Regulation of CXC chemokine expression by H. pylori.

描述（申请人提供）：幽门螺杆菌引起胃部炎症、消化性溃疡疾病和胃癌。最近的临床和体外数据表明，cag致病性岛（PAI）和外炎症蛋白OipA都参与了胃黏膜白细胞介素(IL)-8水平的产生和由此引起的炎症。旨在了解cag pal相关的IL-8信号转导途径的已发表数据提供了不一致的结果，可能是因为没有考虑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启动子中一种新的干扰素刺激响应元件（ISRE）样元件的结合，并且oipA基因产物激活p38有丝分裂原活化蛋白（MAP）激酶级联反应，而cag PAI则没有。我的方法包括使用亲本幽门螺杆菌菌株和oipA、cag PAI、cagA、hopZ或没有极性效应的oipA/cag PAI基因的精确基因缺失突变体，以及补充oipA突变体。AIM 1旨在确定与OipA和cag PAI相关的IL-8基因转录调控机制。我将验证OipA和/或cag PAI激活结合irf到ISRE-like元件，导致IL-8基因转录的假设。我将确定与OipA和cag PAl相关的IL-8启动子上游的MAP激酶途径，并确定IL-8启动子上游的irf - isre - like元件途径、MAP激酶途径和IkappaB-NF-kappaB途径之间是否存在串扰（即它们是连接的还是独立的）。使用的主要方法包括a)荧光素酶报告基因测定，b)电泳迁移率转移测定（EMSA）和超转移测定，e)微亲和分离测定，d) RNA干扰，e) western blot分析。AIM 2利用蒙沙鼠模型研究OipA和cag PAI在体内对胃损伤的影响，以证实和扩展体外实验结果。我将确定OipA和cag PAl对胃炎症的影响、临床结果、CXC趋化因子（KC和IP- 10）的诱导以及与OipA和cag PAl相关的KC和IP- 10启动子的分析。主要使用的方法包括a)实时逆转录聚合酶链反应，b)酶联免疫吸附试验，e) EMSA和supershift试验，d) Western blot分析。这些研究结果将为幽门螺旋杆菌在胃十二指肠疾病发病机制中的作用提供新的见解。

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.