Cryptosporidium infection is now a well-reorganized cause of diarrhea in immunologically healthy and immunocompromised humans throughout the world, largely due to the recognition of a severe form of the infection in patients with AIDS. Diarrheal illness due to Cryptosporidium infection of the gastrointestinal tract in persons with AIDS becomes progressively worse with time and may be a major factor leading to death. At the present time, there is no effective therapy for treating or preventing cryptosporidiosis. Attempts to design treatments for cryptosporidiosis have been largely based on using methods of intervention previously exploited in other disease causing organisms. This approach has not been successful and is limited because it is based on knowledge discovered independently of Cryptosporidium biology. The lack of a basic understanding of parasite biology has been a limiting factor is designing effective means of treating and preventing disease. In our research proposal, we have elected to take a comprehensive, basic biological approach to studying the biology of C. parvum, in order to provide the foundation for rationale design of new therapies for cryptosporidiosis. The goal of our studies is to identify and characterize those genes and gene products, both of the parasite and of the parasite and the host cells, whose mRNAs are specifically expressed in C. parvum-infected cells. The PCR-based technique of differential mRNA display will be used to clone host genes that are down regulated during infection with C. parvum.
In specific aim 2, we will generate monoclonal antibodies specific for C. parvum-infected cells to detect changes in gene expression that are not the result of modulating mRNA levels. These will include those parasite proteins that are synthesized in sporozoites and become expressed in the host cell after penetration and subsequent development. The monoclonal antibodies will be used to clone the cDNAs that experiments to characterize the differentially expressed genes to begin to assign putative functions. These will include determination of nucleic acid sequences and gene origin, host vs. parasite, kinetics of expression, and in localization of the mRNA and protein products. We will focus on characterizing those genes that represent unique aspects of Cryptosporidium biology and host-parasite interaction. Once we have generated the necessary DNA and antibody reagents, we will perform key experiments in vivo, such as in situ localization of mRNA and protein expression, to confirm the conclusions of the in vitro model. These studies will lay the foundation to start to understand specific aspects of the biology of C. parvum and host- parasite interactions.
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