Modulation of inflammation and sepsis by bacterial PAL

细菌 PAL 对炎症和脓毒症的调节

• 批准号: 7234110
• 负责人: Judith Hellman
• 金额: $ 32.76万
• 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/7234110
• 关键词: Adherence; Agonist; Bacteria; Bacterial DNA; Bacterial Outer Membrane Proteins; Biological Assay; Blood; Blood Circulation; Blood Coagulation Disorders; Blood Coagulation Factor; Blood Platelets; Blood Pressure Monitors; Blood Vessels; Blood gas; Bronchoalveolar Lavage Fluid; Cardiac; Cardiovascular system; Catheters; Cell Adhesion Molecules; Cell Communication; Cell Wall; Cells; Chemotaxis; Coagulation Process; Count; Data; Dominant-Negative Mutation; Echocardiography; Endothelial Cells; Escherichia coli; Flagellin; Functional disorder; Heart; Heating; Human; ITGAM gene; Immune response; Inflammation; Inflammatory; Injection of therapeutic agent; Intraperitoneal Injections; Invasive; Knockout Mice; Left; Lipids; Lipoprotein (a); Lipoprotein (a-); Lipoproteins; Lung; Macrophage Activation; Measurement; Measures; Mediating; Modeling; Modification; Mus; Myocardial; Neutrophil Activation; Peptidoglycan; Perfusion; Peritoneal Fluid; Peritonitis; Peroxidase; Process; Proteins; Relative (related person); Role; Sepsis; Structure of parenchyma of lung; TLR4 gene; Testing; Toll-Like Receptor 2; Toll-like receptors; Toxic effect; Transfection; Ventricular; Virulence; Weight; clinically relevant; cytokine; human NOS2A protein; insight; intravenous administration; killings; mRNA Expression; macrophage; microbial; mutant; neutrophil; northern hybridization; peptidoglycan-associated lipoprotein; pressure; response; septic; tool; wet lung

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）： 早期败血症反应是由微生物产物激活的，这些微生物产物通过 Toll 样受体 (TLR) 引发炎症。细菌肽聚糖相关脂蛋白（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，在败血症时会释放到血液中。核心假设是 PAL 会导致炎症和凝血病，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 TLR 激活的其他促炎细菌产物之间的协同作用。 TLR2 在 PAL 脓毒症反应中的作用将使用 TLR2 敲除小鼠进行评估。具体目标 1：探索 PAL 在细胞和全身炎症和凝血中的作用。通过测量粘附分子和凝血因子水平来研究内皮细胞活化。将通过 FACS 分析 CD11b 的表达和趋化性测定来评估中性粒细胞活化。将使用粘附测定来研究中性粒细胞-内皮细胞相互作用。细胞因子水平将用于评估巨噬细胞的激活。注射 PAL 后，将测量小鼠体内细胞因子和凝血因子的循环水平、血小板和中性粒细胞计数以及中性粒细胞活化。具体目标 2：描述 PAL 和 TLR2 对心血管和肺部反应的作用。将使用侵入性和非侵入性手段评估对血管张力和心肌功能的影响，包括超声心动图、左心室电导导管和全身动脉血压监测仪。将通过测量动脉血气、肺湿干重以及分析支气管肺泡灌洗液来评估肺部反应。具体目标 3：评估 PAL 导致脓毒症细菌毒力的机制。将利用缺乏或具有异常 PAL 的细菌突变体。这些研究将测量细胞和系统对热灭活的 PAL 缺失突变体和纯化的未酰化 PAL 的反应。此外，还将进行研究，以比较野生型和 PAL 突变型大肠杆菌对腹膜炎脓毒症模型中野生型和 TLR2 敲除小鼠的影响。这些拟议的研究将使用 PAL 作为工具来深入了解细菌脂蛋白和 TLR2 在脓毒症诱发过程中的作用，包括内皮细胞功能障碍、全身炎症以及心血管和肺功能障碍。