Establishment of a human enteroid model of cryptosporidiosis

隐孢子虫病是一种由隐孢子虫(CP)感染引起的广泛性腹泻疾病。 与高发病率和高死亡率相关的新出现的全球健康问题。全球肠道多中心 研究(GEMS)最近报道这种被忽视的原生动物寄生虫是4种主要腹泻病原体之一 全世界。尽管之前已知会导致艾滋病患者的慢性腹泻和主要的水媒 在西方世界爆发的隐孢子虫病直到最近才引起医学和科学的关注 认识到迫切需要制定有效的治疗策略。当前的治疗方案 到目前为止还没有疫苗，FDA批准的唯一药物硝唑尼德也是有限的 功效。寄生虫通过粪便-口腔途径进入人体，感染宿主肠道上皮细胞。一个 药物开发的主要障碍是对宿主-寄生虫相互作用的了解不足，这主要是由于缺乏 一种生理上相关的疾病模型，概括了人体在活体环境中的天然肠道环境 寄生虫。动物模型不适合人类感染研究，而人类肠道转化细胞 这些株系并不能真实地代表体内宿主与寄生虫的相互作用。在这方面，一项最新的技术产生了 人类肠样，小肠腺衍生的3-D器官样体，管腔周围有一层上皮层， 提供了一个真实再现天然上皮结构和功能多样性的体外模型。 人的肠样和肠样来源的极化单分子层生长在Transwell衬底上 顶端/基底外侧细胞表面作为人类生理相关模型提供了难得的机会 肠道，研究宿主与病原菌的相互作用。因此，我们建议目前的探索性研究 利用隐孢子虫和肠囊虫建立隐孢子虫病体外模型 研究与腹泻疾病相关的宿主-寄生虫相互作用。腹泻主要结果 由于肠道离子和液体转运失调(由于吸收减少和/或分泌增加) 也可能涉及肠道屏障功能的破坏。因此，我们假设一个人类 CP感染的肠样模型将首次能够表征CP引起的细胞调节失调 人体肠道上皮离子转运和屏障功能，并确定新的干预靶点。这个 具体目标包括：1)表征微小弧菌在人体三维肠状突/肠状突衍生的二维组织中的侵袭性 单层及其对上皮屏障结构和功能的影响；2)阐明离子改变的机制 微小弧菌感染人肠系膜/肠系膜后腔内液体的转运和积聚 单层。这种新的隐孢子虫病体外模型应该有助于克服研究中的一个主要障碍 与人类感染相关的宿主-寄生虫相互作用，确定优越的治疗靶点，并建立 用于高通量药物筛选的高效临床前模型，从而为未来的 在药物发现和开发方面的强有力的计划。

Cryptosporidiosis, a widespread diarrheal disease caused by Cryptosporidium (CP) species infection, is an emerging global health problem associated with high morbidity and mortality. The Global Enteric Multicenter Study (GEMS) recently reported this neglected protozoan parasite as 1 of the 4 major diarrheal pathogens worldwide. Although previously known for causing chronic diarrhea in AIDS patients and for major waterborne outbreaks in the Western world, cryptosporidiosis has garnered medical and scientific attention only recently with appreciation of an urgent need to develop effective therapeutic strategies. The current treatment options are severely limited with no vaccines to date and the only FDA approved drug nitazoxanide exhibits limited efficacy. The parasite enters human host via fecal-oral route and infects the host intestinal epithelial cells. A major hurdle in drug development is the poor knowledge of host-parasite interactions primarily due to the lack of a physiologically relevant disease model recapitulating the native human intestinal in vivo environment of the parasite. Animal models are suboptimal for human infection studies, whereas human intestinal transformed cell lines do not truly represent host-parasite interactions in vivo. In this regard, a recent technology to generate human enteroids, small intestinal crypt-derived 3-D organoids with an epithelial layer surrounding a lumen, provided an ex-vivo model truly recapitulating the architecture and functional diversity of the native epithelium. Human enteroids and enteroid-derived polarized monolayers grown on Transwell inserts with distinct apical/basolateral cell surfaces provide exceptional opportunity as physiologically relevant model of human intestine to study host-pathogen interactions. Therefore, we propose the current exploratory studies to establish an ex vivo model of cryptosporidiosis utilizing crypt-derived human enteroids and enteroid-derived monolayers to investigate host-parasite interactions relevant to diarrheal diseases. Diarrhea mainly results from dysregulated intestinal ion and fluid transport (due to decreased absorption and/or increased secretion) and may also involve disruption of intestinal barrier function. Therefore, we hypothesized that a human enteroid model of CP infection will allow, for the first time, characterization of CP-induced dysregulation of epithelial ion transport and barrier function in the human intestine and define novel targets for intervention. The Specific Aims include: 1) Characterize invasion of C. parvum in human 3-D enteroids/enteroid-derived 2-D monolayers and the effects on epithelial barrier structure and function; 2) Elucidate mechanisms of altered ion transport and luminal fluid accumulation following C. parvum infection of human enteroids/enteroid-derived monolayers. This novel ex-vivo model of cryptosporidiosis should help overcome a major hurdle in studying host-parasite interactions relevant to human infection, identify superior therapeutic targets and establish a highly effective pre-clinical model for high throughput drug screening and thereby forming the basis for a future robust program in drug discovery and development.