Other pathogens inhibit release of cytochromecfrom mitochondria (7,10,17,31,50,51) and inhibit caspase activation (25,37). rate and a reduction in the accumulation of meront-containing cells. To test the significance of the moderately proapoptotic state late in the infection, we inhibited apoptosis using pancaspase inhibitor Z-VAD-FMK. This treatment also affected the progression ofC. parvuminfection, as reinfection, normally seen late (24 h to 48 h), did not occur and accumulation of mature meronts was impaired. Control of host apoptosis is complex and crucial to the life ofC. parvum. Apoptosis control has at least two components, early inhibition and late moderate promotion. For a successful infection, both aspects appear to be required. Cryptosporidium parvumis an obligate intracellular apicomplexan parasite and a well-recognized cause of severe diarrheal disease in many mammalian species, including humans (15). It primarily infects epithelial cells of the gastrointestinal tract, resulting in COL4A3BP acute and profuse watery diarrhea that is typically self-limited in immunocompetent individuals but persistent and potentially life threatening in immunocompromised hosts (8,16). Previous in vitro and in vivo studies demonstrated (9) thatC. parvuminfection induces expression of a variety of host immune modulators that activate both the innate and adaptive immune responses, including the accumulation of specific lymphocyte populations within the intestinal villi and increased secretion of several proinflammatory cytokines. Infected cells are structurally affected, as evidenced by polymerization of host cell actins and reorganization of cytoskeleton components (9,13,19,26,34,38). Such host responses are accompanied by blunting of intestinal villi, hyperplasia of crypt cells, and decreased sodium absorption (26,30). On a cellular level,C. parvuminfection of epithelial cells is intracellular but extracytoplasmic and requires the establishment of a parasitophorous vacuole. Apoptosis has been demonstrated to be an important defense mechanism for the host in response to many infections (36,49). Predictably, many parasites possess the ability to modulate host cell apoptosis (11,22,29). Does apoptosis containCryptosporidiuminfection? In natural and experimental infections in vivo, the picture is complex. Apoptosis has been observed in association with infection; however, a simple correlation between pathology and apoptosis does not exist (30,41). In vitro,Cryptosporidiuminduces apoptosis in a small minority of the infected cells (5,33,35), which is not enough to ward off infection.Cryptosporidiumhas apparently evolved countermeasures to keep the host cells in a survival mode. Indeed, the infected cells acquire resistance to various chemical agents that trigger apoptosis (6,28,33,35). Additionally, the host appears to be equipped with a second line of defense also involving apoptosis: uninfected bystander cells die due to FasL secreted from the infected cells (5,6,33,35).Cryptosporidiumactivates the NF-B pathway in the infected cells and survives, while the host can contain the infection by surrounding the infected cells with a zone of apoptosis. Here we report genome-wide expression profiling ofC. parvum-infected intestinal epithelial cells and the functional significance of host cell apoptosis for the infection process. Our results revealed that parasite infection of host epithelial cells is a complex process involving the regulation of 333 host genes. The largest functional group identified from the microarray included 51 genes that are related to cellular apoptosis. Apoptosis gene transcript profiles suggested that host proapoptotic gene expression is actively downregulated early in infection but is favored at late stages. Experimental induction and inhibition of cell apoptosis alteredC. parvuminfection and development, suggesting thatC. parvumactively subverts host apoptosis in a biphasic manner to complete its life cycle. == MATERIALS AND METHODS == == C. parvuminfection assays. == C. parvum(Iowa isolate) oocysts were purchased (Bunch Grass Farm, Drury, ID) and stored at 4C in 2.5% Tepilamide fumarate potassium dichromate for up to 3 months. Monolayers of human ileocecal adenocarcinoma cells (HCT-8; ATCC, Manassas, VA) were cultured in Tepilamide fumarate RPMI 1640 medium supplemented with 10% fetal bovine serum as previously described (43,48). Cells in log phase were plated at 2 106cells per 150- by 25-mm tissue culture dish and infected as previously described (43,48). Briefly, purified oocysts were suspended in phosphate-buffered saline (PBS), sterilized in 33% bleach-PBS for 7 min Tepilamide fumarate on ice, washed in Hanks’ buffered saline solution, and added to HCT-8 cultures. Following a 2-h excystation period at 37C, cells were washed with warm Hanks’.