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（由申请人提供）：隐孢子虫病是未经治疗的HIV/AIDS患者的一种重要机会性感染，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（Crypto）是水媒腹泻暴发的主要原因。在资源有限的世界，它是幼儿腹泻和死亡的主要原因。尽管已经评估了几种药物的抗隐孢子虫功效，但硝唑尼特是目前FDA批准的唯一用于隐孢子虫病的药物。然而，这种药物对免疫功能低下者无效。隐孢子虫病干预措施的发展存在几个限制因素。这些问题包括缺乏在体外连续繁殖Crypto的系统，以及无法对寄生虫进行遗传操作。此外，人类隐孢子虫病的病理生理学和宿主-寄生虫相互作用还没有得到很好的理解。肠上皮细胞是来源于人肠腺干细胞的功能性3-D肠上皮单位，其概括了人肠的结构和功能的整体方面。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的离体、3-D、临床前、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，促进寄生虫的遗传操作，并能够调查隐孢子虫病的病理生理学，宿主-寄生虫相互作用和干预措施。具体目标是开发一种用于隐孢子虫感染和繁殖的离体、3-D、临床前人类肠样模型，并验证其用于隐孢子虫病药物的测试。我们将a）优化隐孢子虫的感染剂量、类肠体的起源组织和分化状态、免疫荧光显微镜观察和定量逆转录PCR定量感染; B）进行超微结构研究以鉴定感染的类肠体中的细胞内阶段和管腔或中膜中的细胞外阶段; c）进行时程研究以确定隐球菌可以在模型中连续繁殖的时间长度，以及d）通过测试药物硝唑尼特和巴龙霉素对肠样组织中隐球菌感染的影响来验证优化的模型。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-寄生虫相互作用，加快寄生虫的遗传操作和测试新的干预措施。长期目标是开发针对隐孢子虫病的有效干预措施。