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Characteristics of Human Corneal-Limbal Epithelial Cells that Exclude Rose Bengal
TEAR FILM & OCULAR SURFACE CHARACTERISTICS OF HUMAN CORNEAL-LIMBAL EPITHELIAL CELLS THAT EXCLUDE ROSE BENGAL. Pablo Argüeso, Sandra SpurrMichaud, Ann Tisdale, Ilene K. Gipson. Schepens Eye Research Institute and Dept. of Ophthalmology, Harvard Medical School, Boston MA USA. Purpose. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. Our aim was to evaluate rose bengal staining in a human corneal-limbal epithelial cell line, designated HCLE, that is known to produce and glycosylate membrane-associated mucins. Methods. HCLE cells and human corneal fibroblasts were grown in media that promote differentiation (includes 10% serum). Immunofluorescence localization of membraneassociated MUC1 and MUC16 mucins and the T-antigen carbohydrate epitope was performed using HMFG-2, OC125 monoclonal antibodies and jacalin lectin, respectively. To assess rose bengal uptake, cell cultures were incubated for 5 min with 0.1 % rose bengal in PBS and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of osmolarity (Na+, Ca+2 and Mg+2) on rose bengal staining in vitro was evaluated by increasing the ion concentration in the rose bengal uptake assay. Results. When cultured in media without serum, the cytoplasm and nucleus of all confluent HCLE cells (lacking the expression of MUC16 but not MUC1), as well as human corneal fibroblasts (which do not express mucins), stained with rose bengal. Culture of HCLE cells in media containing serum resulted in the formation of islands of stratified apical cells that excluded rose bengal. Stratified cells produced the MUC16 mucin and the T-antigen carbohydrate epitope on their apical surfaces. Co-localization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charge. Increasing amounts of mono and divalent cations in the media reduced the cellular area protected against rose bengal uptake. Conclusions. These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated MUC16 mucin and the T-antigen mucin-associated carbohydrate epitope on their apical surfaces, provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of the ocular surface epithelia EFFECT OF ORAL MINOCYCLINE TREATMENT ON MEIBOMIANITIS. Joel D. Aronowicz1, Ward E. Shine1, Deniz Oral1, Jose M. Vargas1, James P. McCulley1. The Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas1. Purpose. To evaluate clinical impact, aqueous tear parameters, meibomian gland drop-out and meibomian gland lipid composition change in patients with primary meibomianitis, before and after initiating treatment with oral minocycline. Methods. Sixteen patients, 11 male and 5 females, mean ages 69 yrs old were enrolled. A prospective, clinic-based study. Patients with clinical meibomianitis were evaluated clinically and by fluorophotometry, evaporometry, meibography and microbiology before beginning oral minocycline and at 3 months on minocycline and 3 months after stopping minocycline. Results. Improvement was observed in clinical signs at the second and third visit. Microflora showed a decrease. Also tear volume (TV) and flow (TF) were decreased (TV: 1.39 r 0.62 Pl vs. 0.79 r 0.43 ҏPl vs. 1.05 r 0.78 ҏPl, P < 0.05; TF: 0.17 r 0.12 Pl/min vs. 0.11 r 0.09 vs. 0.08 r 0.07 ҏPl/min, P < 0.05). Tear evaporation rate did not change over time(0.056 r 0.032 Pl/cm2/min 10-2 vs. 0.054 r 0.031 ҏPl/cm2/min 10-2 vs. 0.053 r 0.026ҏ Pl/cm2/min 10-2, P > 0.05). Meibomian gland drop-out and meibomian secretion turbidity improved over time. Conclusions. There were clinical improvements in meibomianitis signs and these persisted for at least 3 months after discontinuation of treatment. This occurred despite a decrease in aqueous tear production (manifest by a statistically significant decrease in aqueous tear volume and tear flow) with no change in evaporation and an improvement in measurable meibomian gland drop-out and improved clarity of meibomian secretion. Decreased bacterial flora and meibomian gland lipid changes appeared to be associated with clinical improvement. Support: NIH EY12430 and unrestricted grant from Research to Prevent Blindness, Inc.
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FLUOROPHTOMETRY FOR OBJECTIVE MEASUREMENT OF OCULAR SURFACE DISEASE. Penny Asbell, Huma Naikoo, Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York Purpose. Determining efficacy in dry eye trials depends on having well defined endpoints. Current tests typically depend on subjective evaluation by either the patient or examiner, such as degree of symptoms or amount of staining. Objective endpoints that correlate with the signs and symptoms of dry eyes would improve endpoint evaluation. We used fluorophtometry to evaluate patients with dry eyes, as defined by signs and symptoms to determine the repeatability and correlation with exam findings. Patients who had Schirmrers less than 5mm and corneal staining of 1 or more were evaluated by fluorophotometry to determine the degree of corneal permeability. Methods. After a complete eye exam done within the previous month, fluorophotometry of the cornea was performed utilizing the Fluorotron Master ™. Readings were taken before any drops were placed and then at 15minute intervals after placement of 5 microns of 1 % sodium fluorescein dye placed in the inferior cul de sac of each eye. Concentration of fluorescein within the cornea was recorded at each time point and results analyzed and compared to standard eye tests for dry eyes. Similar tests were performed on normal eyes, patients requesting only refraction. Results. Scans of dry eye patients were dramatically different from normal eyes, in that the concentration of fluorescein within the cornea increased 4 to 5 fold in dry eye patients vs. under 1 fold in normal eyes. Preliminary data showed that at 15 minutes, the concentration in 10 dry eyes was 250ng/ml ± 100 SD vs. 62 ng/ml ± 23 SD in normals, and at 60 minutes for dry eyes the concentration was 70 ng/ml ± 28SD vs. 40 ng/ml ± 8 SD. Further analysis and repeatability results to be presented.Conclusions. Objective tests are needed to diagnose, follow dry eye patients and provide clearcut endpoints to evaluate new treatments for dry eyes. Fluorophotometry provides objective measurement of corneal permeability and preliminary analysis suggests it can be correlated with ocular surface changes of the cornea in dry eye patients. Support: NEI#EY01867, Research to Prevent Blindness, Inc. (to Asbell and department) CORNEAL NEOVASCULARIZATION. D. T. Azar, Harvard Medical School, Cornea Service, Massachusetts Eye and Ear Infirmary, Schepens Eye Research Institute, Boston, Massachusetts, USA Corneal vascularization follows severe corneal injuries and infections and is a leading cause of blindness. The balance of anti angiogenic and pro angiogenic factors have been implicated in the regulation of corneal neovascularization (NV). Anti-angiogenic factors are created by the proteolytic cleavage of the precursor molecules to generate functional molecules. Among the factors that regulate corneal neovascularization are bFGF and VEGF (VEGF, VEGF-B, and VEGF-C) and matrix metalloproteinases (MMPs). MMPs are extracellular matrix degrading enzymes whose expression is infuenced by various stimuli in the cornea. The mechanism of these MMPs in corneal neovascularization is not well characterized. MMPs degrade extracelullar matrix proteins to generate antiangiogenic factors or to release proangiogenic factors. Additionally, current data show that MMP-7 is involved in the production of anti-angiogenic molecules and that MMP-14 is a component of the angiogenic response. Matrilysin (MMP-7) is upregulated in the corneal epithelium during wound healing after excimer keratectomy wounds. MMP-7 has been shown to be involved in maintaining corneal avascularity during wound healing. The percentage of the corneal surface occupied by neovascularization after excimer laser keratectomy in the MMP-7-deficient mice is significantly greater than those of wild type controls. Membrane type matrix metalloproteinase (MT1-MMP, MMP-14) is up-regulated and immunolocalized to the stromal keratocytes in the vicinity of invading vessels in models of corneal NV in wild type mice. VEGF transfection results in corneal neovascularization. Corneal NV is diminished in MMP-14 deficient mice after basic fibroblast growth factor (bFGF) stimulation as compared to normal mice. MMP-14 DNA transfected into the corneal stroma of VEGF-LacZ mice resulted in the increased expression of VEGF compared to untreated controls. Future studies of the pathways of corneal neovascularization in inflammatory and non-inflammatory diseases and of the mechanisms of maintenance of corneal avascularity under homeostatic conditions will be valuable to provide therapeutic targets for treating this blinding ocular condition.
THE OCULAR SURFACE / JANUARY, 2005, VOL. 3, NO. 1 / SUPPLEMENT
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FLUOROPHTOMETRY FOR OBJECTIVE MEASUREMENT OF OCULAR SURFACE DISEASE. Penny Asbell, Huma Naikoo, Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York Purpose. Determining efficacy in dry eye trials depends on having well defined endpoints. Current tests typically depend on subjective evaluation by either the patient or examiner, such as degree of symptoms or amount of staining. Objective endpoints that correlate with the signs and symptoms of dry eyes would improve endpoint evaluation. We used fluorophtometry to evaluate patients with dry eyes, as defined by signs and symptoms to determine the repeatability and correlation with exam findings. Patients who had Schirmrers less than 5mm and corneal staining of 1 or more were evaluated by fluorophotometry to determine the degree of corneal permeability. Methods. After a complete eye exam done within the previous month, fluorophotometry of the cornea was performed utilizing the Fluorotron Master ™. Readings were taken before any drops were placed and then at 15minute intervals after placement of 5 microns of 1 % sodium fluorescein dye placed in the inferior cul de sac of each eye. Concentration of fluorescein within the cornea was recorded at each time point and results analyzed and compared to standard eye tests for dry eyes. Similar tests were performed on normal eyes, patients requesting only refraction. Results. Scans of dry eye patients were dramatically different from normal eyes, in that the concentration of fluorescein within the cornea increased 4 to 5 fold in dry eye patients vs. under 1 fold in normal eyes. Preliminary data showed that at 15 minutes, the concentration in 10 dry eyes was 250ng/ml ± 100 SD vs. 62 ng/ml ± 23 SD in normals, and at 60 minutes for dry eyes the concentration was 70 ng/ml ± 28SD vs. 40 ng/ml ± 8 SD. Further analysis and repeatability results to be presented.Conclusions. Objective tests are needed to diagnose, follow dry eye patients and provide clearcut endpoints to evaluate new treatments for dry eyes. Fluorophotometry provides objective measurement of corneal permeability and preliminary analysis suggests it can be correlated with ocular surface changes of the cornea in dry eye patients. Support: NEI#EY01867, Research to Prevent Blindness, Inc. (to Asbell and department) CORNEAL NEOVASCULARIZATION. D. T. Azar, Harvard Medical School, Cornea Service, Massachusetts Eye and Ear Infirmary, Schepens Eye Research Institute, Boston, Massachusetts, USA Corneal vascularization follows severe corneal injuries and infections and is a leading cause of blindness. The balance of anti angiogenic and pro angiogenic factors have been implicated in the regulation of corneal neovascularization (NV). Anti-angiogenic factors are created by the proteolytic cleavage of the precursor molecules to generate functional molecules. Among the factors that regulate corneal neovascularization are bFGF and VEGF (VEGF, VEGF-B, and VEGF-C) and matrix metalloproteinases (MMPs). MMPs are extracellular matrix degrading enzymes whose expression is infuenced by various stimuli in the cornea. The mechanism of these MMPs in corneal neovascularization is not well characterized. MMPs degrade extracelullar matrix proteins to generate antiangiogenic factors or to release proangiogenic factors. Additionally, current data show that MMP-7 is involved in the production of anti-angiogenic molecules and that MMP-14 is a component of the angiogenic response. Matrilysin (MMP-7) is upregulated in the corneal epithelium during wound healing after excimer keratectomy wounds. MMP-7 has been shown to be involved in maintaining corneal avascularity during wound healing. The percentage of the corneal surface occupied by neovascularization after excimer laser keratectomy in the MMP-7-deficient mice is significantly greater than those of wild type controls. Membrane type matrix metalloproteinase (MT1-MMP, MMP-14) is up-regulated and immunolocalized to the stromal keratocytes in the vicinity of invading vessels in models of corneal NV in wild type mice. VEGF transfection results in corneal neovascularization. Corneal NV is diminished in MMP-14 deficient mice after basic fibroblast growth factor (bFGF) stimulation as compared to normal mice. MMP-14 DNA transfected into the corneal stroma of VEGF-LacZ mice resulted in the increased expression of VEGF compared to untreated controls. Future studies of the pathways of corneal neovascularization in inflammatory and non-inflammatory diseases and of the mechanisms of maintenance of corneal avascularity under homeostatic conditions will be valuable to provide therapeutic targets for treating this blinding ocular condition.
THE OCULAR SURFACE / JANUARY, 2005, VOL. 3, NO. 1 / SUPPLEMENT