Regulation of CXC chemokine expression by H. pylori.

描述(申请人提供)：幽门螺杆菌可引起胃炎、消化性溃疡疾病和胃癌。最近的临床和体外数据表明，CAG致病岛(PAI)和外源性炎症蛋白OIPA都参与了胃粘膜白介素8(IL-8)水平的产生和由此引起的炎症。已发表的旨在了解CAG-PAL相关的IL-8信号转导途径的数据提供了不一致的结果，可能是因为没有考虑OIPA的影响。本研究旨在了解幽门螺杆菌感染对IL-8信号转导通路的影响。我假设OIPA基因产物激活干扰素调节因子(IRES)与IL-8启动子中一种新的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丝裂原激活蛋白(MAP)激酶级联，而CAG PAI不激活。我的方法包括使用双亲幽门螺杆菌菌株和OIPA、cag PAI、cagA、HopZ或OIPA/CAG PAI基因的精确缺失突变体，以及补充的OIPA突变体。目的1探讨IL-8基因转录调控与OIPA和CAG-PAI的关系。我将验证这样的假设，即OIPA和/或CAG PAI激活与ISRE样元件结合的IRF，导致IL-8基因转录。我将确定IL-8启动子上游与OIPA和CAG PAL相关的MAP激酶通路。我还将确定在IL-8启动子上游的IRF-ISRE-Ike元件通路、MAP激酶通路和IkappaB-NF-kappaB通路之间是否存在串扰(即它们是关联的还是独立的)。使用的主要方法包括a)荧光素酶报告基因分析，b)电泳迁移率改变分析(EMSA)和超转移分析，e)微亲和分离分析，d)RNA干扰，和e)蛋白质印迹分析。目的2通过建立蒙古沙土鼠胃损伤模型，观察OIPA和CAG PAI对胃损伤的体内作用，以证实和推广体外实验结果。我将确定OIPA和CAG PAL对胃炎症、临床转归、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。主要方法包括a)实时逆转录-聚合酶链式反应，b)酶联免疫吸附试验，e)EMSA和SuperShift试验，d)Western印迹分析。这些研究结果将为幽门螺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.