The aims of the proposed research are to obtain basic information about the cell biology of corneal epithelial adhesion to the stroma and epithelial wound healing that would lead to improved treatment for persistent epithelial defects and recurrent corneal erosions in humans. Another aim is to determine characteristics of the ocular surface epithelial cell glycocalyx, or mucin layer, and ascertain if and how this composition is affected in human ocular surface pathologies such as the dry eye disorders. Corneal epithelial adhesion. The cell-substrate junctions (hemidesmosomes) responsible for adhesion of the basal cells of the corneal epithelium to its basement membrane form over specific sites or components on the basement membrane where anchoring fibrils insert from the stromal surface; epithelial cells recognize these components to reform hemidesmosomes. To identify the basement membrane component that induces hemidesmosome formation, two approaches will be used. By low-voltage scanning electron microscopy, we will determine if any of a group of monoclonal antibodies specific to the epithelial basement membrane bind the epithelial surface of the basement membrane. Such antibodies will be characterized and used to determine if they block hemidesmosome formation. A second approach will recombine extracellular matrix components that will induce hemidesmosome formation in an in vitro assay. Cell motility during corneal epithelial wound healing. An early event within basal cells of the corneal epithelium at a wound margin is loss of hemidesmosomes. Recent data indicate that as cells begin to migrate they synthesize vinculin, a protein known to be in a different type of adhesion junction, the focal contact. Immunolocalization procedures will be used to determine if the vinculin in migrating epithelia is in focal contacts (that contain talin and/or alpha-actinin). Using confocal epifluorescence microscopy, the switch from hemidesmosome to focal contacts at wound margin will be studied to determine if a cell can express both or only one type of junction. Specific extracellular matrix components will be tested to see if they can affect this switch of expression of the individual junctions. Ocular surface glycoproteins. Monoclonal antibodies have been produced that bind exclusively apical cells and/or goblet cells of the entire ocular surface epithelium of the rat. Preliminary data suggest that the antigen(s) is/are either cell surface glycoproteins or surface mucins. Characterization of these antibodies with Western blot analyses and immunoprecipitation methodology will determine that nature and cell of origin of the antigen(s). Hybridomas have been produced from human corneal epithelium that have characteristics in common with the rat antibodies. These will be cloned, characterized, and used to compare, by impression cytology methods, binding of the antibodies to the ocular surface of normal and dry eye patients. Changes in ocular surface glycocalyx, or mucins, may account for tear """"""""dry spots"""""""" and mucous filaments found in dry eye disorders.
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