Establishment of a human enteroid model of cryptosporidiosis

隐孢子虫病人肠样模型的建立

• 批准号: 9928699
• 负责人: Alip Borthakur
• 金额: $ 16.66万
• 依托单位: MARSHALL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-22 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9928699
• 关键词: 3-Dimensional; Acquired Immunodeficiency Syndrome; Affect; Animal Model; Apical; Architecture; Attention; Bicarbonates; Caco-2 Cells; Cell physiology; Cell surface; Cellular Morphology; Chronic diarrhea; Clinical Trials; Cryptosporidiosis; Cryptosporidium; Cryptosporidium parvum; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Diarrhea; Dimensions; Disease model; Dose; Drug Screening; Dysentery; Electrolytes; Elements; Enteral; Enterocytes; Environment; Epithelial; Epithelial Cells; Epithelium; Exhibits; FDA approved; Failure; Fluids and Secretions; Functional disorder; Future; Glucose; Goals; Human; Immunofluorescence Immunologic; Impairment; Infection; Intervention; Intestinal Absorption; Intestines; Ion Transport; Ions; Kinetics; Knowledge; Life; Liquid substance; Mediating; Medical; Membrane; Membrane Proteins; Modeling; Molecular; Morbidity - disease rate; Multicenter Studies; Oral; Oral Rehydration Therapy; Organoids; Outcome; Parasites; Patients; Permeability; Pharmaceutical Preparations; Physiological; Play; Pre-Clinical Model; Proteins; Reporting; Role; Route; SLC26A3 gene; Structure; Surface; Technology; Tight Junctions; Time; Tissues; Transformed Cell Line; Treatment Efficacy; Vaccines; Western World; absorption; cell type; claudin 4; design; diarrheal disease; drug development; drug discovery; global health; human model; imaging study; in vivo; intestinal crypt; luminal membrane; monolayer; mortality; neglect; new therapeutic target; nitazoxanide; novel; occludin; parasite invasion; pathogen; programs; response; therapeutic target; waterborne outbreak

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.

隐孢子虫病是由隐孢子虫属（CP）感染引起的一种广泛传播的肠道疾病， 与高发病率和高死亡率有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种主要的肠道病原体之一 国际吧虽然以前已知会导致艾滋病患者慢性腹泻， 隐孢子虫病在西方世界爆发，直到最近才引起医学和科学的关注 认识到迫切需要开发有效的治疗策略。目前的治疗方案 目前还没有疫苗，而且FDA唯一批准的药物硝唑尼特表现出有限的 功效该寄生虫通过粪-口途径进入人类宿主并感染宿主肠上皮细胞。一 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 的生理学相关疾病模型，其重现了人类肠道的天然体内环境。 寄生虫动物模型对于人类感染研究是次优的，而人类肠转化细胞 线并不真正代表体内宿主-寄生虫相互作用。在这方面，最近的一项技术， 人类肠，具有围绕内腔的上皮层的小肠隐窝衍生的3-D类器官， 提供了一种真实再现天然上皮结构和功能多样性的离体模型。 人肠上皮细胞和肠上皮细胞衍生的极化单层在Transwell插入物上生长， 顶侧/基底侧细胞表面提供了作为人体生理学相关模型的特殊机会 研究宿主-病原体相互作用。因此，我们建议目前的探索性研究， 利用隐窝来源人肠样组织和肠样组织来源的人肠样组织建立隐孢子虫病的离体模型 单细胞层研究宿主-寄生虫相互作用相关的寄生虫病。腹泻主要导致 肠道离子和液体转运失调（由于吸收减少和/或分泌增加） 并且还可能涉及肠屏障功能的破坏。因此，我们假设一个人类 CP感染的肠样模型将首次允许CP诱导的 上皮离子转运和屏障功能，并确定了新的干预目标。的 具体目的包括：1）研究C.人3-D肠样细胞/肠样细胞衍生的2-D 2）阐明了离子通道改变对上皮细胞屏障结构和功能的影响 运输和腔液积聚。人类肠/类肠衍生的细小病毒感染 单层。这种新型的隐孢子虫病离体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，确定上级治疗靶点，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 在药物发现和开发方面的强大计划。