Purpose. new prophylactic agent for preventing contact lens or trauma/injury-associated microbial keratitis. Corneal innate immune responses are key mediators of the host’s defense against microbial infection, including fungal keratitis. Fungal infections of the cornea are frequently caused by species of is a commensal fungus of the normal flora, yet it causes opportunistic infection of the cornea after trauma or surgery, during periods of immunosuppression including prolonged corticosteroid use, and during topical anesthetic abuse.3C6 While keratitis caused by filamentous fungi (and accounts for proportionately more fungal corneal isolates at temperate latitudes.7 To date, there has been no clinically applicable measure for preventing fungal keratitis.8 Hence, there is an urgent need for the development of a prophylactic strategy for prevention of fungal keratitis in populations at high risk, in light of an increasing incidence of fungal keratitis and of a keratitis epidemic associated with contact lens solutions.9,10 Several murine models of fungal keratitis have been reported, including intrastromal corneal injection P7C3-A20 supplier of and biofilm.12 Using these models, it was revealed that innate immunity, primarily mediated by various Toll-like receptors (TLRs) and the MyD88 signaling pathway play a key role in the host response to fungal infection.13,14 However, there appears to be a discrepancy regarding which TLR(s) is involved in the innate defense of the cornea. Whereas deletion of TLR2 or -4 was shown to have no influence on general disease development, they were shown to have a role in controlling growth and replication of contamination in B6 mice.15,16 This treatment diminished the inflammatory response to infection and at the same time enhanced the production of antimicrobial peptides (AMPs) and cytoprotective mediators in a TLR-dependent manner.17,18 We reasoned that, although TLR5 is not a known fungus-recognizing receptor, the protective mechanisms induced after its activation by flagellin may function effectively in Rabbit Polyclonal to IkappaB-alpha controlling fungal contamination. We report for the first time that the topical application of flagellin on wounded cornea is effective in preventing the development of fungal P7C3-A20 supplier keratitis and that the antimicrobial peptide CRAMP (the gene P7C3-A20 supplier product of strain SC5314, a clinical isolate capable of producing experimental keratomycosis, was cultured on YPD agar (Sigma-Aldrich, St. Louis, MO) for 3 days at 25C. Colonies were harvested after 3 days of inoculation and diluted in sterile phosphate-buffered saline (PBS) to yield 2 P7C3-A20 supplier 105 colony-forming units (CFU)/L based on the optical density (OD) at 600 nm, using a predetermined OD600 conversion factor of 1 1 OD = 3 107 CFU/mL. Animals Wild-type (WT) C57BL6 (B6) mice (8 weeks of age; 20C24 g weight) and strain PA01 or PAK, as described earlier.18,20 For P7C3-A20 supplier flagellin pretreatment, mice (= 5/group/treatment) were anesthetized with ketamine and xylazine and placed beneath a stereoscopic microscope at a magnification of 40, and the cornea of the left eye was scratched with three 1-mm incisions made with a sterile 26-gauge needle. Purified flagellin (500 ng in 5 L of PBS) or PBS as the control was applied to the injured corneas. For corneal contamination, the mice were anesthetized, the pretreated corneas were rescratched 6 to 72 hours after the application of flagellin; and a 5-L suspension made up of 1 104 to 106 CFU of strain SC5314 was applied to the surface of the scarified cornea. Clinical Examination For the assessment of clinical scores, the mice were color-coded and examined by two impartial observers daily and photographed at 6 hours and 1, 3, or 5 days after contamination (dpi). Ocular disease was graded in clinical scores ranging from 0 to 12, according to the scoring system developed by Wu et al.21 A grade of 0 to 4 was assigned to each of the following three criteriaarea of opacity,.