An Ex Vivo 3-d Pre-clinical Human Enteroid Model For Cryptosporidium

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.