title: Methods for In Vitro Analysis of Antimicrobial Activity and Toxicity of Anti-keratitis Peptides: Bacterial Viability in Tears, MTT, and TNF-α Release Assays.
abstract: Ease of access to the cornea makes antimicrobial peptides (AMPs) ideal candidates for topical drug application. However, before bringing them to the clinic, it is fundamental to evaluate in vitro: (1) the ability of AMPs to kill bacteria in the presence of human tears, by counting the number of surviving bacteria on agar plates; (2) the potential cytotoxicity of AMPs to mammalian cells by a colorimetric method based on the production of a colored formazan crystals by metabolically active cells; and (3) the ability of AMPs to neutralize the toxic effect of the bacterial cell wall component, lipopolysaccharide (LPS), by measuring the level of the pro-inflammatory cytokine, TNF-α, released from LPS-activated macrophages, using a sandwich enzyme-linked immunosorbent assay.
authors.affiliation: Department of Biochemical Sciences, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy.
authors.forename: Floriana
authors.initials: F
authors.lastname: Cappiello
authors.affiliation: Department of Biochemical Sciences, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy.
authors.forename: Bruno
authors.initials: B
authors.lastname: Casciaro
authors.affiliation: The Ocular Surface Institute, College of Optometry, University of Houston, Houston, TX, USA.
authors.forename: Satya Sree
authors.initials: SS
authors.lastname: Kolar
authors.affiliation: The Ocular Surface Institute, College of Optometry, University of Houston, Houston, TX, USA.
authors.forename: Hasna
authors.initials: H
authors.lastname: Baidouri
authors.affiliation: The Ocular Surface Institute, College of Optometry, University of Houston, Houston, TX, USA.
authors.forename: Alison M
authors.initials: AM
authors.lastname: McDermott
authors.affiliation: Department of Biochemical Sciences, Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy. marialuisa.mangoni@uniroma1.it.
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Mangoni
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects; D003124:Colorimetry / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism; D007634:Keratitis / Q000188:drug therapy / Q000378:metabolism* / Q000382:microbiology*; D008070:Lipopolysaccharides / Q000276:immunology; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D009500:Neutralization Tests; D013666:Tears / Q000382:microbiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha
keywords: Colony counts; Colorimetric MTT assay; LPS detoxification; Sandwich immunosorbent assay; Tear collection