Pathways and pathogens affecting gastric epithelial repair in vivo

描述（由申请方提供）：消化性溃疡疾病仍然是美国医疗保健系统和全球范围内的一个重大负担，在很大程度上是由胃病原体幽门螺杆菌的存在或摄入治疗药物的上消化道毒性引起的。我们工作的基本原理是，改进或替代的治疗策略可以基于对胃防御机制的理解，这些机制可以对抗这些挑战。基于初步研究，我们假设，胃粘膜环境，包括细胞内和细胞外Ca2+的作用，是胃表面上皮修复的中央调节器。本申请的目的是确定胃肠道毒性双膦酸盐药物，H。幽门螺杆菌病原体，或这些应激源的组合。我们特别要问的是急性H.幽门螺杆菌和双膦酸盐药物是通过干扰宿主Ca2+信号传导，这对修复很重要。我们开创了光学技术，可以真实的实时创建焦点损伤并持续量化修复。我们的工作重点是修复局灶性病变在体内将外推到实验评价胃炎和溃疡，以提高相关性的胃病理临床观察。在本申请中，我们介绍了新的能力，图像细胞内钙动员在体内的胃表面上皮细胞，和细胞外钙动员在胃腔的粘液微环境。我们的第一个目的是研究钙依赖性信号传导在胃局灶性病变修复过程中的作用。实验是基于初步的研究结果，表明磷脂酶C，蛋白激酶C，胰高血糖素，细胞内和细胞外的钙动员所需的有效的胃修复在完整的胃。我们将定义这些促进胃修复的钙依赖性信号传导途径之间的层次和顺序，并询问GI毒性双膦酸盐药物是否通过中断这种Ca2+信号传导来干扰胃上皮修复。我们的第二个目的是检验H.幽门影响胃修复。实验是基于荧光标记的H。幽门螺杆菌在几分钟内专门聚集在病灶部位，并减缓该病灶的修复。我们还观察到H的选择性积累。肉眼可见溃疡部位的幽门。我们会问如果H。pylori由于细菌化学感应而在损伤部位积聚，使用H. pylori突变体在化学感应基因中有缺陷，并操纵粘膜间隙中潜在的化学引诱物的水平。我们将评估这种积累对溃疡愈合和双磷酸盐胃炎进展的影响。这些结果将为了解发病机制的早期因素提供一个独特的窗口，这些因素有可能导致预防性干预。 公共卫生相关性：胃溃疡和消化性溃疡疾病是美国医疗保健系统中的一个重大负担，在很大程度上是由胃病原体幽门螺杆菌的存在或摄入的治疗药物（如阿司匹林或用于治疗骨质疏松症的双膦酸盐药物）的毒性引起的。本项目探讨了最初暴露于H引起胃刺激的细胞和分子机制。幽门螺杆菌或双磷酸盐，以及胃对这种损伤作出反应而迅速自愈的显著能力。

DESCRIPTION (provided by applicant): Peptic ulcer disease remains a significant burden in the US healthcare system and worldwide, caused in large part by the presence of the gastric pathogen Helicobacter pylori or the upper GI toxicity of ingested therapeutic drugs. The rationale for our work is that improved or alternative therapeutic strategies can be based on understanding the mechanisms of gastric defense rallied against such challenges. Based on preliminary studies, we hypothesize that the juxtamucosal environment, including a role for both intracellular and extracellular Ca2+, is a central regulator of gastric repair of the surface epithelium. The objective of this application is to identify the underlying mechanisms for inhibited gastric epithelial repair in response to the GI- toxic bisphosphonate drugs, the H. pylori pathogen, or a combination of these stressors. We specifically ask if the pathogenesis from acute H. pylori and bisphosphonate drugs is via their interference with host Ca2+ signaling, which we show to be important for repair. We have pioneered optical technologies that allow real- time creation of focal damage and continual quantification of repair. Our work focusing on the repair of focal lesions in vivo will be extrapolated to experiments evaluating gastritis and ulceration to enhance relevance to gastric pathologies observed clinically. In this application, we introduce the novel ability to image intracellular calcium mobilization in vivo in the gastric surface epithelium, and extracellular calcium mobilization in the juxtamucosal microenvironment of the gastric lumen. Our first aim is to examine the role of calcium-dependent signaling during the repair of focal gastric lesions. Experiments are based on preliminary findings that show phospholipase C, protein kinase C, prostaglandins, and both intracellular and extracellular calcium mobilization are required for efficient gastric repair in the intact stomach. We will define the hierarchy and sequence among these calcium-dependent signaling routes that promote gastric repair and ask if the GI-toxic bisphosphonate drugs interfere with gastric epithelial repair by interrupting this Ca2+ signaling. Our second aim examines how the presence of H. pylori affects gastric repair. Experiments are based on preliminary observations that fluorescently-labeled H. pylori accumulate specifically at the site of a focal lesion within minutes, and slow the repair of that lesion. We also observe selective accumulation of H. pylori at sites of macroscopic ulceration. We will ask if H. pylori accumulates at damage sites due to bacterial chemosensing, using H. pylori mutants defective in chemosensing genes and manipulating the level of potential chemoattractants in the juxtamucosal space. We will evaluate the impact of this accumulation on ulcer healing and on progression of bisphosphonate gastritis. The outcomes will provide a unique window into understanding early factors in pathogenesis, which have potential to lead to preventive interventions. Public Health Relevance: Gastritis and peptic ulcer disease are a significant burden in the US healthcare system, caused in large part by the presence of the stomach pathogen Helicobacter pylori or the toxicity of ingested therapeutic drugs (such as aspirin, or the bisphosphonate drugs used to treat osteoporosis). This project explores the cellular and molecular mechanisms underlying the stomach irritation caused by initial exposure to H. pylori or bisphosphonates, and the remarkable ability of the stomach to heal itself quickly in response to such insults.