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）： 早期的化粪池反应被通过Toll样受体（TLR）引发炎症的微生物产物激活。细菌肽聚糖相关的脂蛋白（PAL）是无处不在的革兰氏阴性细菌外膜蛋白。 PAL是一种有效的TLR2激动剂，在败血症中释放到血液中。中心假设是PAL有助于炎症和凝血病，以及败血症中的心血管和肺功能障碍。研究将探索单独使用PAL的影响，并将测试通过各种TLR激活的PAL和其他促炎细菌产品之间的协同作用。 TLR2在对PAL的作用中的作用将使用TLR2基因敲除小鼠进行评估。特定目标1：探索PAL在细胞和全身炎症和凝结中的作用。通过测量粘附分子和凝结因子水平，将研究内皮细胞活化。中性粒细胞激活将通过FACS分析评估CD11b和趋化测定法的表达。将使用依从性测定研究中性粒细胞 - 内皮细胞相互作用。细胞因子水平将用于评估巨噬细胞激活。用PAL注射后，将在小鼠中测量细胞因子和凝结因子，血小板和中性粒细胞的循环水平。特定目标2：表征PAL和TLR2在心血管和肺部反应中的作用。对血管张力和心肌功能的影响将使用侵入性和非侵入性手段进行评估，包括超声心动图，左心室电导导管以及全身性动脉血压监测器。肺反应将通过测量动脉血气，肺湿重量和支气管肺泡灌洗液分析来评估肺反应。特定目的3：评估PAL在脓毒症中有助于细菌毒力的机制。缺乏或具有异常PAL的细菌突变体将被使用。这些研究将测量对热焊接的PAL脱落突变体的细胞和全身反应，并纯化未囊化的PAL。此外，将进行研究以比较脓肿模型中野生型和pal型大肠杆菌对野生型和TLR2敲除小鼠的影响。这些提出的研究将使用PAL作为一种工具，以深入了解细菌脂蛋白和TLR2在败血症诱导的过程中的作用，包括内皮细胞功能障碍，广义炎症以及心血管和肺功能障碍。