Browsing by Subject "Corneal epithelium"
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Item CYTOSOLIC PHOSPHOLIPASE A2 ALPHA INHIBITORS ATTENUATE APOPTOSIS OF THE CORNEAL EPITHELIAL CELLS AND MITIGATE ACANTHAMOEBA KERATITIS(2013-04-12) Tripathi, TrivendraPurpose: The aim of this study was to explore if MIP-133 from Acanthamoeba castellanii trophozoites induces apoptosis of Chinese hamster corneal epithelial cells (HCORN) in vitro via cPLA2ɑ pathway, and to determine the efficacy of cPLA2ɑ inhibitors to alleviate AK in vivo. Methods: HCORN were incubated with or without MIP-133 at doses of 7.5, 15, and 50µg/ml for 6, 12, and 24hrs. Inhibition of cPLA2ɑ was carried out by pre-incubating HCORN for 1hr with cPLA2ɑ inhibitors (10µM MAFP and 20µM AACOCF3) with or without 15µg/ml MIP-133 for 24hrs. Chinese hamsters were injected subconjunctivally with MIP-133 at dosage 40µg/40µl and eyes were removed 3 days after infection. Chinese hamsters were infected with parasite-laden contact lens and treated with cPLA2ɑ inhibitors (AACOCF3 and CAY10650) topically 50µg/5µl three times a day for 14 days post-infection. Expression of cPLA2ɑ at mRNA and enzyme levels was examined by RT-PCR and cPLA2ɑ enzyme assay. Apoptosis was determined by DNA fragmentation assay. CXCL2 expression was examined by RT-PCR and ELISA. In vivo infections were examined by clinical severity of disease scored on a scale of 0 to 5 based on corneal infiltration, corneal neovascularization, and corneal ulceration. Clinical pathology of cornea was examined by histopathology. Results: MIP-133 induces significant increase in cPLA2ɑ and CXCL2 levels from corneal cells. Moreover, cPLA2ɑ inhibitors, MAFP and AACOCF3, significantly reduce cPLA2ɑ and CXCL2 from these cells (P<0.05). Additionally, cPLA2ɑ inhibitors significantly inhibit MIP-133-induced apoptosis in HCORN cells (P<0.05). Subconjunctival injection of purified MIP-133 produced cytopathic properties characteristic of MIP-133. Treatments of cPLA2ɑ inhibitors showed significantly less severe keratitis and decreased clinical pathology as compared with control animals. Conclusions: Results suggest that cPLA2ɑ inhibitors therapeutically attenuate apoptosis of the corneal epithelial cells and mitigate AK.Item PROTEASE-ACTIVATED RECEPTOR 2 (PAR2) IS UPREGULATED BY ACANTHAMOEBA PLASMINOGEN ACTIVATOR (APA) AND INDUCES PROINFLAMMATORY CYTOKINE IN HUMAN CORNEAL EPITHELIAL CELLS(2014-03) Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, HassanAcanthamoeba keratitis (AK) is a vision-threatening disease caused by pathogenic strains of Acanthamoeba. The main risk factors for AK are wearing eye contact lenses, corneal injuries, and contact with contaminated water. Diagnosis of AK is not straightforward and treatment is very demanding because of Acanthamoeba cysts resist to most antimicrobial agent. Our project is based on the observations that the innate immune system plays an important role in AK, and exploring the sequential pathogenic cascades of AK and targeting the therapeutic approaches to determine new remedies to prevent and treat AK. We have shown that Acanthamoeba trophozoites secrete cytopathic serine proteases, MIP-133 and aPA. Role of MIP-133 in the pathogenesis of AK via cPLA2α pathway has been explored; however, aPA interaction to corneal epithelial cells in disease progression is still unknown. In the present study, we have shown that aPA specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway and PAR2-antagonists may be a therapeutic target in AK. Purpose (a): Acanthamoeba plasminogen activator (aPA), is a serine protease elaborated by Acanthamoeba trophozoites, facilitates invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA induces proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptor PAR2 pathway. Methods (b): A. castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system and aPA activity was determined by zymography assays. HCE cells were incubated with or without aPA (100µg/ml), PAR1-agonists (Thrombin, 10µM; TRAP-6, 10µM), and PAR2-agonists (SLIGRL-NH2, 100µM; AC55541, 10µM) for 24 hours. Inhibition of PAR1 and PAR2 involved pre-incubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH79797, 60μM) and PAR2 (FSLLRY-NH2, 100μM) and then incubated with or without aPA, Thrombin, TRAP-6, SLIGRL-NH2, and AC55541 for 24 hours. Expression of PAR1 and PAR2 was examined by qRT-PCR, flow cytometry, and immunocytochemistry. IL-8 expression was quantified by qRT-PCR and by ELISA. Results (c): PAR1 and PAR2 surface protein were expressed in HCE cells. aPA and PAR2-agonists significantly upregulated PAR2 expression (~1-2 times) (P<0.05). PAR2-antagonist significantly inhibited aPA and PAR2-agonists-induced PAR2 expression (~2-5 times) (P<0.5) in HCE cells. PAR1-agonists, but not aPA, significantly upregulated PAR1 expression, which was significantly inhibited by PAR1-antagonist (~30-45 times) in HCE cells. aPA and PAR2-agonists, but not PAR1-agonists, stimulated IL-8 production, which is significantly diminished by PAR2-antagonist (~2-10 times) (P<0.5). PAR1-antagonist did not diminish aPA-induced IL-8 production in HCE cells. Conclusions (d): aPA specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway and PAR2-antagonists may be a therapeutic target in AK.