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Presented by: Chris Raske

Presented by: Chris Raske. Cryptosporidium Parvum. A protozoan parasite which belongs to the class Coccidia. Undergoes a complex life cycle with alternating asexual and sexual reproductive cycles. It is one of the most common non-viral causes of diarrhea in many animals as well as humans.

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Presented by: Chris Raske

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  1. Presented by: Chris Raske

  2. Cryptosporidium Parvum • A protozoan parasite which belongs to the class Coccidia. • Undergoes a complex life cycle with alternating asexual and sexual reproductive cycles. • It is one of the most common non-viral causes of diarrhea in many animals as well as humans. • The first reported human cases were in 1976, and continues to infect people worldwide. In 1993 there was an outbreak in Milwaukee which infected over 400,000 people.

  3. Life Cycle

  4. Cryptosporidium Parvum • In immunocompetent individuals cryptosporidium causes transient diarrhea, however in immunosuppressed individuals, infection results in more persistent and severe diarrhea which can be fatal. (ex. AIDS) • Cryptosporidium generally infects the apical layer of endothelial cells of the intestine, and normally does not invade deeper layers of the gastrointestinal mucosa. • Upon infection, the epithelial cells release proinflamitory cytokines including neutrophil chemoattractants, which are at least partly responsible in attracting effector cells.

  5. Cryptosporidium Parvum • Mice deficient in B cells are able to fully eliminate the parasite which suggests antibodies are not required for its elimination. • However, SCID mice and mice unable to make MHC class II molecules are susceptible to infection by C. parvum. • These results suggest that cell-mediated immunity is the most important factor in recovering from C. parvum infection.

  6. Infection Leads to Cell Lysis • After excystation in the intestine, the parasite invades epithelial cells where it remains in a parasitophorous vacuole (PV) directly beneath the apical membrane of the cell. • It has been shown that cell lysis occurs at these parasitophorous vacuoles at the apical membrane. • However, the mechanism of cell death was previously unknown.

  7. Researcher’s Objective • Are the cells undergoing cell death through apoptotic pathways or necrosis? • DNA condensation? • DNA fragmentation? • If it is an apoptotic pathway, what are the molecular mediators? Could a caspase inhibitor alter cryptosporidium’s pathology?

  8. Apoptotic DNA Condensation and Fragmentation • During Apoptosis, the cell cycle arrests and DNA remains in a condensed form. • Apoptotic DNA fragmentation is a result of caspase dependent pathways. • Caspase-activated DNA fragmenting factor (CAD) is activated when it is cleaved by caspases and released from its inhibitor (ICAD). • Upon activation CAD enters the nucleus and degrades DNA

  9. In a previous study researchers induced apoptosis in liver cells with Antibodies against Fas, then inhibited the apoptosis pathway with a caspase inhibitor. Caspase Inhibitors

  10. Apoptosis

  11. Results Apoptotic nuclear condensation during infection: Both the number of cells infected and the number of apoptotic cells increased with higher concentrations of oocysts. Methods: Cytofluorimetry PI staining

  12. Results Morphology of infected cells: DAPI staining to observe chromatin condensation (A) HCT-8 cells without C. parvum (B) HCT-8 cells with C. parvum

  13. Results DNA Fragmentation: There is considerable DNA fragmentation when approximately 20% of cells are infected. However, in the presence of a caspase inhibitor (zVAD) the amount of fragmentation is decreased.

  14. Results Z-VAD effect on nuclear condensation of infected cells: zVAD inhibited most of the DNA condensation, however it’s presence increased the % of infected cells.

  15. Conclusions • C. parvum’s infection of epithelial cells does induce apoptosis. Both DNA condensation and fragmentation were observed shortly after infection. • DNA condensation and fragmentation were inhibited with a caspase inhibitor (z-VAD-fmk), which suggests a caspase dependent pathway. In addition the caspase inhibitor increased the percentage of infected cells.

  16. Future Studies • A previous study showed C. parvum also caused apoptosis in an infected biliary epithelial cell line. Does C. parvum cause apoptosis in all epithelial cells and what is the exact mechanism? • Intrinsic or extrinsic pathway or both? • What are the consequences of inhibiting apoptosis in these epithelial cells in vivo? • Does it benefit the host or the pathogen? • During C. parvum infection the disruption of the intestinal barrier allows absorption of large molecules including lactulose and mannitol leading to more severe diarrhea. • Could apoptosis be contributing to this increased permeability?

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