Analysis of Factors that Provide Protection Against Acanthamoeba Keratitis

Abstract

Abstract: : Purpose: It is thought that tears of normal individuals contain parasite–specific IgA that provides protection against Acanthamoeba keratitis (AK). In view of the fact that factors other than IgA may also be involved in providing protection against AK, the goal of the present study was to shed light on the possible factors present in human secretions, which provide protection against Acanthamoeba–induced cytopathic effect (CPE). Methods: In vitro CPE assays were used to assess protective effect of human tears on Acanthamoeba–induced CPE on corneal epithelial cells. Human tears and milk share many similarities. For example, both (i) provide protection against infection by a wide variety of pathogenic organisms and, (ii) share many chemical constituents including secretory IgA, lactoferrin, and lysozymes. Therefore, in the present study, after initial experiments with tears, in–depth studies requiring large quantities of starting material were carried out using milk. Results: Acanthamoeba parasites produced potent CPE on corneal epithelial cells within 18 hours. Both human tears and delipidated milk were potent inhibitors of Acanthamoeba–induced CPE. Experiments with milk fractions separated based on molecular weight revealed that the component(s) responsible for providing protection is of molecular weight > 100–kDa. In some experiments, prior to CPE assays, parasites or epithelial cells were preincubated with milk for 30 minutes and excess milk components were removed by washing the cells in buffer. Acanthamoeba induced CPE was almost completely inhibited if parasites were pretreated with milk suggesting that the inhibitory component binds to the parasites. In contrast, pretreatment of epithelial cells with milk was not protective. Western blotting of parasite extracts which had been pretreated with milk revealed that human milk contains parasite–specific IgA suggesting that, as expected, IgA present in milk may be one of the components responsible for providing protection. However, milk depleted of IgA also provided significant protection against Acanthamoeba–induced CPE indicating that protective factors other than IgA are also present. Conclusions: Tears and other human secretions such as milk contain factors that provide protection against Acanthamoeba–induced CPE. Identification of these factors is crucial to understanding of pathogenic mechanisms of Acanthamoeba keratitis.

Keywords: Acanthamoeba • cornea: tears/tear film/dry eye • keratitis