RICKETTSIA-INDUCED TRANSCRIPTIONAL ACTIVATION

说明(改编自申请人的摘要)： 此应用程序旨在了解内皮细胞 细胞-立克次体相互作用引发细胞反应 立克次体病的发病机制。血管细胞内感染 感染立克次体的内皮细胞引起基因改变 转录导致促凝剂和促炎因子的表达 分子。这些内皮细胞的反应可能在 与人类感染相关的病理变化，导致 这种疾病被称为落基山斑点热(RMSF)。初步研究 提示立克次体感染导致细胞活化。 转录因子核因子-kB(NF-kB)，控制表达 在许多可诱导基因中，涉及对炎症刺激的早期反应。 建议的研究将集中在立克次体诱导核因子-kB的机制上。 激活。该提案分为两个具体目标。第一 具体目标将集中在表征核因子-kB的激活 发生在培养的内皮细胞感染立克次体的过程中， 并将包括对活化动力学的研究以及分子 活化络合物的表征。第二个具体目标是 利用两种实验方法探索细胞内信号转导 在立克次体诱导的核因子-kB激活中起作用的通路。去探索 在诱导这种细胞中起重要作用的生物体的特征 为研究核因子-kB的活性，建立了一套研究核因子-kB活性的系统 寄主细胞胞质。通过绕过进入过程，该系统将允许 对立克次体的广泛操纵仍然允许直接 立克次体与宿主细胞信号机制的相互作用。抗肿瘤药物 关键的调节分子也将用于完整的细胞中，以识别 细胞内感染的“靶点”导致激活。这个 研究人员推测，立克次体诱导的核因子-kB激活是 通过有机体与宿主细胞成分的相互作用来调节，但 这种反应可能与已知的生理诱导剂略有不同 关于动力学和异构体的专一性。由于主要事件在 RMSF的病程是广泛的微血管血栓形成，从中获得的启示 这些研究可能不仅在内皮细胞方面提供有价值的见解 与立克次体疾病有关，但一般与血栓性疾病有关。

DESCRIPTION (Adapted from the applicant's abstract): Studies described in this application are designed to understand mechanisms by which endothelial cell-rickettsia interactions elicit cellular responses critical to the pathogenesis of rickettsial disease. Intracellular infection of vascular endothelial cells with R. rickettsii results in changes in gene transcription leading to expression of procoagulant and proinflammatory molecules. These endothelial cell responses are likely important in the pathologic changes associated with human infection, which results in the disease known as Rocky Mountain Spotted Fever (RMSF). Preliminary studies indicate that R. rickettsii infection results in activation of the transcription factor, nuclear factor -kB (NF-kB), which controls expression of many inducible genes involved in early responses to inflammatory stimuli. The studies proposed will focus on mechanisms of R. rickettsii induced NF-kB activation. The proposal is divided into two specific aims. The first specific aim will concentrate on characterization of NF-kB activation which occurs during infection of cultured endothelial cells with R. rickettsii, and will include study of the kinetics of activation as well as molecular characterization of the activated complex. The second specific aim will utilize two experimental approaches to explore intracellular signaling pathways operative in R. rickettsii-induced NF-kB activation. To explore characteristics of the organism important in eliciting this cellular response, a system was developed to study activation of NF-kB in isolated host cell cytoplasm. By bypassing the entry process, this system will allow extensive manipulation of the rickettsia organisms yet still allow direct interaction of rickettsia with host cell signaling machinery. Inhibitors of key regulatory molecules will also be used in intact cells to identify intracellular "targets" of infection resulting in activation. The investigators hypothesize that R. rickettsii induced NF-kB activation is mediated via interaction of the organism with host cell components, but that the response likely differs somewhat from known physiologic inducers with regard to kinetics and isoform specificity. Since a primary event in the course of RMSF is extensive microvascular thrombosis, insights gained from these studies may provide valuable insights not only in endothelial cell involvement in rickettsial disease, but in thrombotic disease in general.