An Ex Vivo 3-D Pre-Clinical Human Enteroid Model for Cryptosporidium

隐孢子虫离体 3D 临床前人类肠样模型

• 批准号: 9090036
• 负责人: Honorine D Ward
• 金额: $ 20.63万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9090036
• 关键词: 3-Dimensional; AIDS/HIV problem; Animal Model; Autologous; Cell Culture Techniques; Cells; Cessation of life; Child; Chronic; Collaborations; Colon; Complex; Cryptosporidiosis; Cryptosporidium; Development; Diarrhea; Disease; Disease Outbreaks; Dose; Duodenum; Enteroendocrine Cell; Epithelial; Epithelial Cells; FDA approved; Functional disorder; Future; Goals; Goblet Cells; Health; Human; Imagery; Immune; Immunocompromised Host; Immunofluorescence Immunologic; In Vitro; Infection; Intervention; Intestinal parasite; Intestines; Investigation; Lead; Length; M cell; Malnutrition; Medicine; Microscopic; Modeling; Opportunistic Infections; Paneth Cells; Parasites; Paromomycin; Patients; Pharmaceutical Preparations; Research; Resources; Reverse Transcription; Sporozoites; Staging; Stem cells; Structure; Study models; System; Testing; Time; Tissues; Undifferentiated; United States; Vaccines; Villus; base; college; drug candidate; drug testing; effective intervention; effective therapy; enteric pathogen; extracellular; genetic manipulation; ileum; intestinal crypt; jejunum; microbiome; nitazoxanide; pre-clinical; stem; success; therapy development; wasting; waterborne

 DESCRIPTION (provided by applicant): Cryptosporidiosis is a significant opportunistic infection in untreated HIV/AIDS patients in whom it can lead to severe, chronic and ultimately fatal diarrhea and wasting. In the United States, Cryptosporidium (Crypto) is a major cause of waterborne outbreaks of diarrhea. In the resource-constrained world, it is a leading cause of diarrhea and death in young children. Although several drugs have been evaluated for anti- cryptosporidial efficacy, Nitazoxanide is currently the only FDA-approved drug for cryptosporidiosis. However, this drug is ineffective in the immunocompromised. There are several constraints to development of interventions for cryptosporidiosis. These include the lack of a system for continuous propagation of Crypto in vitro and the inability to genetically manipulate the parasite. Further, the pathophysiology of human cryptosporidiosis and host-parasite interactions are not well understood. Enteroids are functional 3-D intestinal epithelial units derived from human intestinal crypt stem cells and which recapitulate integral aspects of the structure and function of human intestine. Our preliminary studies indicate that Crypto can indeed infect enteroids. We propose to develop an ex vivo, 3-D, pre-clinical, human enteroid model for Crypto. Our hypothesis is that this model will support infection and continuous propagation of Crypto, facilitate genetic manipulation of the parasite and enable investigation of the pathophysiology, host-parasite interactions and interventions for cryptosporidiosis. The specific aim is to develop an ex vivo, 3-D, pre-clinical, human enteroid model for Crypto infection and propagation and validate it for testing of drugs for cryptosporidiosis. We will a) optimize the infective dose of Crypto sporozoites, tissue of origin and differentiation status of te enteroids, microscopic visualization by immunofluorescence and quantification of infection by quantitative reverse transcription PCR; b) perform ultrastructural studies to identify the intracellular stages in infected enteroids and extracellular stages in the lumen or media; c) perform time course studies to determine the length of time that Crypto can be continuously propagated in the model and d) validate the optimized model by testing the effect of the drugs Nitazoxanide and Paromomycin on Crypto infection in the enteroids. In future studies the model can be used to study the pathophysiology and host-parasite interactions of human cryptosporidiosis, expedite genetic manipulation of the parasite and test new interventions. The long term goal is to develop targeted and effective interventions for cryptosporidiosis.

 描述(申请人提供)：隐孢子虫病是未经治疗的艾滋病毒/艾滋病患者中的一种严重的机会性感染，可导致严重、慢性和最终致命的腹泻和消瘦。在美国，隐孢子虫是引起水媒腹泻暴发的主要原因。在资源有限的世界里，它是导致幼儿腹泻和死亡的主要原因。尽管已经对几种药物进行了抗隐孢子虫疗效评估，但硝唑尼德是目前FDA批准的唯一一种治疗隐孢子虫病的药物。然而，这种药物对免疫功能低下的人无效。隐孢子虫病干预措施的发展有几个制约因素。这些问题包括缺乏在体外持续繁殖Crypto的系统，以及无法从基因上操纵这种寄生虫。此外，人类隐孢子虫病的病理生理学和宿主-寄生虫的相互作用还不是很清楚。肠样体是来源于人类肠道隐窝干细胞的功能性三维肠道上皮细胞，它概括了人类肠道结构和功能的完整方面。我们的初步研究表明，Crypto确实可以感染肠道。我们建议开发一种用于Crypto的体外、三维、临床前的人类肠样模型。我们的假设是，该模型将支持隐孢子虫的感染和持续繁殖，促进对寄生虫的遗传操作，并使研究隐孢子虫病的病理生理、宿主-寄生虫相互作用和干预措施成为可能。其具体目的是开发一种用于隐孢子虫感染和繁殖的体外、三维、临床前的人类肠样模型，并验证其是否用于测试治疗隐孢子虫病的药物。我们将a)优化隐子孢子的感染剂量、TE肠样体的起源和分化状态、免疫荧光显微镜观察和定量逆转录聚合酶链式反应(QRT-PCR)定量感染；b)进行超微结构研究，以确定感染肠小体的细胞内阶段和腔或介质中的细胞外阶段；c)进行时程研究，以确定Crypto可以在模型中持续繁殖的时间长度；d)通过测试药物硝唑尼德和帕罗霉素对肠道中隐孢子虫感染的影响来验证优化模型。在未来的研究中，该模型可用于研究人类隐孢子虫病的病理生理学和宿主与寄生虫的相互作用，加快对寄生虫的遗传操作，并测试新的干预措施。长期目标是开发针对隐孢子虫病的有针对性的有效干预措施。