The coherent transmission of light through the cornea aids in forming clear images on the retina. Permanent alterations in the cells or matrix molecules of this highly organized tissue resulting from corneal dystrophies, corneal injuries or other abnormal conditions affect visual acuity. An immunological approach will be used to characterize cell-surface makeup of the corneal epithelial and stromal cells and their extracellular matrices under normal and pathological conditions. Monoclonal antibodies (MAbs) derived from the hybridomas developed by the fusion of mouse myeloma cells with spleen cells of mice immunized with (i) membrane fractions of cells in culture or cells derived from the tissues or (ii) partially purified extracellular components will be used for characterizing the cell surfaces and the extracellular matrices. The profile of cell-surface antigens and the extracellular matrix components will be studied immunohistochemically using these MAbs in (1) the normal rabbit cornea, (2) regenerating corneas (in healing of experimental wounds) and (3) tissue cultures under a variety of conditions. An indirect immunoperoxidase technique or an immunofluorescent technique will be employed for these studies. The topographic distribution and ultrastructural localization of the antigens will be investigated using the MAbs of specific interset. Cell-surface antigens and extracellular matrix components in human corneal cells from normal and dystrophic eyes will be analyzed immunohistochemically. The surface antigen profile of debrided corneal epithelium from recurrent corneal erosions will also be investigated. Based on the results of the immunohistochemical analysis, antigens that are of significant interest will be selected for their further biochemical characterization. Proteins or glycoproteins will be radioactively labeled using selective procedures for labeling. Labeled antigens will then be precipitated from the complex mixture of antigens using specific antibodies and precipitates analyzed by SDS-polyacrylamide gel electrophoresis. In addition, proteins blotted from SDS-gels onto nitrocellulose papers will be reacted with antibody to identify the specific antigens. Affinity purified antigens using antibody-affinity columns will be further analyzed for their molecular size, chemical composition, polypeptide composition and peptide maps derived from CNBr or enzyme treatments. These studies will give an insight into the biochemical alterations of the cell surface and extracellular matrix associated with cellular migration, proliferation and quiescence and also different pathological states of the cornea.
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