Mechanisms of gastric mucosal response to H. pylori infection at acidic pH

描述(由申请者提供)：本建议书描述了一项为期四年的基础科学培训计划，旨在发展儿科胃肠病学术生涯。首席研究员伊丽莎白·A·马库斯博士是加州大学洛杉矶分校的儿科胃肠病学临床讲师，预计2013年7月1日的头衔是助理教授，拥有普通儿科和儿科胃肠病学的董事会认证。她在洛杉矶儿童医院完成了儿科住院医生的工作。她参与了基础科学研究，在医学院、住院医生和团契期间研究胃部病原体幽门螺杆菌的酸驯化机制和细菌生理学。目前的建议纳入了一个新开发的和不同的研究重点，研究细菌和酸性pH对胃粘膜的影响。本提案中概述的计划将为申请者提供良好的研究环境和受保护的时间，以获得实现成为独立调查员的目标所需的技能。这位导师乔治·萨克斯博士在胃生理学、胃酸分泌和与幽门螺杆菌酸适应相关的细菌因素方面是公认的专家。萨克斯博士有指导研究生和博士后研究员的丰富历史，这些研究生和博士后研究员后来成为了独立调查人员。共同导师David Scott博士将贡献幽门螺杆菌、显微镜、动物模型和真核细胞系统方面的专业知识。共同导师Charalabos Potulakis博士将提供炎症和粘膜免疫学方面的专业知识。一个咨询委员会将监督职业发展，并提供免疫学、质谱学和上皮生理学方面的额外培训。儿科已经承诺为申请人提供75%的受保护研究时间。加州大学洛杉矶分校提供了丰富的研究和学术环境，这将促进研究独立性的发展。拟议的研究重点是幽门螺杆菌如何与胃酸协同作用，能够损害胃粘膜并引发晚期疾病的发展。幽门螺杆菌感染在世界范围内非常普遍，至少会引起胃炎。其中一些感染者发展为胃或十二指肠溃疡疾病、胃萎缩和癌症。随着新出现的抗生素耐药性和患者对复杂治疗方案的依从性问题，治疗变得越来越困难。尽管已经研究了多种细菌和宿主因素，但尚不确定这些细菌是如何逃避免疫系统，导致终身感染的，也不知道是什么因素导致了晚期疾病的发展。这项建议将使用与宿主环境具有生理相似性的体外和体内模型系统来确定上皮变化和免疫反应的变化。使用SILAC(细胞培养中氨基酸稳定同位素标记)技术的定量质谱学将被用来研究细胞连接中蛋白质的变化，首先是对酸性的响应，然后是对幽门螺杆菌感染的响应。候选蛋白质或途径将被抑制，以确认参与其中。共聚焦显微镜将被用来进一步研究在酸性pH的共同培养中的细胞连接。将表征细胞层电阻和渗透率的变化。参与Th1和Th17免疫反应的介体将在幽门螺杆菌感染和酸性pH的背景下进行研究。幽门螺杆菌基因与 在酸性和沙土鼠模型中增加表达将被作为免疫反应的潜在调节器进行研究。一种新的酸阻滞剂对感染沙土鼠的有效抑酸作用将被用来确定对细菌负荷、炎性渗透和细胞因子产生的影响。预计这项工作将增加对胃损伤机制的了解，并将导致针对感染及其对宿主的短期和长期后果的新治疗靶点的开发。

DESCRIPTION (provided by applicant): This proposal describes a four-year basic science training program for the development of a career in academic Pediatric Gastroenterology. The principal investigator, Dr. Elizabeth A. Marcus, a Clinical Instructor in Pediatric Gastroenterolog at the University of California, Los Angeles with a projected title of Assistant Professor as of 7/1/13, is board-certified in General Pediatrics and Pediatric Gastroenterology. She completed Pediatrics residency at Children's Hospital Los Angeles. She participated in basic science research, studying the acid acclimation mechanisms and bacterial physiology of the gastric pathogen Helicobacter pylori throughout medical school, residency, and fellowship. The current proposal incorporates a newly developed and divergent research focus, studying the effect of the bacteria and acidic pH on the gastric mucosa. The program outlined in this proposal will provide the applicant with an excellent research environment and protected time to attain the skills needed to achieve her goal of becoming an independent investigator. The mentor, Dr. George Sachs, is a recognized expert in gastric physiology, acid secretion, and bacterial factors associated with H. pylori acid acclimation. Dr. Sachs has a strong history of mentoring graduate students and postdoctoral fellows who have progressed to become independent investigators. Co-mentor Dr. David Scott will contribute expertise on H. pylori, microscopy, animal models, and eukaryotic cell systems. Co-mentor Dr. Charalabos Pothoulakis will provide expertise on inflammation and mucosal immunology. An advisory committee will monitor career development and provide additional training in immunology, mass spectrometry and epithelial physiology. The Department of Pediatrics has already committed 75% protected research time to the applicant. UCLA provides a rich research and academic environment that will foster the development of research independence. The proposed research focuses on how H. pylori, in coordination with gastric acidity, is able to injure the gastric mucosa and trigger development of advanced disease. H. pylori infection is highly prevalent worldwide and at a minimum causes gastric inflammation. Some of those infected progress to develop gastric or duodenal ulcer disease, gastric atrophy, and cancer. Treatment is becoming more difficult with emerging antibiotic resistance and problems with patient compliance with a complex treatment regimen. It is not definitively known how the bacteria are able to evade the immune system, leading to lifelong infection, or what factors contribute to development of advanced disease, although multiple bacterial and host factors have been studied. This proposal will use in vitro and in vivo model systems with physiologic similarities to the host environment to determine epithelial changes and alterations in immune response. Quantitative mass spectrometry using SILAC (Stable Isotope Labeling by Amino acids in Cell culture) technology will be used to study protein changes in the cell junction in response first to acidity, then to H. pylori infection. Candidate proteins or pathways will be inhibited to confirm involvement. Confocal microscopy will be used to further study the cell junctions in co-culture with acidic pH. Cell layer resistance and permeability changes will be characterized. Mediators involved with the Th1 and Th17 immune responses will be studied in the context of H. pylori infection and acidic pH. H. pylori genes with increased expression in acid and in a gerbil model will be studied as potential modulators of immune response. Potent acid inhibition with a novel acid blocker in infected gerbils will be employed to determine the effect on bacterial load, inflammatory infiltrate, and cytokine production. It is anticipated that this work will add to the understanding of the mechanisms of gastric injury and will lead to development of novel treatment targets for both the infection and its short and long term consequences to the host.