EHRLICHIACIDAL MECHANISM BY MACROPHAGE CA++ MOBILIZATION

埃立克体能引起人类埃立克体病和过敏等疾病 埃立克体病。埃立克次体是一组独特的立克次体，是专性的 巨噬细胞或粒细胞的胞内寄生虫。它们会成倍增加 不与溶酶体融合的膜结合的液泡。的目标是 这个项目是为了找出巨噬细胞激活剂是否会杀死 以及如果是这样的话，如何才能完全消除埃利希菌 宿主细胞。我们将检验以下具体假设。 假设1：埃利希菌感染阻止了钙离子流入巨噬细胞， 因此，能够在感染的巨噬细胞中动员钙离子的药物会杀死 埃利希亚科。目标1：通过检查剂量和时间来检验假设1：a) 巨噬细胞清除埃立克体对这些反应的反应 试剂，b)通过测量细胞内钙离子，以及c)通过检查 钙通道阻滞剂或钙拮抗剂的作用。 假设2：钙离子动员诱导的埃立克体杀伤是介导的 通过蛋白激酶C、钙调蛋白、磷脂酶A2和/或cAMP或cGMP。目标 2：通过a)评估蛋白激酶C的作用来检验假设2 钙调素拮抗剂、钙离子依赖性磷脂酶A2 抑制物，以及增加杀灭埃立克体上cAMP和cGMP的药物 钙离子激动剂的影响，以及b)磷酸二酯酶的测定， 蛋白激酶C、磷脂酶A2活性和/或cAMP、cGMP。 假设3：巨噬细胞中的钙离子动员通过以下方式破坏埃立克体 溶酶体与含有埃立克氏菌的液泡融合的启动 伴随着微管蛋白的聚合和重组增加 感染的巨噬细胞胞浆内的肌动蛋白。目标3：通过a检验假设3) 检查用电子密度计标记的溶酶体的融合 含有埃利希亚科的液泡酸性磷酸酶细胞化学，由b) 微丝和微管的荧光标记，以及c) 检测微管和微丝破碎剂的效果。

Ehrlichiae cause diseases such as human ehrlichiosis and sennetsu ehrlichiosis. Ehrlichiae are a unique group of rickettsiae and are obligate intracellular parasites of macrophages or granulocytes. They multiply in the membrane-bound vacuoles which do not fuse with lysosomes. The goal of this project is to find whether macrophage activating agents kill ehrlichiae and if so how ehrlichiae can be completely eliminated from the host cells. We will test the following specific hypotheses. Hypothesis 1: Ehrlichial infection blocks Ca2+ influx into the macrophage, thus the agents which can mobilize Ca 2+, in infected macrophages kill ehrlichiae. AIM 1: Tests hypothesis 1: a) by examining dose- and time- responses of macrophages in eliminating ehrlichiae in response to these agents, b) by measuring intracellular Ca2+ ion, and c) by examining the effects of Ca2+ channel blockers or Ca2+ antagonists. Hypothesis 2: Ehrlichial killing induced by Ca2+ mobilization is mediated by protein kinase C, calmodulin, phospholipase A2 and/or cAMP, or cGMP. AIM 2: Tests hypothesis 2 by a) evaluating the effects of protein kinase C inhibitors, calmodulin antagonists, Ca2+-dependent phospholipase A2 inhibitors, and agents which increase cAMP and cGMP on the ehrlichiacidal effects of Ca 2+ mobilizing agents, and b) measuring phosphodiesterase, protein kinase C, phospholipase A2 activity and/or cAMP and cGMP. Hypothesis 3: Ca2+ mobilization in the macrophage destroys ehrlichiae by initiation of lysosomal fusion with ehrlichia-containing vacuoles which is accompanied by increased polymerization and reorganization of tubulin and actin in the infected macrophage cytoplasm. AIM 3: Tests hypothesis 3 by a) examining the fusion of lysosomes labeled with electron densetracers or acid phosphatase cytochemistry with ehrlichiae-containing vacuoles, by b) fluorescent labeling of microfilaments and microtubules, and by c) examining the effect of microtubule and microfilament disrupting agents.