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Background: Most clinical trials of sepsis treatment modalities fail at their primary objective of establishing superiority over placebo when added to background standard of care. While there is no definitive explanation for the high failure rate, it might be stated that our attempts to insert a new therapeutic agent into standard of care encounters severe problems with definition of exactly what stage is ongoing, and what are the criteria for progression or resolution from that time point onwards. Clearly there is need for a means of defining steps in the septic process that would apply to individuals, and to better define the course of sepsis in each patient after they are enrolled in a trial. Methods: For core model development, 30 septic patients were studied for time-related progression in relation to biomarkers, employing a Load Model in a neural net algorithm in MatLab. Causative bacterial infections were linked to primary infection sites. In order to minimize overparameterization, the model was allowed to estimate outputs using the best three input parameters. Bacterial load was tracked from origin using clinical and microbiologic data to provide an estimate at the start of sepsis. The bacterial load as well as clinical and laboratory parameters were model inputs with the output parameter being organ failures and/ or mortality. Results: At onset of sepsis, human bacterial load estimates ranged from between 10 8 and 10 11 CFU, which is consistent with inocula in animal models of sepsis. Sepsis proceeds to organ failures and mortality in a series of steps that are initially linked to bacterial load and inflammatory response, followed by coagulopathy, ischemia, oxygen deprivation in organs and tissues, and culminating in organ failures. The later stages of sepsis are all driven by metabolic parameters, and there seems to be little benefit to blocking inflammation at later stages. Substrate and oxygen deficiencies must be addressed first. Conclusion: Neural net progression models based on biomarkers and physiological markers are able to describe the evolution of sepsis to septic shock, organ failures, and provide some evidence that mortality may be a consequence of the stages of sepsis. Overall, these models appear useful to the task of sorting out organ failure endpoints and mechanisms in individual patients with sepsis progression across sepsis to septic shock. P2 Extracellular matrix turnover, angiogenesis and endothelial function in acute lung injury: relationship to pulmonary dysfunction and outcome

Introduction: During the course of systemic inflammation, most of the immune cell types get activated to a certain degree as part of, or contributing to, the cascade of physiopathological events. Whether for some cells, classically phagocytes of the innate immune system, it is clear that direct sensing of pathogen-associated molecular patterns leads to activation initiating systemic inflammation, the picture is not so clear for natural killer (NK) cells. While NK cells have been shown to express toll-like receptors (TLR), the role of these receptors on NKs during systemic inflammation has not been directly addressed. Methods: To directly assess the role of TLR expression on NK cells we used an adoptive transfer model in which NKs purified from the spleens of WT, TLR4KO and TLR2/4DKO mice were transferred intravenously to RAG2-/γc-/-(devoid of T, B and NK cells). Five days after reconstitution the mice were challenged intraperitoneally with conventional or TLR-grade lipopolysaccharide (LPS). Immune cell activation and production of IFNγ by NK cells was determined after 6 hours by FACS analysis. Results: We observed no differences in reconstitution of the recipient mice with NK cells from different backgrounds suggesting no difference in trafficking and survival of the transferred cells. At 6 hours after LPS challenge, WT, TLR4KO or TLR2/4DKO NK cells recovered from the spleen and lungs of RAG2-/γc-/mice showed comparable levels of CD69 activation marker expression. Intracellular labeling for IFNγ in NK cells also revealed no significant differences. Conclusion: Whether there is a role for direct TLR signaling on NK cells remains the objective of further investigations; however, our data show that in the course of a systemic inflammatory process, like endotoxinemia, the expression of TLR2 and TLR4 by NK cells makes no difference in terms of their activation and secretion of IFNγ P2 Role of 6-hour, 12-hour, and 24-hour lactate clearance in mortality of severe sepsis and septic shock patients.