Immunology Core

免疫学核心-摘要 腹泻病是五岁以下儿童死亡的第二大原因。了解 参与细菌发病机制和宿主对肠道病原体的反应的过程对于改善 治疗和制定有效的预防策略。这些研究由于缺乏可靠的 模型，完全概括了复杂的细胞和分子的事件和相互作用，发生在 人类的内脏免疫学核心（IC）将建立和完善人肠上皮细胞的共培养模型， 和先天免疫细胞来重建组织结构，细胞相互作用，和协调的反应， 人类的内脏这种生理相关的系统将产生新的见解，协调行动， 上皮细胞和免疫细胞在肠道感染。该委员会还将支持 通过提供实施免疫共培养的技术专长，加强了项目。IC有两个主要目标。 目标1.作为核心开发目标，IC将：1）通过接种优化肠免疫细胞共培养物 胶原蛋白包被的多孔支架（即Alvetex®）上的上皮细胞单层， 接触和迁移。2)建立一个多细胞模型，由与以下细胞共培养的上皮细胞单层组成： 不同的先天性吞噬细胞：单核细胞衍生的巨噬细胞（MF），多形核中性粒细胞（PMN）， 和从人外周血分离的树突状细胞（DC）。吞噬细胞不仅具有抗微生物作用， 功能，但也启动适应性免疫。我们已经成功地将人类吞噬细胞单独移植到 肠样单层。为了更好地重建上皮和先天免疫细胞的个体和协同功能， 我们将联合收割机将这三种主要的吞噬细胞谱系与肠样细胞单层结合。组织结构和整合， 细胞表型、活力和功能将通过组织学、共聚焦显微镜、流式细胞术和 微阵列技术吞噬活性，迁移和细胞因子和趋化因子的产生将是 检查以确定细胞功能。3)建立上皮细胞和TCRgd+ T细胞共培养模型 从人的外周血中提取粘膜屏障内的TCRgd+ T细胞，也称为上皮内 淋巴细胞（IEL）被快速动员并部署多种先天免疫功能以响应病原体。 细胞表型将通过流式细胞术表征，功能将通过细胞因子产生和细胞周期来评估。 细胞毒性将通过组织学和显微镜检查组织结构。将监测细胞移动 使用粒子跟踪系统/软件和共聚焦显微镜。免疫产生的最佳条件 将确定共培养物，并制定方案，供研究项目使用。 目标2.作为支持服务Aim，IC将编写详细的协议来实现上述方法 并将提供培训和技术援助，以促进个别项目的试验工作。 总的来说，这项工作将产生关于肠道感染和免疫的新知识， 预防性和治疗性开发工作。

IMMUNOLOGY CORE – ABSTRACT Diarrheal disease is the second leading cause of death in children under five years of age. Understanding the processes involved in bacterial pathogenesis and host response to enteric pathogens is essential to improve treatment and develop effective prevention strategies. Such studies have been hindered by the lack of reliable models that fully recapitulate the complex cellular and molecular events and interactions that take place in the human gut. The Immunology Core (IC) will establish and refine co-culture models of human intestinal epithelial and innate immune cells to recreate the tissue structure, cell interaction, and coordinated responses of the human gut. This physiologically relevant system will generate new insights into the coordinated actions of epithelial and immune cells during enteric infections. The IC will also support the studies performed by the projects by providing technical expertise for implementation of immune co-cultures. The IC has two main Aims. Aim 1. As a core developmental Aim, the IC will: 1) Refine the enteroid-immune cell co-culture by seeding epithelial cell monolayers on a collagen-coated porous scaffold (i.e. Alvetex®) that will allow direct cell-to-cell contact and migration. 2) Establish a multi-cellular model consisting of epithelial cell monolayers co-cultured with different innate phagocytic cells: monocyte-derived macrophages (MF), polymorphonuclear neutrophils (PMN), and dendritic cells (DC) isolated from human peripheral blood. Phagocytic cells not only perform anti-microbial functions but also initiate adaptive immunity. We have successfully engrafted human phagocytes individually to enteroid monolayers. To better recreate individual and synergistic function of epithelial and innate immune cells, we will combine these three main phagocytic lineages with enteroid monolayers. Tissue structure and integration, cell phenotype, viability, and functionality will be assessed by histology, confocal microscopy, flow cytometry and microarray technology. Phagocytic activity, transmigration and cytokine and chemokine production will be examined to determine cell function. 3) Develop a co-culture model containing epithelial cells and TCRgd+ T cells from human peripheral blood. TCRgd+ T cells within the mucosal barrier, also known as intraepithelial lymphocytes (IEL), are rapidly mobilized and deploy diverse innate immune functions in response to pathogens. Cell phenotype will be characterized by flow cytometry, and function will be assessed by cytokine production and cell cytotoxicity. Tissue structure will be examined by histology and microscopy. Cell movement will be monitored with particle tracking system/software and confocal microscopy. Optimal conditions for the generation of immune co-cultures will be defined, and protocols produced and made available to the research projects. Aim 2. As a support service Aim, the IC will write detailed protocols to implement the methods described above and will provide training and technical assistance to facilitate the experimental work of the individual projects. Collectively, the work proposed will generate new knowledge on enteric infection and immunity that can inform prophylactic and therapeutic development efforts.