Pathogenesis of E. coli and Shigella infections in human enteroid models

摘要 人类肠样体的发展为研究人类肠道提供了巨大的机会 目的：探讨感染性肠道疾病的发病机制。我们建议将来自以下方面的大量专业知识结合起来 三个在肠道疾病研究方面有着悠久而令人印象深刻的历史的机构来利用这一点 研究腹泻病四大病因致病机制的有力模型：三种病机 致泻性大肠杆菌(肠聚集性、肠出血性和产肠毒素大肠杆菌)和志贺氏菌。 来自马里兰大学、约翰·霍普金斯大学和弗吉尼亚大学的调查人员将 在此计划项目拨款(PPG)中进行协作。本次PPG的领导班子由三人组成 国际知名的肠道疾病专家，辅以微生物学方面的培训， 胃肠病学、分子生理学和儿科传染病。他们将指导一个多学科的 具有细胞生物学、分子发病机制和粘膜免疫学专业知识的合作研究团队。这个 总体目标是增加对这四种疾病的病理生理学和潜在治疗方法的了解。 重要的病原体。拟议的研究将使用正常的人类迷你肠道，称为肠样或 克隆，生长在单层上，以开发模拟人类疾病的模型。审查 所研究疾病的共同病理生理学方面将有助于整合这些项目。这些方面 包括粘蛋白、称为Spates的细菌蛋白酶、肠毒素和分泌的细胞因子的作用，并在 除了每一种感染特有的病理生理方面。此外，参与的细胞的贡献 先天免疫将通过肠样/结肠样细胞与人巨噬细胞的共培养来检测， 中性粒细胞和树突状细胞。这些使用人类迷你肠道的研究提供了揭示 对正常人类肠道而非动物模型的疾病病理生理学方面的见解 以及到目前为止一直使用的癌细胞模型。这四个项目中的每个项目都专注于一种特定的病原体： 肠聚集性大肠埃希菌、志贺氏菌、肠出血性大肠埃希氏菌、产肠毒素大肠埃希菌。除了管理人员 核心，有一个肠样核心，提供人类肠样/结肠样和生长介质，指示所有 关于如何生产肠样/结肠型单层和测量细胞因子的免疫学核心的项目 和趋化因子，并分离人类巨噬细胞、中性粒细胞和树突状细胞。肠样体和 免疫学核心共同开发先天免疫细胞和 项目将使用的肠样/结肠样病变。调查人员将每月联合定期进行互动 实验室会议。拟议中的项目将对肠道疾病引起的疾病产生许多重要的新见解 这些重要的病原体。

Abstract The development of human enteroids as a model to study the human intestine offers tremendous opportunities to study the pathogenesis of infectious enteric disease. We propose to combine the substantial expertise from three institutions with long and impressive histories in the investigation of enteric diseases to exploit this powerful model to study the pathogenesis of four major etiologic agents of diarrheal disease: three pathotypes of diarrheagenic E.coli (enteroaggregative, enterohemorrhagic and enterotoxigenic E. coli) and Shigella. Investigators from the University of Maryland, Johns Hopkins University and the University of Virginia will collaborate in this Program Project Grant (PPG). The leadership team of this PPG comprises three internationally renowned experts in enteric diseases, with complementary training in microbiology, gastroenterology, molecular physiology, and pediatric infectious diseases. They will direct a multidisciplinary team of co-investigators with expertise in cell biology, molecular pathogenesis, and mucosal immunology. The overall goal is to increase understanding of the pathophysiology and potential treatments of these four important pathogens. The proposed studies will use normal human mini-intestines, called enteroids or colonoids, grown on monolayers to develop models that mimic human disease. Examination of pathophysiologic aspects common to the diseases studied will serve to integrate the projects. These aspects include the role of mucins, bacterial proteases called SPATES, enterotoxins and secreted cytokines and are in addition to pathophysiologic aspects specific to each infection. In addition, the contribution of cells involved in innate immunity will be examined by co-culture of the enteroids/colonoids with human macrophages, neutrophils and dendritic cells. These studies using human mini-intestines offer the possibility of revealing insights in disease pathophysiology that are specific to normal human intestine rather than the animal models and cancer cell line models used until now. Each of the four projects focuses on a specific pathogen: enteroaggregative E. coli, Shigella, enterohemorrhagic E. coli, entertoxigenic E. coli. Besides an Administrative Core, there is an Enteroid Core that provides human enteroids/colonoids and growth media, instructs all projects on how to produce enteroid/colonoid monolayers and an Immunology Core that measures cytokines and chemokines, and isolates human macrophages, neutrophils and dendritic cells. The Enteroid and Immunology Cores work together to develop co-culture systems of innate immune cells and enteroids/colonoids that will be used by the projects. The investigators will regularly interact by monthly joint laboratory meetings. The proposed project will yield many significant new insights into enteric disease caused by these important pathogens.