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Joseph C Manarang, Hasna Baidouri, Satya Sree N Kolar, Maria L Mangoni, Alison M McDermott; Antimicrobial Peptides and Acanthamoeba: Peptide expression and anti-amoebicidal activity via time-lapse imaging. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6266.
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© ARVO (1962-2015); The Authors (2016-present)
To determine human corneal epithelial cell antimicrobial peptide (AMP) expression in response to Acanthamoeba castellani (A. castellani) and to visualize the trophocidal activity of AMPs using time-lapse live cell imaging.
Telomerase modified human corneal epithelial cells (hTCEpi) were exposed to 2X105/ml A. castellani trophozoites for up to 24 hours and AMP (cathelicidin LL-37, human beta defensins (hBD) -1, -2, -3 and -4) mRNA expression determined by RT-PCR. AMP expression by A. castellani trophozoites was also investigated. For visualization of trophocidal activity, 1X105/ml A. castellani trophozoites stained with Ethidium homodimer-1 were exposed to 1-500 μg/ml of AMPs (LL-37, hBD-1, -2, -3, insect AMP Cecropin B and amphibian AMPs Magainin II and Esculentin1-21). Serial bright field and fluorescence images were taken every 3 minutes for 6 hours using a Deltavision Deconvolution microscope.
As expected human AMPs were not expressed by A. castellani. hTCEpi exhibited low to no expression of hBD-2, -3, -4, and low to moderate expression of hBD-1 and LL-37 (n=3) under basal conditions. Exposure of hTCEpi to A. castellani trophozoites upregulated expression of hBD-1 5-fold and downregulated LL-37 by approximately 50% (n=2). Live cell imaging showed that LL-37 and hBD-1 were not amoebecidal even at 200μg/ml, the highest concentration tested. hBD-2 caused some trophozoite rupture with deposition of cellular debris at 200μg/ml, although multiple viable and dividing trophozoites were still observed. hBD-3 resulted in minimal cellular debris deposition at 100μg/ml. Esculentin1-21 exhibited complete trophocidal activity at 250μg/ml. Cecropin B exhibited partial trophocidal activity at 500μg/ml, whereas Magainin II exhibited complete killing at 500μg/ml, with no deposition of cellular debris in the background.
hBD-1 was upregulated in corneal epithelial cells, whereas LL-37 was downregulated. Though hBD-1 expression was increased, this and the other human AMPs showed minimal activity against A. castellani at the concentrations tested. The non-human AMPs Esculentin1-21, Cecropin B, and Magainin II exhibited potent amoebicidal activity at higher concentrations. These findings suggest that some AMPs have the potential to serve as amoebicidal agents for acanthamoeba keratitis and as disinfectants in contact lens care solutions.
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