INTRACELLULAR INFECTION OF HUMAN ALVEOLAR MACROPHAGES

描述 (改编自申请者摘要)嗜肺军团菌和分枝杆菌 退伍军人病和结核病的病原体--结核病 分别是寄生于细胞内的兼性病原体 人肺泡巨噬细胞和外周血单核细胞在肺脏部位的募集 感染。宿主对这些感染的有效反应取决于 细胞介导的免疫和细胞因子网络调节这种反应。这个 支持这一提议的总体假说是肺炎链球菌和麻黄杆菌。 结核病对人肺泡巨噬细胞细胞因子应答的影响 通过诱导允许的宿主细胞因子促进自身的生存 解除吞噬细胞的保护性抗微生物机制。这个 具体目标是：1)确定人细胞因子激活的作用 肺泡巨噬细胞在决定肺耐受或消除中的作用 嗜肺性乳杆菌和结核分枝杆菌的细胞内感染；2) 确定感染的肺泡巨噬细胞的凋亡是否会降低 细胞内嗜肺乳杆菌和结核分枝杆菌的生存能力；3)至 转导A基因的嗜肺乳杆菌强毒株的选育和使用 维多利亚绿色荧光蛋白基因共定位于细胞内 人肺泡巨噬细胞宿主的细菌和特定成分 在被感染的细胞中的反应。具体目标的实验方法 将使用已建立的体外模型，在该模型中，人肺泡巨噬细胞 感染嗜肺乳杆菌或结核分枝杆菌。细胞因子的作用 将使用选定的组合来测量细胞内生长 重组细胞因子和封闭抗体。特定的交互作用 活细菌和细胞壁糖脂诱导的细胞因子信号 通过阻断补体受体、CD14和LBP进行评估。巨噬细胞 将通过细胞形态、Annexin V结合和 DNA片段化分析和细菌活性的相关变化将 被量化。将评估受感染细胞对单个细胞的反应 应用流式细胞术和荧光显微镜共定位绿色 用选定的标记表达荧光蛋白的细菌菌株 主场防守。这些研究将提供对人类的重要见解 宿主对嗜肺乳杆菌和结核分枝杆菌感染的反应。一次彻底的 对这些反应的理解对于制定 军团病的合理免疫治疗途径 疾病和结核病，这将对 免疫功能低下患者和耐药感染的治疗 菌株。(摘要结束)

DESCRIPTION (Adapted from applicant's abstract) Legionella pneumophila and Mycobacterium tuberculosis, the etiologic agents of Legionnaires' disease and tuberculosis respectively, are facultative intra-cellular pathogens which parasitize human alveolar macrophages and blood monocytes recruited to the site of lung infection. An effective host response to these infections depends on cell-mediated immunity, and cytokine networks modulate this response. The overall hypothesis driving this proposal is that L. pneumophia and M. tuberculosis subvert the human alveolar macrophage cytokine response to facilitate their own survival by inducing permissive host cytokines which disarm the protective antimicrobial mechanisms of the phagocyte. The specific aims are: 1) to define the role of cytokine activation of human alveolar macrophages in determining the tolerance or elimination of intracellular infection by L. pneumophila and M. tuberculosis; 2) to determine whether apoptosis of infected alveolar macrophages diminishes the viability of intra-cellular L. pneumophila and M. tuberculosis; 3) to develop and use a virulent L. pneumophila strain transduced with the A victoria green fluorescent protein gene to co-localize intracellular bacteria and specific components of the human alveolar macrophage host response in infected cells. The experimental approach to the specific aims will use an established in vitro model in which human alveolar macrophages are infected with L. pneumophila or M. tuberculosis. Effects of cytokines on intracellular growth will be measured using selected combinations of recombinant cytokines and blocking antibodies. Specific interactions signaling cytokine induction by live bacteria and cell wall glycolipids will be assessed by blocking complement receptors, CD14, and LBP. Macrophage apoptosis will be assessed by cellular morphology, Annexin V binding, and by DNA fragmentation assays, and associated changes in bacterial viability will be quantitated. Individual cell response by infected cells will be assessed by flow cytometry and fluorescent microscopy co-localization of green fluorescent protein expressing bacterial strains with selected markers of host defense. These studies will provide important insights into the human host responses to L. pneumophila and M. tuberculosis infections. A thorough understanding of these responses will be necessary for the development of rational immunotherapeutic approaches to the treatment of Legionnaires' disease and tuberculosis, which will be increasingly important for the treatment of immunocompromised patients and infections with drug-resistant strains. (End of Abstract)