title: Methods for In Vivo/Ex Vivo Analysis of Antimicrobial Peptides in Bacterial Keratitis: siRNA Knockdown, Colony Counts, Myeloperoxidase, Immunostaining, and RT-PCR Assays.
abstract: Antimicrobial peptides (AMPs) are essential components of the innate immune response. They have direct killing ability as well as immunomodulatory functions. Here, we describe techniques to identify specific AMPs involved in the protection against microbial keratitis, a vision threatening infection of the cornea of the eye which is the most serious complication of contact lens wear. Specifically we detail the use of siRNA technology to temporarily knockdown AMP expression at the murine ocular surface in vivo and then describe ex vivo assays to determine the level of bacteria, relative number of neutrophils, and levels of cytokines, chemokines, and AMPs in infected corneas.
authors.affiliation: The Ocular Surface Institute, College of Optometry, University of Houston, 4901 Calhoun Road, Houston, TX, 77204, USA.
authors.forename: Satya Sree
authors.initials: SS
authors.lastname: Kolar
authors.affiliation: The Ocular Surface Institute, College of Optometry, University of Houston, 4901 Calhoun Road, Houston, TX, 77204, USA.
authors.forename: Hasna
authors.initials: H
authors.lastname: Baidouri
authors.affiliation: Department of Biochemical Sciences "A. Rossi Fanelli", Istituto Pasteur-Fondaziona Cenci Bolognetti, Sapienza University of Rome, Rome, Italy.
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Mangoni
authors.affiliation: The Ocular Surface Institute, College of Optometry, University of Houston, 4901 Calhoun Road, Houston, TX, 77204, USA. AMcdermott@Central.UH.EDU.
authors.forename: Alison M
authors.initials: AM
authors.lastname: McDermott
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000378:metabolism; D001424:Bacterial Infections / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000382:microbiology*; D058491:Bacterial Load; D016207:Cytokines / Q000378:metabolism; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D054884:Host-Pathogen Interactions / Q000235:genetics / Q000276:immunology; D018836:Inflammation Mediators / Q000378:metabolism; D007634:Keratitis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000382:microbiology*; D051379:Mice; D050296:Microbial Viability; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D023181:Antimicrobial Cationic Peptides; D016207:Cytokines; D018836:Inflammation Mediators; D034741:RNA, Small Interfering; D009195:Peroxidase
keywords: Antimicrobial peptide; Inflammation; Keratitis; Neutrophil; Pseudomonas