We shall attempt to elucidate the mechanism of the development of Acanthamoeba keratitis by examining the plasma membrane molecules of corneal epithelium and Acanthamoeba. In project 1 we shall (1) identify and characterize those rabbit corneal epithelial cell surface glycoconjugates which play a role in the host-parasite interactions, (2) pinpoint the sites of parasite attachment on Acanthamoeba-reactive cell surface glycoconjugates of rabbit corneal epithelium, (3) prepare monoclonal and polyclonal antibodies against those Acanthamoeba and corneal epithelial cell surface glycoconjugates which play a role in the host-parasite interactions, (4) determine using immunohistochemical techniques, whether Acanthamoeba-reactive glycoconjugate, found on rabbit corneal epithelial cells in culture are present on normal human and rabbit corneas in vivo, and (5) determine whether it is possible to block the adherence of Acanthamoeba to human and rabbit corneas using the above antibodies and by various saccharides, exoglycosidases and lectins. Since the neuraminidase of many pathogens, including influenza virus and parasite Trypanosoma cruzi, plays a role in the pathogenesis of infection, we shall, in project 2, try to determine whether Acanthamoeba neuraminidase plays a role in the pathogenesis of Acanthamoeba keratitis. In this study, we shall (1) isolate and characterize the neuraminidase produced by human corneal isolates of Acanthamoeba and establish whether the enzyme is structurally related to the neuraminidases of other parasites, especially T cruzi, (2) identify those rabbit corneal epithelial cell surface glycoconjugates which serve as substrates for Acanthamoeba neuraminidase and determine whether asialation of corneal epithelial cell surface glycoconjugates by the parasite enzyme exposes the parasite attachment sites and (3) determine whether anti-neuraminidase antibodies and non-immune inhibitors of neuraminidases inhibit or enhance the adherence of Acanthamoeba to human and rabbit corneal epithelium and thereby establish whether Acanthamoeba neuraminidase, like the T cruzi enzyme, regulates the infection by a negative control mechanism. It is hoped that the proposed studies will lead to a better understanding of the molecular basis of Acanthamoeba keratitis and to the development of more effective therapy for this sight threatening and difficult to treat disease.
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