Regulation of CXC chemokine expression by H. pylori.

描述（由申请人提供）：幽门螺杆菌引起胃炎症、消化性溃疡和胃癌。最近的临床和体外研究表明，cag致病岛（派）和外部炎症蛋白OipA都参与了胃粘膜白细胞介素（IL）-8水平的产生和由此产生的炎症。旨在理解cag PAl相关IL-8信号转导途径的已发表数据提供了不一致的结果，这可能是因为未考虑OipA的作用。本研究旨在了解幽门螺杆菌感染刺激的IL-8信号转导通路。我假设oipA基因产物激活干扰素（IFN）调节因子（IRES）与IL-8启动子中的新型干扰素刺激反应元件（ISRE）样元件的结合，并且oipA基因产物激活p38促分裂原活化蛋白（MAP）激酶级联，而cag派则不激活。我的方法包括使用父母的H。pylori菌株和oipA、cag派、cagA、hopZ或oipA/cag派基因的无极性效应的精确基因缺失突变体，以及互补的oipA突变体。目的1探讨OipA和cag派对IL-8基因转录的调控机制。我将测试假设OipA和/或cag派激活结合IRFs的ISRE样元件，导致IL-8基因转录。我将鉴定与OipA和cag PAl相关的IL-8启动子上游的MAP激酶途径。我还将确定在IL-8启动子上游的IRF-ISRE样元件途径、MAP激酶途径和IkappaB-NF-kappaB途径之间是否存在串扰（即，是相互联系还是相互独立）。使用的主要方法包括a）荧光素酶报告基因测定，B）电泳迁移率变动测定（EMSA）和超变动测定，e）微亲和分离测定，d）RNA干扰，和e）蛋白质印迹分析。目的2通过观察OipA和cag派对长爪沙鼠胃损伤的影响，进一步证实和推广上述体外实验结果。本发明人将确定OipA和cag PAL对胃炎症、临床结果、CXC趋化因子（KC和IP- 10）诱导的影响，以及与OipA和cag PAL相关的KC和IP- 10启动子的分析。待使用的主要方法包括a）真实的时间逆转录-聚合酶链反应，B）酶联免疫吸附测定，e）EMSA和supershift测定，以及d）Western印迹分析。这些研究结果将为H.幽门螺杆菌在胃十二指肠疾病发病中的作用。

DESCRIPTION (provided by applicant): Helicobacter pylori causes gastric inflammation, peptic ulcer disease, and gastric cancer. Recent clinical and in vitro data indicate that both the cag pathogenicity island (PAI) and the outer inflammatory protein, OipA are involved in production of gastric mucosal interleukin (IL)-8 levels and the resulting inflammation. Published data aimed at understanding the cag PAl-associated IL-8 signal transduction pathway have provided inconsistent results possibly because the effect of OipA was not taken into account. This proposed study seeks to understand the IL-8 signal transduction pathway stimulated by H.pylori infection. I hypothesize that the oipA gene product activates the binding of interferon (IFN) regulatory factors (IREs) to a novel interferon-stimulated responsive element (ISRE)-like element in the IL-8 promoter and that the oipA gene product activates p38 mitogen-activated protein (MAP) kinase cascades, which the cag PAI does not. My approach involves use of parental H. pylori strains and precise gene deleted mutants of oipA, cag PAI, cagA, hopZ or oipA/cag PAI genes without polar effects, as well as complemented oipA mutants. AIM 1 seeks to determine the mechanisms for regulation of IL-8 gene transcription in relation to OipA and cag PAI. I will test the hypothesis that OipA and/or cag PAI activates binding IRFs to the ISRE-like element, leading to IL-8 gene transcription. I will identify the MAP kinase pathways upstream of the IL-8 promoter related to OipA and the cag PAl. I will also determine whether there is cross-talk among the IRF-ISRE-Iike element pathway, the MAP kinase pathway, and IkappaB-NF-kappaB pathway upstream of the IL-8 promoter (i.e., are they linked or independent). Primary methods to be used include a) a luciferase reporter gene assay, b) electrophoretic mobility shift assays (EMSA) and supershift assay, e) microaffinity isolation assay, d) RNA interference, and e) western blot analysis. AIM 2 seeks to confirm and extend the in vitro results by investigation of the in vivo effect of OipA and the cag PAI on gastric injury using the Mongolian gerbil model. I will determine the effect of OipA and cag PAl on gastric inflammation, clinical outcome, CXC chemokine (KC and IP- 10) induction and analyses of KC and IP- 10 promoter related to OipA and the cag PAL Primary methods to be used include a) real time reverse transcription-polymerase chain reaction, b) enzyme-linked immunosorbent assay, e) EMSA and supershift assay, and d) Western blot analysis. The results from these studies will provide new insights into the role of H. pylori in the pathogenesis of gastroduodenal disease.