Arginase, Nitric oxide, and the Defective Immune Response to Helicobacter pylori

描述（由申请人提供）：本提案的总体目标是提供一种培训工具，使申请人能够为成功的职业生涯做好准备，作为生物医学研究的独立调查员。该研究的重点是细菌病原体幽门螺杆菌，尽管存在强烈的先天性和适应性粘膜免疫反应，但它仍会在宿主的一生中持续存在于人类胃中。长期的目标是阐明这种酶在诱导型一氧化氮（NO）合酶（iNOS）依赖的宿主防御H。幽门。这一假说的假设是，L-精氨酸（L-Arg）的代谢酶II是致病性的H。通过限制iNOS依赖性宿主防御和通过产生多胺促进炎症来抑制幽门螺杆菌感染。具体目标是：目标1，体外：目的：证实L-Arg在巨噬细胞对H.幽门。我们认为，iNOS酶II有效地与iNOS竞争L-Arg，并通过降低细胞内L-Arg浓度来抑制iNOS翻译和NO合成。使用用抗肿瘤酶抑制剂和抗肿瘤酶II特异性siRNA/shRNA处理的RAW 264.7鼠巨噬细胞，以及从抗肿瘤酶II-/-和野生型小鼠分离的胃巨噬细胞，我们将评估：I. iNOS表达、蛋白质翻译和活性;和ii.杀死H。幽门螺杆菌;和B.）凋亡和多胺水平。目的2，体内研究：探讨L-精氨酸酶Ⅱ在H.幽门感染我们将使用两个模型的H。pylori感染，其中用H.幽门螺杆菌感染4个月，以及小鼠被攻击48小时的急性模型。使用这两种系统，野生型小鼠将用β-内酰胺酶抑制剂处理，并将β-内酰胺酶II-/-和野生型小鼠进行比较。在这些时间点，将评估以下内容：A.）H.胃组织中的幽门螺杆菌定植、组织学和iNOS表达; B.）离体胃巨噬细胞iNOS表达、NO生成和凋亡这些研究有望为H.幽门感染与公共卫生的相关性：H。幽门螺杆菌感染大约50%的世界人口和20-40%的美国人。它导致所有个体的慢性胃炎，是世界上胃腺癌的主要原因，也是非药物诱导的消化性溃疡疾病的主要原因。由于所有感染者中有10-20%会出现临床表现，因此疾病负担是巨大的。并非所有患者都可以成功地用抗生素治疗，并且没有适当的筛选策略;因此，非常需要更好地了解本研究中所追求的宿主免疫应答失败的原因。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to provide a training vehicle that will allow the applicant to be prepared for a successful career as an independent investigator in biomedical research. The research is focused on the bacterial pathogen Helicobacter pylori, which persists in the human stomach for the life of the host despite a vigorous innate and adaptive mucosal immune response. The long-term goal is to elucidate the role of the enzyme arginase in inducible nitric oxide (NO) synthase (iNOS)-dependent host defense against H. pylori. The hypothesis of this proposal is that L-arginine (L-Arg) metabolism by arginase II is pathogenic in H. pylori infection by restricting iNOS-dependent host defense and by contributing to the inflammation by generation of polyamines. The specific aims are: Aim 1, in vitro: To demonstrate that L-Arg metabolism by arginase II has important biological effects in the macrophage response to H. pylori. We propose that arginase II effectively competes with iNOS for L-Arg and inhibits iNOS translation and NO synthesis by decreasing the intracellular concentration of L-Arg. Using RAW 264.7 murine macrophages treated with arginase inhibitors and arginase II-specific siRNA/shRNA, and gastric macrophages isolated from arginase ll-/- and wild-type mice, we will assess: A.) i. iNOS expression, protein translation, and activity; and ii. killing of H. pylori; and B.) apoptosis and polyamine levels. Aim 2, in vivo: To determine the biological importance of L-Arg metabolism by arginase II in H. pylori infection. We will use two models of H. pylori infection, a chronic model in which mice are challenged with H. pylori for four months, and an acute model in which mice are challenged for 48 hours. Using both systems, wild-type mice will be treated with arginase inhibitors, and arginase ll-/- and wild-type mice will be compared. At these time points, the following will be assessed: A.) H. pylori colonization, histology, and iNOS expression in gastric tissues; B.) iNOS expression, NO generation, and apoptosis in isolated gastric macrophages. It is expected that these studies will provide new insights into the immunopathogenesis of H. pylori infection. Relevance to public health: H. pylori infects approximately 50% of the world's population and 20-40% of Americans. It causes chronic gastritis in all individuals and is the major cause of gastric adenocarcinoma in the world and the primary cause of peptic ulcer disease that is not drug-induced. Because 10-20% of all of those infected will present with clinical manifestations, the disease burden is enormous. Not all patients can be successfully treated with antibiotics, and there is no screening strategy in place; therefore, greater understanding of the reasons for the failed host immune response, as is being pursued in this study, are greatly needed.