Early septic responses are activated by microbial products that initiate inflammation through Toll-like receptors (TLRs). Bacterial peptidoglycan-associated lipoprotein (PAL) is a ubiquitous Gram-negative bacterial outer membrane protein. PAL is a potent TLR2 agonist that is released into the blood in sepsis. The central hypothesis is that PAL contributes to inflammation and coagulopathy, and to cardiovascular and pulmonary dysfunction in sepsis. Studies will explore effects of PAL alone and will test for synergy between PAL and additional pro-inflammatory bacterial products that activate through various TLRs. The role of TLR2 in septic responses to PAL will be evaluated using TLR2 knockout mice.
Specific Aim 1 : Explore the role of PAL in cellular and systemic inflammation and coagulation. Endothelial cell activation will be studied by measuring adhesion molecule and coagulation factor levels. Neutrophil activation will be assessed by FACS analysis for expression of CD11b and chemotaxis assays. Neutrophil-endothelial cell interactions will be studied using adherence assays. Cytokine levels will be utilized to evaluate macrophage activation. Circulating levels of cytokines and coagulation factors, platelet and neutrophil counts, and neutrophil activation will be measured in mice following injection with PAL.
Specific Aim 2 : Characterize the roles of PAL and TLR2 on cardiovascular and pulmonary responses. Effects on vascular tone and on myocardial function will be assessed using invasive and non-invasive means, including echocardiography, left ventricular conductance catheters, and systemic arterial blood pressure monitors. Pulmonary responses will be assessed by measuring arterial blood gases, lung wet-to-dry weights, and by analysis of bronchoalveolar lavage fluid.
Specific Aim 3 : Evaluate mechanisms by which PAL contributes to bacterial virulence in sepsis. Bacterial mutants that lack or have abnormal PAL will be utilized. These studies will measure cellular and systemic responses to heatkilled PAL-deletion mutants and to purified unacylated PAL. In addition, studies will be performed to compare effects of wild-type and PAL-mutant E. coli bacteria on wild-type and TLR2 knockout mice in a peritonitis sepsis model. These proposed studies will use PAL as a tool to gain insight into the role of bacterial lipoproteins and of TLR2 in sepsis-induced processes, including endothelial cell dysfunction, generalized inflammation, and cardiovascular and pulmonary dysfunction.
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