INTRACELLULAR INFECTION OF HUMAN ALVEOLAR MACROPHAGES

描述 （改编自申请人摘要）嗜肺军团菌和分枝杆菌 结核病、军团病和结核病的病原体 分别是兼性细胞内病原体， 人肺泡巨噬细胞和血单核细胞募集到肺的部位 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应答。 的 提出这一建议的总体假设是L. pneumoniae和M. 结核病破坏人肺泡巨噬细胞的细胞因子反应， 通过诱导允许的宿主细胞因子促进其自身的存活， 解除吞噬细胞的保护性抗菌机制 的 具体的目的是：1）确定细胞因子激活在人类 肺泡巨噬细胞在确定耐受性或消除 胞内感染L. pneumophila和M.结核病; 2） 确定感染的肺泡巨噬细胞的凋亡是否减少了 细胞内L. pneumophila和M.结核病; 3） 开发和利用一种强毒L.用A. 维多利亚绿色荧光蛋白基因共定位于细胞内 细菌和人肺泡巨噬细胞宿主的特定成分 感染细胞的反应。 具体目标的实验方法 将使用已建立的体外模型，其中人肺泡巨噬细胞 感染L. pneumophila或M.结核 细胞因子的作用 对细胞内生长的影响将使用以下物质的选定组合来测量： 重组细胞因子和阻断抗体。 特异性相互作用 活细菌和细胞壁糖脂的信号细胞因子诱导将 通过阻断补体受体、CD14和LBP进行评估。 巨噬 细胞凋亡将通过细胞形态学、膜联蛋白V结合和 DNA片段化测定以及细菌活力的相关变化将 要量化。 将评估受感染细胞的个体细胞应答 通过流式细胞术和荧光显微镜共定位绿色 表达荧光蛋白的细菌菌株，所述细菌菌株具有选择的 宿主防御 这些研究将提供重要的见解，人类 寄主对L. pneumophila和M.肺结核感染。 的透彻 了解这些反应将是必要的发展 合理的免疫治疗方法 疾病和结核病，这将是越来越重要的， 治疗免疫功能低下的患者和耐药性感染 菌株 (End摘要）

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)