INTESTINAL INFLAMMATION ORCHESTRATED BY PATHOGENS

沙门氏菌相关性胃肠炎和 炎症性肠病的慢性状态），如溃疡性结肠炎 和克罗恩病，在组织学上的特征是多形性 白细胞（PMN）迁移进入和穿过上皮衬里的 肠子这些事件导致上皮的急性炎症， 随后上皮功能障碍。中性粒细胞移入 肠隐窝和隐窝扩张的形成是疾病的指示 严重程度，并在临床上用于评估IBD的活性。目前还不清楚 是什么触发了中性粒细胞在肠上皮中的定向运动。 为此，我们最近已经证明上皮细胞本身可以 将这些信号发送给下面的PMN，这些信号由肠道调节。 植物群，如S.鼠伤寒。本提案的长期目标 是为了研究上皮细胞的分子机制， 对微生物病原体的反应可以向PMN发出信号，并协调它们的 定向迁移。一旦我们开始理解这种跨细胞的基础 信号传导在促进S.鼠伤寒发病机制， 有可能开发出新的治疗策略， 改善IBD。具体目标最终指向 实现这一目标，有三个方面。具体目标1旨在 确定S的性质。鼠伤寒毒力因子，并确定其 有助于粘膜炎症的上皮协调。 具体地说，我们将描述如何S。鼠伤寒沙门氏菌SipA、SopB和SopA 分泌的蛋白质干扰信号通路， 通过这些表达的粘膜炎症的上皮协调 使用腺病毒表达载体在上皮细胞中表达蛋白质。功能 这些蛋白质表达对促炎性细胞因子协调作用的影响 控制PMN跨上皮迁移的事件将与 共聚焦和电子显微镜的形态学后果。具体 目的2是为了确定信号转导级联，导致 促炎化学引诱物PEEC的释放，并将采用几种 不同的方法，其中包括确定S之间的关系。 鼠伤寒侵袭和PEEC的顶端上皮释放，检查 JNK途径的作用，决定了小GTP酶（cdc 42， rac-1和Arf 6）表达对S.鼠伤寒沙门氏菌诱导PMN 通过显性抑制突变体表达的跨上皮迁移 腺病毒表达载体，检测磷脂信号传导的作用， 并确定S.鼠伤寒沙门氏菌诱导PEEC分泌 与它们诱导细胞内钙离子增加的能力相关， 模型肠上皮细胞。具体目标3旨在描述 最近发现的促炎性中性粒细胞化学引诱物。的第一部分 这一目标将阐明PEEC的结构，利用HPLC纯化，NMR 分析，质谱和序列分析，而第二部分， 这一目标将明确PEEC与其他中性粒细胞化学引诱物的关系 包括其在PMN化学引诱物层次中的排名，将决定 PEEC是否能够激活其他免疫型细胞以及评估 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.