Presented by: Chris Raske

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• Presented by Chris Raske

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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.

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Life Cycle
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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.

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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.

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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.

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Researchers 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 cryptosporidiums pathology?

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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

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Caspase Inhibitors

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

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Apoptosis
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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
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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
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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.
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Results
Z-VAD effect on nuclear condensation of infected
cells
zVAD inhibited most of the DNA condensation,
however its presence increased the of infected
cells.
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Conclusions

• C. parvums 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.

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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?