Cryptosporidium parvum is a coccidian parasite which causes severe diarrhea and malabsorption in patients with AIDS for which there is no effective therapy. The lack of a well defined in vitro model of infection has severely hampered research into the biology of cryptosporidial invasion of the host epithelial cell which in turn, presents obstacles to the development of therapeutic agents. The training objectives of this proposal will focus on immunologic inhibition of Cryptosporidium infection in an in vitro model. In view of the lack of a suitable small intestinal cell line, we have developed an in vitro model of Cryptosporidium infection using a subclone of the HT29 human colon carcinoma cell line (HT29.74), which morphologically resembles small intestinal enterocytes. Electron microscopy has confirmed the intracellular nature of the infection and asexual replication of Cryptosporidium in the HT29.74 cell line. These findings resemble those occurring during in vivo infection of the human enterocyte. We propose to evaluate whether and how different immunologic agents such as hyperimmune cow colostrum, polyclonal and monoclonal antisporozoite antibodies inhibit cryptosporidial infection of the human HT29.74 enterocyte cell line. This model will be used to examine the underlying mechanisms of inhibition of cryptosporidial infection by assessment of the sequential events: parasite binding, internalization into the host cell, and intracellular developmental stages of the parasite.
The specific aims of the proposal are: 1) To evaluate antibody and non antibody components of hyperimmune cow colostrum to determine the specific constituent which inhibits Cryptosporidium infection of HT29.74 cells, and to explore the modulatory effects of anticryptosporidial polyclonal and monoclonal antibodies to inhibit in vitro infection of HT29.74 cells; 2) To determine whether the inhibitory activities of colostrum or specific polyclonal and monoclonal antibodies are mediated through changes in a) sporozoite binding to the host cell surface, b) sporozoite penetration of the host cell, and c) parasite development within the host cell; and 3) To characterize the specific surface sporozoite antigens recognized by colostral, polyclonal and monoclonal antibodies which are inhibitory to Cryptosporidium infection of HT29.74 cells and to determine whether a particular target antigen on the parasite is central to attachment to the enterocyte. The proposed studies will show how immunologic agents modify the interaction of Cryptosporidium with the host enterocyte and should facilitate development of new and effective therapies.
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