INTESTINAL INFLAMMATION ORCHESTRATED BY PATHOGENS

沙门氏菌相关性胃肠炎的活动期和 炎症性肠病的慢性状态)，如溃疡性结肠炎 和克罗恩病，组织学上以多形核细胞为特征 白细胞(PMN)迁移到和穿过上皮衬里。 肠子。这些事件导致上皮细胞的急性炎症和 随之而来的上皮功能障碍。中性粒细胞向外迁移的程度 肠隐窝和隐窝脓肿的形成是疾病的征兆 严重程度，临床用于评估IBD的活动性。目前还不清楚 是什么触发了中性粒细胞在肠道上皮的定向移动。 为此，我们最近表明，上皮细胞本身可以 将这些信号发送到底层PMN，这些信号由肠道调节 植物区系，如鼠伤寒沙门氏菌。这项提议的广泛的长期目标 是为了研究上皮细胞在体内的分子机制 对微生物病原体的反应可以向PMN发出信号，并协调其 定向迁移。一旦我们开始了解这种跨细胞的基础 信号在促进鼠伤寒沙门氏菌致病过程中的重要作用 有可能开发针对治疗的新的治疗策略 治疗和改善IBD。具体的目标最终是针对 实现这一目标，有三个方面。特定目标1旨在 确定鼠伤寒沙门氏菌毒力因子的性质并确定其 对粘膜炎症的上皮性协调的贡献。 具体来说，我们将描述鼠伤寒沙门氏菌SipA、SopB和SOPA是如何 分泌的蛋白质干扰信号通路，从而导致 这些蛋白在黏膜炎症中的表达与上皮细胞的协调 用腺病毒表达载体在上皮细胞中表达蛋白质。功能性 这些蛋白的表达对促炎性反应的影响 控制PMN跨上皮迁移的事件将与 共聚焦显微镜和电子显微镜的形态结果。特定的 Aim 2的设计目的是识别导致 促炎症化学诱导剂PEEC的释放，并将使用几种 不同的方法，包括确定S. 小鼠侵袭和PEEC根尖上皮释放的检测 JNK途径的作用，决定了小GTP酶(cdc42， Rac-1和Arf6)在鼠伤寒沙门氏菌诱导PMN能力中的表达 利用显性抑制突变体表达的跨上皮游走 腺病毒表达载体，检测磷酸肌醇信号转导的作用， 以及确定鼠伤寒沙门氏菌是否具有刺激PEEC分泌的能力 与它们诱导细胞内钙离子增加的能力有关 模型肠上皮细胞。《特定目标3》旨在描述一种 最近发现了促炎性中性粒细胞趋化因子。第一部分 这一目标将利用高效液相纯化、核磁共振等手段来阐明PEEC的结构。 分析、质谱分析和序列分析，而第二部分 这一目标将定义PEEC与其他PMN化学诱导剂的关系 包括它在PMN化学吸引剂层次结构中的排名，将决定 PEEC是否能够激活其他免疫类型的细胞并评估 PEEC在炎症中的作用。

The active phase of both Salmonella-associated gastroenteritis and chronic states of inflammatory bowel disease IBD), such as ulcerative colitis and Crohn's disease, is characterized histologically by polymorphonuclear leukocyte (PMN) migration into and across the epithelial lining of the intestine. These events result in acute inflammation of the epithelium and subsequent epithelial dysfunction. The degree of PMN transmigration into intestinal crypts and the formation of crypt abscesses is indicative of disease severity and is used clinically to evaluate the activity of IBD. It is unclear what triggers directional movement of PMN across the intestinal epithelium. Towards this end, we have recently shown that epithelial cells themselves can send such signals to underlying PMN and these signals are regulated by enteric flora, such as S. typhimurium. The broad long term objectives of this proposal are to investigate the molecular mechanism by which epithelial cells in response to microbial pathogens can signal to PMN and orchestrate their directed migration. Once we begin to understand the basis of such transcellular signaling important in promoting disease flares of S. typhimurium pathogenesis, it may be possible to develop novel therapeutic strategies aimed at treatments for and ameliorating IBD. The specific aims are ultimately directed at achieving this goal, and are three-fold. Specific Aim 1 is designed to determine the nature of S. typhimurium virulence factors and define their contribution to the epithelial orchestration of mucosal inflammation. Specifically, we will delineate how S. typhimurium SipA, SopB, and SopA secreted proteins interfere with the signaling pathways which lead to epithelial orchestration of mucosal inflammation by expression of these proteins in epithelial cells using adenoviral expression vectors. Functional effect of expression of these proteins on orchestration of proinflammatory events which govern PMN transepithelial migration will be correlated with morphological consequences by both confocal and electron microscopy. Specific Aim 2 is designed to identify the signal transduction cascades which lead to the release of the proinflammatory chemoattractant PEEC and will employ several different approaches which include determining the relationship between S. typhimurium invasion and the apical epithelial release of PEEC, examination of the role of the JNK-pathway, determining the effects of small GTPase (cdc42, rac-1, and Arf6) expression on the ability of S. Typhimurium to induce PMN transepithelial migration by expression of dominant inhibitory mutants using adenoviral expression vectors, examining the role of phosphinositide signaling, and determining whether the ability of S. typhimurium to elicit PEEC secretion correlates with their ability to induce an increase in intracellular calcium in model intestinal epithelia. Specific Aim 3 is designed to characterize a recently identified pro-inflammatory PMN chemoattractant. The first part of this aim will elucidate the structure of PEEC utilizing HPLC purification, NMR analysis, mass spectrometry and sequence analysis, while the second part of this aim will define PEEC's relationship to other PMN chemoattractants including its ranking in the PMN chemoattractant hierarchy, will determine whether PEEC is able to activate other immune-type cells as well as assess the role of PEEC in inflammation.