The wet surface of the eye is crucial for light refraction. Drying diseases and pathogen invasion often occur on this exposed surface. Mucins of two types-secreted and membrane associated-are hypothesized to be vital for maintenance of the wet surface. Membrane associated mucins present in a dense layer on the apical cells of the corneal epithelium at the tear film interface are also hypothesized to be a barrier to pathogen and large molecule penetrance. Building on the characterizations of membrane associated mucins of the ocular surface and on the systems developed for their study in our previous funding period, we propose to investigate the function of this class of mucins at the surface of the eye in six aims.
Aim I : We will determine if membrane-associated mucins are involved in surface membrane microplicae formation on corneal surfaces.
Aim II : We will determine if membrane-associated mucins give rise to the changes on surface cells indicative of apical cell differentiation of the ocular surface epithelia.
Aim III : We will determine if membrane-associated mucins function as disadhesives, preventing cell or bacterial adhesion.
In Aims I -III, the studies will be done using a human corneal limbal epithelial cell line, along with siRNA technology, field emission immuno-scanning electron microscopy, and real time PCR. Data from the cell line will be correlated to that of wild type and mice null for membrane associated mucins.
Aim I V: We will determine if membrane associated mucins form a barrier to penetrance of large molecules and pathogens along the glycocalyx present on the tips of the microplicae of the corneal epithelium, and if the secreted goblet cell mucin MUC5AC is partitioned outside that barrier. Rapid freeze, freeze substitution followed by Immuno-electron microscopy will be used to localize pathogens and MUC5AC in relation to membrane associated mucins.
Aim V : We will determine if membrane associated mucins protecting the eye ? from fluid loss and dry eye, using mice null for membrane associated mucins housed in a controlled ? environment chamber, which reliably produces decreased tear volume in mice.
Aim V I: We will determine if the alteration of MUC16 seen previously in dry eye is a result of a decrease in mucin gene expression, shedding rate, and/ or post-translational modification, and if the extent of these alterations correlates with severity of dry eye. ? ?
|Gipson, Ilene K; Mandel, Ulla; Menon, Balaraj et al. (2017) Generation and characterization of a monoclonal antibody to the cytoplasmic tail of MUC16. Glycobiology 27:920-926|
|Robert, Marie-Claude; Arafat, Samer N; Spurr-Michaud, Sandra et al. (2016) Tear Matrix Metalloproteinases and Myeloperoxidase Levels in Patients With Boston Keratoprosthesis Type I. Cornea 35:1008-14|
|Gipson, Ilene K (2016) Goblet cells of the conjunctiva: A review of recent findings. Prog Retin Eye Res 54:49-63|
|Gipson, Ilene K; Spurr-Michaud, Sandra; Tisdale, Ann (2016) Human conjunctival goblet cells express the membrane associated mucin MUC16: Localization to mucin granules. Exp Eye Res 145:230-234|
|Menon, B B; Kaiser-Marko, C; Spurr-Michaud, S et al. (2015) Suppression of Toll-like receptor-mediated innate immune responses at the ocular surface by the membrane-associated mucins MUC1 and MUC16. Mucosal Immunol 8:1000-8|
|Marko, Christina Kaiser; Tisdale, Ann S; Spurr-Michaud, Sandra et al. (2014) The ocular surface phenotype of Muc5ac and Muc5b null mice. Invest Ophthalmol Vis Sci 55:291-300|
|Gipson, Ilene K; Spurr-Michaud, Sandra; Tisdale, Ann et al. (2014) Comparison of the transmembrane mucins MUC1 and MUC16 in epithelial barrier function. PLoS One 9:e100393|
|Arafat, Samer N; Suelves, Ana M; Spurr-Michaud, Sandra et al. (2014) Neutrophil collagenase, gelatinase, and myeloperoxidase in tears of patients with stevens-johnson syndrome and ocular cicatricial pemphigoid. Ophthalmology 121:79-87|
|Spurr-Michaud, Sandra J; Gipson, Ilene K (2013) Methods for culture of human corneal and conjunctival epithelia. Methods Mol Biol 945:31-43|
|Marko, Christina K; Menon, Balaraj B; Chen, Gang et al. (2013) Spdef null mice lack conjunctival goblet cells and provide a model of dry eye. Am J Pathol 183:35-48|
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