Modulation of inflammation and sepsis by bacterial PAL

细菌 PAL 对炎症和脓毒症的调节

• 批准号: 6821965
• 负责人: Judith Hellman
• 金额: $ 31.45万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6821965
• 关键词: bacterial proteins; blood lipoprotein; blood pressure; cell cell interaction; chemotaxis; clinical research; echocardiography; flow cytometry; genetically modified animals; heart function; human subject; immune response; immunoregulation; inflammation; laboratory mouse; leukocyte activation /transformation; lung lavage; molecular pathology; neutrophil; peptidoglycan; protein structure function; respiratory function; septicemia; toll like receptor; virulence

DESCRIPTION (provided by applicant): 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.

描述(由申请人提供)： 早期的败血症反应是由微生物产物激活的，微生物产物通过Toll样受体(TLRs)启动炎症。细菌肽多糖相关脂蛋白(PAL)是一种普遍存在的革兰氏阴性细菌外膜蛋白。PAL是一种有效的TLR2激动剂，在脓毒症时释放到血液中。中心假说是，PAL参与炎症和凝血障碍，以及败血症时的心血管和肺功能障碍。研究将探索PAL单独的作用，并将测试PAL与通过各种TLRs激活的其他促炎细菌产物之间的协同作用。将使用TLR2基因敲除小鼠评估TLR2在PAL败血症反应中的作用。具体目的1：探讨PAL在细胞和全身炎症及凝血中的作用。将通过测量黏附分子和凝血因子水平来研究内皮细胞的激活。中性粒细胞的活化将通过流式细胞仪分析CD11b的表达和趋化试验来评估。中性粒细胞与内皮细胞的相互作用将使用黏附试验进行研究。细胞因子水平将被用来评估巨噬细胞的激活。在注射PAL后，将测量小鼠循环中细胞因子和凝血因子的水平，血小板和中性粒细胞计数，以及中性粒细胞的激活。具体目标2：研究PAL和TLR2在心血管和肺反应中的作用。对血管张力和对心肌功能的影响将使用有创和非侵入性手段进行评估，包括超声心动图、左心室电导导管和全身动脉血压监测仪。肺反应将通过测量动脉血气、肺湿重/干重和分析支气管肺泡灌洗液来评估。具体目标3：评估PAL在脓毒症中促进细菌毒力的机制。缺乏PAL或具有异常PAL的细菌突变体将被利用。这些研究将测量细胞和系统对热致死的PAL缺失突变体和纯化的未酰化PAL的反应。此外，还将在腹膜炎脓毒症模型中比较野生型和PAL突变大肠杆菌对野生型和TLR2基因敲除小鼠的影响。这些拟议的研究将使用PAL作为一种工具，以深入了解细菌脂蛋白和TLR2在脓毒症诱导的过程中的作用，包括内皮细胞功能障碍、全身性炎症以及心血管和肺功能障碍。