The pathogenesis of diabetic foot infections in these patient populations appears to be related to ulcers and underlying peripheral neuropathy being the most important risk factors as well as existing diabetic multiorgan dysfunction risk. The major risk factors for DFS are still unclear but existing data suggest poor glycaemic control is associated with respective poor outcome. However, in most studies, the possible immunocompromising role of DFS microbiota is not regarded as an influential factor. Moreover, many studies ignore micribiota's role in sepsis development in DFS patients. Objectives: The aim of this study is to evaluate micribiota's influence on non-specific host resistance and its role in sepsis development in DFS patients. Methods: Study involves 107 DFS patients (PEDIS grades 2-3, IDSA moderate to severe infection score). Microbiota obtained intraoperatively evaluated using standard aerobic and anaerobic techniques, phagocytic activity and phagocytic index of neutrophils and monocytes isolated in the two-step ficoll-verographin 1077/1093 density-gradient centrifugation. Results: It is established that conditionally pathogenic bacteriapathogens of purulent-necrotic processes of soft tissues under DFS show suppressive effect on the phagocytosis of neutrophil granulocytes systems, decreasing phagocytic activity, phagocytic quantity and phagocytic capacity of peripheral blood of patients. The highest level of suppression of phagocytic activity was associated with S. aureus (75.58%), S. pyogenes (92.36%), R. melaninogenicus (74.75%), and P. aeruginosa (34.55%). Lowest immunosuppressive activity found in E. coli (16.13%), S. haemolyticus (17.80%), E. cloacae (18.39%), S. intermedius (19.01%).

Differentiated usage of ointments in the treatment of infected wounds Fuss J (1), Voloboyeva A (2) (1) Department of surgery, Regional Central hospital in Pustomyty, Ukraine, (2) Department of Intensive Care, Hospital §8, Lviv, Ukraine Introduction: Differentiated selection of optimal means for local treatment, depending on the stage of the wound process, its features and complications has a significant effect on the timing, purification, filling granulation and preparation of wounds to the operation of skin plastics [1, 2, 3] . In this regard, the effect of differentiated use of ointments on the treatment of wounds in the second phase was studied [4, 5] . Objectives: Study the differentiated use of ointments for the treatment of infected wounds Methods: The results of the treatment of 50 patients who were admitted to the burn department for extensive purulent wounds for treatment and preparation for operations in skin plastic surgery were studied. In the first phase of the wound process, ''Baneocin'' (Sandoz) was used, in the second stage, the ''Sulfargin Ò '' (Grindex) was used. The comparison group consisted of 50 patients with similar areas and depths of wounds, which used ''Bactroban'' (GlaxoSmithKine) for treatment. Results: The use of ''Baneocin'' in patients with the first phase of the wound process did not cause an increase in pain, most (43 out of 50) reported a decrease in pain intensity. As a rule, the 5th day of treatment in the wounds marked the appearance of granulation, which allowed the transition to the use of ''Sulfargin''. In the comparison group, the application of ''Bactroban'' in the first phase of the wound process did not cause an increase in pain, at the same time, the reduction in the intensity of pain associated with bandage was noted in only 2 out of 50 patients. The appearance of granulation was observed at 6-8 days of treatment. The microbial wound landscape was represented primarily by St. epidermidis, E. coli and Ps. aeruginosa ( Table 1) . As can be seen from the table, the total antibiotic efficacy of mupirocin was slightly greater than 1/3, while neomycin was effective in more than cases. Using the Sulfargin provided rapid wound preparation to skin plastic surgery. Conclusions: 1. Local treatment of purulent wounds should be conducted with differentiated use of drugs, which allows adequately affect the phases of the wound process. 2. In the first phase of the wound process, the use of ointment Baneocin provides a reduction in the duration of the first phase of the wound process on average for 1 day.

debris, delimited by leukocyte shaft, consisting mainly of neutrophilic granulocytes in a state of severe dystrophy. For a long time, persistent neutrophilic infiltration of drugs is determined, which persists for 2 weeks of treatment. On the 14th day, in the majority of microscopic preparations, tissue debris was subjected to lysis and rejection. In preparations taken from infiltrate lymphohistiocytic elements prevail, in 56% of the micropreparations neutrophilic granulocytes constitute a significant proportion of cells. By day 21, lymphohistiocytic infiltration of varying severity is present in the wounds. There was no formation of argyrophil and collagen fibers, inhibition of fibroblast proliferation and maturation of granulation tissue was noted. In addition, it was noted slow resorption of tissue debris. In the study of microscopic specimens from the wounds of the main group, neutrophilic tissue infiltration was also maintained during the first 2 weeks of treatment during treatment. In dynamics, neutrophilic infiltration was observed in 32% of biopsy specimens. By day 14, lymphocytes, plasma cells, eosinophils, and macrophages prevailed in the preparations. On the 21st day after the start of treatment, the predominance of fibrous structures and the maturation of the connective tissue were observed in wound biopsy, indicating the beginning of regeneration. Thus, the presented histological data indicate a lengthening of the inflammation phase of the wound process on the background of diabetes mellitus, which is manifested by an increase in the neutrophil content leukocytes on the 14th day. The use of a vacuum system led to a more rapid cleansing of wounds during the treatment process.

Conclusions: The use of vacuum therapy in the complex treatment of purulent wounds contributed to a markedly rapid cleansing of wounds and, accordingly, a reduction in their area and depth, and an acceleration of the process of granulation and epithelialization compared with traditional treatment methods. 

The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3)'. JAMA 315

Assessment of Global Incidence and Mortality of Hospital-Treated Sepsis. Current Estimates and Limitations'. American Journal of Respiratory and Critical Care Medicine 193

Intensive Care Medicine 44

Procalcitonin Predicts Real-Time PCR Results in Blood Samples from Patients with Suspected Sepsis

A double-blind, placebo-controlled, randomised, multicentre, proof-of-concept and dose-finding phase II clinical trial to investigate the safety, tolerability and efficacy of adrecizumab in patients with septic shock and elevated adrenomedullin concentration (AdrenOSS-2)

Aquaporin 5 gene promoter-1364A/C polymorphism associated with 30-day survival in severe sepsis

AQP5-1364A/C polymorphism and the AQP5 expression influence sepsis survival and immune cell migration: a prospective laboratory and patient study

Aquaporin 5 -1364A/C Promoter Polymorphism Is Associated with Pulmonary Inflammation and Survival in Acute Respiratory Distress Syndrome

Genetic variants of intron region of aquaporin AQP5 gene and development of pulmonary edema in lung infection complicated by septic shock development

55-1.63) compared to matched pairs. Furthermore, patients with former septic shock (OR: 7.49, 5.97-9.39), sepsis (OR: 6.86, 6.53-7.21) as well as severe infections (OR: 6.84, 6.38-7.33) experienced a markedly greater risk of care dependency within 1 year after hospital discharge. Strikingly, patients treated in specialized aftercare facilities showed a significantly increased 5-year survival after suffering from sepsis or sepsis shock

Gawlytta R (1,2), Scherag A (2,3)

Adult patients (n = 83) on average 4 months after intensive care of (severe) sepsis or septic shock were included (median age 64 years, 60% male). PTSD symptom severity was assessed by three different self-report measures: two versions of the Posttraumatic Stress Scale (PTSS-10, PTSS-14), and the Posttraumatic Stress Disorder Checklist for DSM-5 (PCL-5). A clinical PTSD diagnosis was derived by using the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5). Results: Ten patients (12%) were diagnosed with PTSD. PTSS-10, PTSS-14, and PCL-5 revealed good reliability and concurrent validity. PTSS-14 showed the best accuracy in screening patients at risk for PTSD after intensive care with 80% sensitivity and 92% specificity at the recommended cutoff of 40

Internet-based cognitive-behavioral writing therapy reduces post-traumatic stress after intensive care in patients and their spouses: First results of the REPAIR trial

Niemeyer H (3), Böttche M (3,4), Scherag A (2,5)

Immunosuppressive activity of chronic wounds microflora associated with DFS is an important, though under evaluated pathogenesis factor leading to increased severity and sepsis

Associating SIRS, sepsis, cardio-vascular insufficiency and antiphospholipid syndrome in patients with diabetes type II complicated by diabetic foot syndrome

Sydorchuk OI (3), Vakarchuk AV

Identification and phenotypic characterization of the most frequent bacterial etiologies in chronic skin ulcers

Application of VAC-system in the treatment of diabetic foot syndrome Fuss J (1), Voloboyeva A (2)

Department of surgery, Bukovinian state medical university

Lavery L. Diabetic Foot Study Consortium. Negative pressure wound therapy after partial diabetic foot amputation: a multicentre, randomised controlled trial

Soft Tissue Reconstructive Options for the Ulcerated or Gangrenous Diabetic Foot

Gründling M (1), Vollmer M

Because of high mortality, prolonged hospital stays and increasing costs, invasive Candida infections of critically ill patients are of particular interest. Notably, surgical non-neutropenic septic patients are at high risk due to their risk profile

were analyzed. Candida Score [11] and Ostrosky-Zeichner's clinical decision rule [12] were constructed for every patient at the time of diagnosing sepsis. The predictive power of the two classifiers was evaluated and compared with a receiver operating characteristic. The primary endpoint was the presence of an invasive candidiasis. A logistic regression model was developed to identify significant risk factors and for prediction of invasive Candida infection. Results: The prevalence of invasive candida infections was 14.3% (89 cases). For the Candida Score a sensitivity of 89.4%, a specificity of 30.8%, a positive predictive value of 17.1% and a negative predictive value of 94.8% was found. Ostrosky-Zeichner's clinical decision rule performed with a sensitivity of 35.3%, a specify of 81.2% a positive predictive value of 23.1% and a negative predictive value of 88.7%. In the logistic regression model 5 major risk factors were found: age

The OR was 1.41 (95% CI 1.05-1.89) if three out of four minor risk factors (acute renal and circulatory failure, metabolic acidosis, mechanical ventilation) were present. The new rule (combining the major and minor risk factors) showed a higher area under the curve (AUC 0,798) than Candida Score (AUC 0,636) and Ostrosky-Zeichner

Neither the Candida Score nor the clinical decision rule (Ostrosky-Zeichner et al.) can be deemed to be significant for an empirical decision regarding antifungal therapy in critically ill patients at the time of sepsis diagnosis. The possible new rule has to be evaluated in an independent test group. There is still a need for more precise diagnosis in this particular subgroup of patients to identify the ones who benefit from empirical antifungal therapy and to avoid an overuse of antifungal therapy (this could cause increasing antifungal resistance, healthcare-costs and toxic side effects from antifungal therapy on patients

Clinical factors affecting costs in patients receiving systemic antifungal therapy in intensive care units in Greece: Results from the ESTIMATOR study

Septic shock attributed to Candida infection: Importance of empiric therapy and source control

Empirical Micafungin Treatment and Survival Without Invasive Fungal Infection in Adults With ICU-Acquired Sepsis, Candida Colonization, and Multiple Organ Failure: The EMPIRICUS Randomized Clinical Trial

Multicenter retrospective development and validation of a clinical prediction rule for nosocomial invasive candidiasis in the intensive care setting

Mikheev A (1), Gavryliuk O (1), Sydorchuk A (1), Bodnaruk O (1), Sydorchuk O

Not getting from mother Ig M, the newborn is induced by low protection against gram-negative bacteria. In newborns, the relative number of leukocytes and lymphocytes is reduced, their secretory activity and functional capacity of other immunocompetent cells, resulting in the formation of immunodeficiency state of varying degrees, which promotes the generalization of microbial infections. Objectives: The aim of the work was to study the taxonomic composition of microbiota purulent-septic processes in newborns and the establishment of microecological indicators of the ecosystem ''hostmicrobiome'' in purulent-septic process, transformed into a generalized infection

Prediction of Early-Onset Neonatal Sepsis (EONS) in pregnant women with Preterm Premature Rupture of membranes (PPROM) by vaginal microbiome analysis: a pilot project

) Department of Neonatology and Pediatric Intensive Care, Clinic for Child and Adolescent Medicine

22 mother-neonate pairs). Study design, an interim analysis of the study data and first sequencing results will be reported. Up to date results confirm the feasibility of the pilot trial. Conclusions: The PEONS pilot trial investigates the vaginal microbiota composition in patients with PPROM with regard to the development of an EONS. Based on preliminary results, the microbiota composition may predict an EONS and may correspond to the fetal microbiota. How far microbiota composition may predicts an EONS

Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin

) Department of Neonatology and Pediatric Intensive Care, Clinic for Child and Adolescent Medicine

Overall, the risk-rate of developing an early-onset neonatal sepsis after PPROM is no less than 14-22%

Up to now such discriminatory sepsis biomarker is not established in the diagnosis of EONS. Objectives: The hypotheses of the PEONS-CAAP48 project as nested co-study to the PEONS pilot trial are: 1. Is CAAP48 a putative biomarker to assess EONS early in umbilical cord blood of neonates born by pregnant women with PPROM? 2. Is the diagnostic performance of CAAP48 superior to the established biomarkers as CRP, IL-6, PCT or white blood cell count? Methods: The prospective multicenter PEONS pilot trial enrolls full age pregnant woman with PPROM event between 22 ? 0 and 34 ? 0 weeks of gestation (n = 65). Development of an EONS is defined as primary endpoint (group 1: EONS criteria met n = 15, group 2: EONS criteria not met n = 50). CAAP48 analysis is performed in antepartum (maternal blood), peripartum (maternal blood, umbilical cord blood) and postpartum (neonatal blood) serum samples by mass spectrometry according to a proof-of-concept. This is to perform a structured evaluation of CAAP48 as a potential pathogen derived marker within the exceptional and well characterized cohort of PPROM patients and their neonates. Results: Recruitment process started effectively

Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin

C-terminal alpha-1 antitrypsin peptide: a new sepsis biomarker with immunomodulatory function

Gender disparities in pediatric infection and sepsis in Germany

Schwarzkopf D (1,2), Thomas-Rueddel DO (1,2), Reinhart K (1,2,3)

Methods: Patients B 19 years treated with infection and sepsis between 2010 and 2015 were identified in a nation-wide database (DRG statistics) based on ICD-10 coded primary and secondary hospital discharge diagnoses. Sepsis was identified by 27 explicit sepsis ICD-10-codes, severe sepsis by ICD-10 codes R65.1 and R57.2. To identify infection-related hospitalizations, ICD-9 infection codes were adapted from Angus et al. (Crit Care Med 2001) and translated into ICD-10. We calculated population-based incidences based on nationwide population data of the Federal Statistical Office Germany for 2010-2015. Results: Between 2010 and 2015, 2.76 million children with infectious diseases were treated in German hospitals, of which 94.910 had sepsis (including 12.083 severe sepsis cases). In children aged \ 1 year, the incidence rate of infection (per 100.00 population) was 27% higher in males compared to females

Incidence rates of sepsis and severe sepsis were approx. 40% higher in male than in female children 1 year. Case fatality rates for sepsis were found to be higher in females than in males in young infancy (4.35% vs. 3.69%), but approximated and reversed late adolescence. Most pronounced differences in severe sepsis case fatality were found in children aged \ 1 year (males: 17

Planer L 036

Introduction: While septic shock or cardio-vascular insufficiency (CVI) is a major cause of death in sepsis, there is no clear concept concerning the role of immune disorders in its pathogenesis in sepsis backed by various metabolic and cardiovascular comorbidities. Objectives: The aim of the study was to evaluate the levels of antiphospholipid antibodies dependently of chronic CVI stages and complications appearance in patients with verified non-inflammatory thrombotic vessels pathology (NITVP) and metabolic disorders (diabetes type II complicated by diabetic foot syndrome) as a malicious background for sepsis. Methods: One hundred fifty four patients with NITVP and diabetes type II complicated by diabetic foot syndrome (DFS) were involved into the study (64 men and 90 women, mean age 58.4 ± 4.1 years): 1st group included 25 patients with CVI II, 2nd group-20 patients with CVI III, complicated with cerebral ischemic stroke and AMI control group included 10 patients with CVI I. All patients were tested for presence of NITVP and CVI by Doppler ultrasonography, duplex scanning, angiography, Echo-CG, Reoencephalography, BP and ECG Holter-monitoring, clinical laboratory examination. For research purpose the antibodies to cardiolipine (AKL) isotypes IgG and IgM were typified with the standard immune-enzyme method (ELISA), titres of antibodies to myocardial, renal, cerebral and vascular intimae antigens were investigated. Standard treatment and surgical procedures according to IDF Clinical Practice Recommendations were applied. All patients had sepsis or SIRS at least once during the course of disease, making it possible to establish connections with comorbidity and septic shock risk. Results: Isotype of IgMG-AKL antibodies' increasing was revealed in 5 patients of 1st group. Isotypes of IgG-AKL and IgM-AKL antibodies were increased in 5 and 4 patients of the 2nd group, accordingly. The titres of antibodies to myocardial, renal, cerebral and vascular intimae antigens were significantly higher in the patients of 1st and 2nd groups comparatively to the control group (p \ 0.05). Level of antibodies to target-organs antigens most closely correlated to the CVI in 2nd group (the highest correlation indices). Smaller, but still strong correlation coefficients of CVI to myocardial, renal, cerebral and vascular intimae antigens antibodies were in 1st group. Mortality among patients due to sepsis was as follows: 8 (32%) patients in group 1, 11 (55.0%) patients in group 2, and 1 (10.0%) in control.Conclusions: Data obtained in the study shows importance of comorbidity as a major factor predicting sepsis-related mortality. IgG-AKL and IgM-AKL or antiintimae, antirenal, antimyocardial and anticerebral antibodies were respective to CVI showing dependences between vascular failure, complications appearance and immune disorders in patients with diabetes type II complicated by sepsis. Objectives: To evaluate the impact of surgical approach on respiratory distress beyond pulmonary complications, oxygenation indexes (OI) of patients after right-sided thoracotomy for esophagectomy (ESO-group) are compared with those of patients after major lung resection (PULM-group). Methods: Retrospective single-center analysis of 181 patients after two-staged esophagectomy (n = 83) and major lung resection (n = 98). OI (PaO 2 /FiO 2 ) was calculated perioperatively until postoperative day ten. FiO 2 of patients without mechanical ventilation was estimated being 30%. OI \ 300 mmHg indicated respiratory distress. Discharge from intensive care unit indicated adequate respiratory function (OI C 300 mmHg). Mann-Whitney-U and Pearson's-v 2 test were used for two-group comparisons, Kaplan-Meier estimator calculated cumulative incidences. Results: The rate of reduced OI was higher initially in PULM-group (p = 0.009), and, although the thoracic part of esophagectomies was shorter as of pulmonary resections (p \ 0.0001), patients from ESOgroup suffered more frequently from reduced OI with statistical significance at all postoperative days. In line, cumulative incidence of reduced OI was higher in ESO-group versus PULM-group (p \ 0.001). The rate of postoperative pneumonia was higher in ESOgroup (22.9% versus 10.2%, p = 0.025) but misses significance (p = 0.1) in Kaplan-Meier analysis. Conclusions: Postoperative respiratory dysfunction depends on the surgical procedure. Beneath thoracotomy, duration of single-lung ventilation and perioperative inflammation other causes should be considered. A major difference between the two reported surgical procedures is impairment of parasympathetic branches of the vagal nerve, which may cause respiratory impairments by pulmonary edema formation.Introduction: A developing local infection in a diabetic foot is only a factor that aggravates the course of the diabetic foot and does not play an independent role in the development of this pathology [1, 2, 3] . One of the new methods used in the treatment of wounds is vacuum therapy or VAC therapy (vacuum -assisted closure). Vacuum therapy is a highly effective method of treating wounds, based on prolonged local effects of negative pressure on the wound [4, 5] . Negative pressure wound healing is an innovative technique that accelerates wound healing and allows successfully treat wounds that cannot be cured by other methods [6] . Objectives: Improving the results of treatment of patients with neuroischemic and neuropathic form DFS. Methods: Studies were conducted on the basis of the Department of General Surgery. The work is based on the results of treatment of 114 patients with purulent-necrotic complications of diabetic foot syndrome from 2015 to 2018. Of these, 23 patients with type 1 diabetes, 91-with type 2 diabetes. Men-49, women-65. The age of patients ranged from 38 years to 64 years. The study included patients with wounds after small operations performed and necroectomies within the foot with a depth of injury-grades II-III. According to the Wagner classification. In the structure of purulent-necrotic lesions of the feet, there was a predominance of phlegmon of the foot in 88 (77.1%) patients. Next in frequency is the gangrene of the toes of 26 (22.9%) patients. Patients were divided into two groups: control-traditional therapy was applied using ointment dressings and staged necrectomy-57 patients and the basic group-57 patients, vacuum therapy was used. The surface area of wounds ranged from 4.5 to 11.8 cm 2 and averaged 8.15 ± 1.1 cm 2 . In determining the microbial landscape of the wound flora, it was found that in most cases mixed flora was observed, most often represented by St. aureus, Ps. aeruginosa and E. coli. Results: In order to determine the effectiveness of this technique, we used such criteria as: the dynamics of the microbial landscape and the wound process, the timing of the wound defect closure. On the background of the treatment, there was a gradual decrease in the number of sowing microorganisms from wounds. So, in the dynamics in the control group, the frequency of detection of St. epidermidis decreased in a week by 20%, in 14 days-by 45%, and in the main group-by 26.1% and 65.2%, respectively. Cultivation of St. aureus in the control group after a week from the start of treatment decreased by 12.5%, after 14 days-by 37.5%, and in the basic group-by 25% and 75%, respectively. Wound healing in diabetes is characterized by lengthening of the inflammatory phase and inhibition of proliferation. In order to detect these changes and assess the impact of the use of vacuum therapy, we studied microscopic specimens of wound biopsy specimens. Biopsy samples were taken on days 7, 14, and 21 after the start of treatment. In the control group, in microscopic preparations taken by the end of 7 days from the start of treatment in the area of the wound defect clearly defined areas of tissue Conclusions: Gender-specific infection and sepsis incidence and case fatality rates differ between age groups. Trends observed in young infants, such as an elevated sepsis incidence in males and an increased sepsis mortality in females, disappeared or reversed in adolescence. Further research is needed to understand these differences between genders, in particular regarding predisposing comorbidities, underlying risk factors such as prematurity, and characteristics of hospital treatment.