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Eddie Dominguez Micro 790 March 5 2015
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Today’s adventure entails… Overview of Entamoeba histolytica Colon epithelial tissue Extracellular Matrix Matrix Metalloproteinase Paper – Previous work – Techniques
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Entamoeba histolytica Parasite Disease: Amebiasis Key Source: contaminated food/drinking water Deaths/year: 70,001 worldwide Incdence in U.S.: 1,800/year, mainly travelers/immigrants Symptoms: diarrhea, dysentery, amebic colitis, liver abscesses
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Atlas of Human Infectious Diseases
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E. histolytica Life Cycle Ingestion of cysts Excystation in small intestine Division of quadrinucleate cyst into 4 and then 8 trophozoites Trophozoites move to colonize the colon Excystation Excretion of cysts Ingestion of cysts by the patient Wikipedia
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E. histolytica Life Cycle Ingestion of cysts Excystation in small intestine Division of quadrinucleate cyst into 4 and then 8 trophozoites Trophozoites move to colonize the colon Excystation Excretion of cysts Ingestion of cysts by the patient Wikipedia
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Colon Tissue Glycoforum. 1999UNSW Embryology iupui.edu
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Extracellular Matrix Frantz et al. J Cell Sci 2010
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Matrix Metalloproteinase (MMP) Tan and Liu. American Journal of Physiology – Renal Physiology 2012
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MMPs Wikipedia IL-1β
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Previous findings include… Well documented that cysteine proteases are a requirement in intestinal barrier disruption (Que and Reed, 2000; Moncada et al. 2006) 86 genes encoding putative proteases are present in the E. histolytica genome (Tillack et al., 2007) Trophozoites silenced for CP-A5 failed to trigger a host inflammatory response or invade the colonic lamina propria (Bansel et al., 2009) Pro-mature CP-A5 binds colonocyte and triggers cytokine secretion (Hou et al., 2010) MMPs-1 and -3 were overexpressed in patients with intestinal amoebiasis (Peterson et al., 2011)
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Cysteine protease A5 (CP-A5) Secreted protease by the trophozoite Cleaves the major mucus component mucin 2 Contains a Arg-Gly-Asp (RGD) motif that can bind directly to α v β 3 integrin on colonocytes (colon epithelial cells) and triggers a NFκB-mediated pro- inflammatory response (TNF-α and IL- 1β) Thibeaux et al. Cellular Microbiology 2012
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Techniques Western Blot qRT-PCR Immunofluorescence and confocal microscopy Multiphoton (MPM) and second-harmonic generation imaging microscopy (SHIM) Collagen degradation assays
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Second-Harmonic Generation Imaging Microscopy Scientific Volume Imaging
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Big Question Are human MMPs involved in the destruction of the ECM of the lamina propria during intestinal invasion by E. histolytica?
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Does Human MMP play a role in E. histolytica invasion?
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Human MMP enzymatic activity plays a role in E. histolytica invasion Trophozoites pretreated with marimastat results in tissue invasion Tissue pretreated with marimastat results in no tissue invasion
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Is Human MMP activity responsible for collagen remodeling? Fibrillar collagen: Green Endogenous cells: Red WT trophozoites: Purple
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Human MMP activity responsible for collagen remodeling Fibrillar collagen: Green Endogenous cells: Red WT trophozoites: Purple Trophozoites pretreated with marimastat results in collagen remodelling Tissue pretreated with marimastat results in no collagen remodelling
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Is CP-A5 involved in MMP expression?
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CP-A5 is involved in MMP expression
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In what tissue is MMP-3 and -1 localized upon amoebic invasion?
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MMP-3 and -1 are localized at the epithelial cell layer and lamina propria
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Active MMP-3 and -1 is present during colonic invasion
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Active MMP-1 is present during colonic invasion
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C119 is important for catalytic activity in CP-A5
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Who activates MMP-1?
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CP-A5 does not activate MMP-1
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Who activates MMP-1 and MMP-3?
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CP-A5 activates Pro-MMP-3 which in turn activates Pro-MMP-1
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WT-CP-A5 restores invasion
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Role of CP-A5
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Thibeaux et al. Cellular Microbiology 2012 MMP-1 MMP-3 Model for E. histolytica tissue invasion MMPs
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Summary E. histolytica utilizes the hosts immune system during intestinal invasion CP-A5 instigates this process by eliciting the - secretion of pro-inflamatory cytokines (THF-α and IL-1β), which then triggers the synthesis of MMPs CP-A5 then activates MMP-3 who in turn activates MMP-1 which then degrades the ECM. Upon degradation of the ECM, trophozoites are able to invade into the lamina propria
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