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MUC16 is a large transmembrane mucin expressed on the apical surfaces of the epithelium covering the ocular surface, respiratory system and female reproductive tract. The transmembrane mucin is overexpressed by ovarian carcinomas, it is one of the most frequently used diagnostic markers for the disease and it is considered a promising target for immunotherapeutic intervention. Immunodetection of the mucin has to date been through antibodies that recognize its exceptionally large ectodomain. Similar to other membrane anchored mucins, MUC16 has a short cytoplasmic tail (CT), but studies of the biological relevance of the C-terminal domain of MUC16 has been limited by lack of availability of monoclonal antibodies that recognize the native CT. Here, we report the development of a novel monoclonal antibody to the CT region of the molecule that recognizes native MUC16 and its enzymatically released CT region. The antibody is useful for immunoprecipitation of the released CT domain as demonstrated with the OVCAR3 ovarian cancer cell line and can be used for detailed cytolocalization in cells as well as in frozen sections of ocular surface and uterine epithelium. more...

Purpose To investigate the tear levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinase-1 in eyes after Boston keratoprosthesis type I (B-KPro) implantation and to correlate these markers with the established B-KPro prognostic categories. Methods Tear washes were collected from 40 patients (7 with autoimmune disease, 2 with chemical burn, and 31 with other noncicatrizing diagnoses). Tear levels of MMPs, MPO, and tissue inhibitor of metalloproteinase-1 were quantified using multianalyte bead-based enzyme-linked immunosorbent assays. The total MMP activity was determined using a fluorimetric assay. The analytes were compared to the underlying diagnosis and other clinical factors. Results The MMP-8, MMP-9, and MPO levels were markedly elevated in the eyes with B-KPro (80 ± 31, 291 ± 77, and 244 ± 33 pg/μg, respectively). Chemical burn was associated with significantly higher tear MMP-8 (474 ± 376 pg/μg) and MMP-9 levels (1300 ± 635 pg/μg) compared with noncicatrizing diseases (MMP-8: 41 ± 15 pg/μg, P = 0.02 and MMP-9: 196 ± 57 pg/μg, P = 0.02) and higher MMP-9 levels compared with autoimmune diseases (MMP-8: 96 ± 65 pg/μg, P = 0.21 and MMP-9: 306 ± 196 pg/μg, P = 0.04). Similar analyte levels were observed in the B-KPro eye and the contralateral non-B-KPro eye of patients with bilateral diseases. MMP-8, MMP-9, and total MMP activities correlated strongly with each other. Conclusions In the eyes with B-KPro, tear MMP-8 and MMP-9 levels seem to be related to the underlying ocular surface pathology and not significantly influenced by the presence of the prosthesis. more...

MUC16 is an extraordinarily large 22,152 amino acid membrane spanning mucin that has been shown to be present in the glycocalyx of the apical cells of the human cornea and conjunctiva where it interfaces with the tear film. The ectodomain of the molecule has been demonstrated in tears, where it has been presumed to be from surface epithelial cells. Data presented here from multiple assays, including immunohistochemistry, immunoelectron microscopy, in situ hybridization, and RT-PCR of RNA isolated from goblet cells isolated by laser capture microdissection, demonstrate that the membrane tethered mucin is also expressed by conjunctival goblet cells both in humans and in mice. The mucin is present in mucin granules and appears to be localized to the mucin granule membrane. Correlation analyses of the amounts of the goblet cell secreted mucin MUC5AC and the amounts of MUC16 and of MUC1 another membrane tethered mucin ectodomain found in human tear samples demonstrated that MUC5AC amounts correlated to the amounts of MUC16 but not to MUC1. These data suggest that goblet cells are a second source of the mucin in tears. The function of the membrane tethered mucin in the mucin granule remains to be determined. more...

Membrane-associated mucins (MAMs) expressed on the ocular surface epithelium form a dense glycocalyx that is hypothesized to protect the cornea and conjunctiva from external insult. In this study, the hypothesis that the MAMs MUC1 and MUC16, expressed on the apical surface of the corneal epithelium, suppress Toll-like receptor (TLR)-mediated innate immune responses was tested. Using an in vitro model of corneal epithelial cells that are cultured to express MAMs, we show that reduced expression of either MUC1 or MUC16 correlates with increased message and secreted protein levels of the proinflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α) following exposure of cells to the TLR2 and TLR5 agonists, heat-killed Listeria monocytogenes and flagellin, respectively. As mice express Muc1 (but not Muc16) in the corneal epithelium, a Muc1(-/-) mouse model was used to extend in vitro findings. Indeed, IL-6 and TNF-α message levels were increased in the corneal epithelium of Muc1(-/-) mice, in comparison with wild-type mice, following exposure of enucleated eyes to the TLR2 and TLR5 agonists. Our results suggest that the MAMs MUC1 and MUC16 contribute to the maintenance of immune homeostasis at the ocular surface by limiting TLR-mediated innate immune responses. more...

Objective To investigate the levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in tears of patients with Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP). Design Prospective, noninterventional cohort study. Participants Four SJS patients (7 eyes), 19 OCP patients (37 eyes), and 20 healthy controls who underwent phacoemulsification (40 eyes). Methods Tear washes were collected from all patients and were analyzed for levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 using multianalyte bead-based enzyme-linked immunosorbent assays. Total MMP activity was determined using a fluorometric assay. Correlation studies were performed between the various analytes within study groups. Main Outcome Measures Levels of MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MPO, and TIMP-1 (in nanograms per microgram of protein) and total MMP activity (in relative fluorescent units per minute per microgram of protein) in tears; MMP-8–to–TIMP-1 ratio; MMP-9–to–TIMP-1 ratio; and the correlations between MMP-8 and MMP-9 and both MMP and MPO. Results MMP-8, MMP-9, and MPO levels were elevated significantly in SJS and OCP tears (SJS>OCP) when compared with controls. The MMP activity was highest in SJS patients, whereas OCP patients and controls showed lower and similar activities. The TIMP-1 levels were decreased in SJS and OCP patients when compared with those in controls, with levels in OCP patients reaching significance. The MMP-8–to–TIMP-1 and MMP-9–to–TIMP-1 ratios were markedly elevated in SJS and OCP tears (SJS>OCP) when compared with those of controls. Across all study groups, MMP-9 levels correlated strongly with MMP-8 and MPO levels, and MMP-8 correlated with MPO, but it did not reach significance in SJS patients. There was no relationship between MMP-7 and MPO. Conclusions Because MMP-8 and MPO are produced by inflammatory cells, particularly neutrophils, the correlation data indicate that they may be the common source of elevated enzymes, including MMP-9, in SJS and OCP tears. Elevated MMP-to-TIMP ratios and MMP activity suggest an imbalance in tear MMP regulation that may explain the predisposition of these patients to demonstrate corneal melting and chronic complications associated with persistent inflammation. Myeloperoxidase in tears may be a sensitive and specific marker for the quantification of ocular inflammation. more...

Membrane-tethered mucins are large glycoproteins present in the glycocalyx along the apical surface of all wet-surfaced epithelia of the body, including that of the ocular surface. Originally thought to function only in epithelial surface lubrication and hydration, data now indicate that the mucins are multifunctional molecules, each having unique as well as common functions. This review summarizes current knowledge regarding the three major membrane mucins of the ocular surface, MUC1, MUC4, and MUC16. The mucins vary in their ocular surface distribution, size, structural motifs, and functions. The ectodomains of each are released into the tear film and are, thus, a component of the soluble mucins of the tear film. Both animal and in vitro models for their study are herein described, as are alterations of the mucins in ocular surface disease. more...

To determine in vitro whether titanium is superior in corneal cell compatibility to standard polymethyl-methacrylate (PMMA) for the Boston Keratoprosthesis (KPro).Human corneal-limbal epithelial (HCLE) cells were cultured 24, 48, 72, 96, 120, 144, or 168 hours in culture plates alone (controls) or with PMMA or titanium discs. Experiments were performed in triplicate and repeated (final n = 6). To determine if a soluble, toxic factor is emitted from materials, concurrent experiments at 48 and 144 hours were performed with discs placed in Transwell Supports, with HCLE cells plated beneath. As an additional test for soluble factors, cells were incubated 24 hours with disc-conditioned media, and number of viable cells per well was quantified at each timepoint by proliferation assay. To determine if delayed cell proliferation was attributable to cell death, HCLE cell death was measured under all conditions and quantified at each timepoint by cytotoxicity assay. The effects of material on HCLE cell proliferation over time was determined by repeated measures ANOVA. P0.05 was statistically significant.HCLE cell proliferation was greater in wells with titanium discs compared to PMMA. Differences between the test discs and control non-disc cocultures were statistically significant over time for both cell proliferation (P = 0.001) and death (P = 0.0025). No significant difference was found using Transwells (P = 0.9836) or disc-conditioned media (P = 0.36).This in vitro HCLE cell model demonstrates significantly increased cell proliferation and decreased cell death with cell/titanium contact compared to cell/PMMA contact. Moreover, differences are unlikely attributable to a soluble factor. Prospective in vivo analysis of the two KPro biomaterials is indicated. more...

Conjunctivitis outbreaks have occurred in the US in which nontypeable (NT) Streptococcus pneumoniae (Pnc) strains have been identified as the etiologic agent; however, the pathogenesis of Pnc conjunctivitis has not been extensively evaluated. Here we assessed the adhesive and invasive properties of 13 NT US conjunctivitis outbreak strains (cPnc) using an immortalized human conjunctival epithelial cell (HCjE) line expressing high or low levels of mucin as a surrogate for in vivo ocular surface events. Studies reveal differential binding efficiencies (up to 18-fold) among cPnc strains to HCjE cells and reduced or little adherence efficiency to high mucin-expressing (HME-HCjE). Additionally, in the presence of exogenous mucin there is considerable inhibition (20% to approximately 100%) of bacterial binding to the HCjE cells. Invasion assays suggest that the cPnc are internalized in HCjE, and less in HME-HCjE cells. Microarray analysis of cPnc isolates revealed an up-regulation of Pnc neuraminidases, and treatment of HME-HCjE cells with exogenous neuraminidase resulted in a 2-13-fold enhancement in cPnc binding. The results indicate that mucin acts as a protective barrier in vitro and that neuraminidases, which can degrade mucin, may be contributing factors leading to bacterial adherence, a first step in the pathogenesis of this transmissible infection. more...

Purpose This study was designed to determine whether long-term tolerant contact lens (CL) wear causes changes in the expression of mucin mRNA by the conjunctival epithelium and mucin protein content in tears and to determine whether specific mucins adhere to contact lenses. Methods Twenty long-term (> or = 5 years ) and tolerant CL wearers (2 with hard and 18 with soft contact lenses) were compared with 23 non-CL wearers. One hour after CL removal, tear fluid was collected after instillation of 60 microL of sterile water onto the ocular surface, and protein concentration was determined. Impression cytology was performed on the bulbar temporal region of conjunctiva to collect cells for RNA isolation. Real-time polymerase chain reaction was performed using TaqMan primer and probes for MUC1, 4, 5AC, and 16. ELISA was performed on the collected tears to detect MUC5AC and the mucin carbohydrate epitope H185. For the analysis of adherent mucins on CL, discarded daily-wear contact lenses were collected, rinsed, and incubated overnight at 4 degrees C in mucin isolation buffer. Immunoblot analysis of adherent mucins was performed to detect MUC1, 4, 5AC, 16, and H185. Results No significant changes in the levels of mucin mRNA from impression cytology samples were detected when comparing CL and non-CL wearers. The amount of total protein in tears collected from CL wearers (39.9 +/- 27.2 microg) was significantly less than that from non-CL wearers (95.1 +/- 73.8 microg, P = 0.001). The level of MUC5AC mucin and the H185 epitope in tears per unit protein in CL wearers was not significantly different from non-CL wearers. Low levels of membrane-associated mucins, the secreted mucin MUC5AC, and the carbohydrate epitope, H185, were detected in protein extracts from discarded CLs. Compared with MUC1, 4, and 5AC, there was less MUC16 adherent to the CLs. Conclusion Neither mucin mRNA expression by conjunctival epithelia nor mucin content per unit protein in tears was altered by long-term tolerant CL wear; however, the amount of protein in the tears was significantly less. Shed membrane-associated mucins and the goblet cell mucins adhere to CLs. more...

Purpose To assess the clinical characteristics, incidence, and pathologic correlation of corneal epithelial adhesion abnormalities encountered during LASIK. Design Prospective noncomparative interventional case series. Participants Five hundred consecutive eyes of 268 patients undergoing primary LASIK procedures by one surgeon utilizing the Moria LSK One microkeratome and VISX Star S-2 excimer laser. Methods Corneal epithelial adhesion was assessed immediately preoperatively using a cellulose surgical sponge (adhesion test), and the incidence, extent, and location of epithelial defects occurring during the microkeratome incision of the corneal flap were recorded. Epithelial specimens from 7 corneas requiring debridement of dysadhesive epithelium were examined by transmission electron microscopy. Main outcome measures The characteristics of the study population (age, gender, contact lens use, relevant ocular surface or systemic disease, refractive error, keratometry, pachymetry) and the microkeratome-related variables (head selection and vacuum level) were compared with the results of the preoperative epithelial adhesion test, the development of intraoperative epithelial effects (size and location), and the postoperative outcome. Results Epithelial defects were sustained by 51 corneas (10.2%), and among these, 31 (6.2%) were microdefects and 20 (4.0%) were macrodefects. The adhesion test was positive (indicative of compromised adhesion of epithelium to stroma) in 20 (64.5%) corneas with microdefects, but false negatives (epithelial defect despite negative adhesion test) occurred in 11 cases (35.4%). The adhesion test was positive in 16 (80%) of corneas having macrodefects, with 4 (20%) false negatives. Thus, the overall positive predictive value of the adhesion test was 59%, and the percentage of positive prediction was 92% (Bayes' theorem). Among all other outcome measures assessed, only corneal flap thickness seemed a potential risk factor, as 40 (78.4%) epithelial defects were associated with the creation of a 180-μm-thick flap, whereas 10 (19.6%) were associated with a 160-μm-thick flap and only 1 (2%) occurred with a 130-μm flap. These trends were not, however, statistically significant ( P = 0.15, Fisher exact test). Transmission electron microscopy of all epithelial debridement specimens disclosed consistent abnormality of the basement membrane adhesion complex, as thickened and multilaminated basement membrane remained adherent to the intact epithelial sheet. Conclusions: Corneal epithelial dysadhesion and defects occurring in the course of LASIK surgery may be associated with an intrinsic compromise of the basement membrane adhesion complex, as evidenced clinically by the adhesion test and demonstrated pathologically by duplicated basement membrane. more...

Purpose. Dry eye syndrome is prevalent in post-menopausal women, and post-menopausal women secrete less mucus in their reproductive tracts. Using a mouse model, the purpose of this study was to determine if estrogen and/or progesterone regulates Muc4 and Muc5AC gene expression in the ocular surface epithelia, as the hormones do in reproductive tract epithelia. Methods. Adult C57BL/6 mice were ovariectomized, and 19 days later, pellets containing estrogen, progesterone, or a combination were inserted subcutaneously. Ocular surface and reproductive tract tissues were harvested following seven days of hormone treatment. A control group consisted of ovariectomized mice that received no hormone treatment. Real-time reverse transcription-polymerase chain reaction was used to determine the tissue expression levels of mucin mRNA of each treatment group relative to the control. Muc4 mRNA expression levels were determined for the reproductive tract, and both Muc4 and Muc5AC expression levels were determined for the ocular surface epithelia. Muc4 and Muc5AC gene expression in ocular surface and Muc4 in reproductive tract epithelia was demonstrated by In Situ hybridization, and Muc4 and Muc5AC protein was demonstrated in the epithelia of animals in the experimental groups. Results. The mRNA expression levels of Muc4 and Muc5AC and the immunofluorescence localization pattern in the ocular surface epithelia were not significantly different in any hormone treatment group when compared to the control ovariectomized group. By comparison, mice that were administered estrogen had a significant increase of Muc4 mRNA in the reproductive tract epithelia, progesterone given in combination with estrogen antagonized the upregulatory effects of estrogen in the reproductive tract, and the amount of Muc4 mRNA in the reproductive tract of progesterone-treated animals was not different from ovariectomized controls. Immunofluorescence localization of Muc4 in the reproductive tract epithelia of the experimental groups correlated to message levels, with lack of Muc4 protein detected in the control and progesterone groups. Conclusion. In comparison to reproductive tract epithelia, Muc4 and Muc5AC are not hormonally regulated by estrogen or progesterone in the ocular surface epithelia of mice. These data demonstrate that regulation of epithelial mucin genes is tissue specific. more...

As wounds in stratified epithelia close, the numbers of cells at the leading edge of migration decreases. It is not known whether cells at the leading edge are continually replaced or whether some retain their position at the leading edge over time. Replication-deficient adenovirus carrying the green fluorescent protein gene was applied to corneal epithelial wounds in mice and it was found that they primarily infect the leading edge cells of healing epithelium. Eighteen hr after viral transduction, green fluorescent protein expressing cells were located in the apical layer at varying distances behind the leading edge. These data indicate that leading edge cells are replaced during healing of stratified epithelia. more...

Mucins are vital for maintenance of a healthy, wet ocular surface. Once only thought to be secreted by goblet cells, mucins are now also known to be of the membrane-associated type. Stratified ocular surface epithelia express at their apical-tear fluid surface a repertoire of membrane-associated mucins including MUC1, MUC4, MUC16. These mucins are concentrated on the tips of the microplicae, forming a dense glycocalyx at the epithelial tear film interface. A major mucin of the secretory class is the goblet-cell-derived gel-forming mucin MUC5AC. A small soluble mucin, MUC7, is expressed by the lacrimal gland acini. Our hypothesis of the role/distribution of the secreted and membrane-associated mucins at the ocular surface is that the secreted mucins are soluble in the tear fluid, and are moved about and shunted to the nasolacrimal duct and by the eyelids during blinking. Thus, in the tears, the secreted mucins act as clean-up/debris removing multimeric networks that at the same time, through their hydrophilic nature, hold fluids in place and harbor defense molecules secreted by the lacrimal gland. Membrane-associated mucins, on the other hand, form a dense barrier in the glycocalyx at the epithelial tear film interface. This barrier prevents pathogen penetrance and is a lubricating surface that allows lid epithelia to glide over the corneal epithelia without adherence. The secreted mucins move easily over the glycocalyx mucins because both have anionic character that creates repulsive forces between them. Little is known regarding regulation of expression and glycosylation of mucins by ocular surface epithelia. Since ocular surface drying diseases alter both goblet cell and mucin production, and production and glycosylation of membrane-associated mucins, studies of mucin gene regulation and glycosylation may yield treatment modalities for these diseases. more...

To compare conjunctival goblet cell numbers as well as epithelial turnover in patients with non-Sjögren syndrome--associated keratoconjunctivitis sicca (NSS-KCS) and those with SS-KCS before and after 6 months of treatment with topical cyclosporine A (CsA) ophthalmic emulsion.Conjunctival biopsy specimens from 16 patients with NSS-KCS and 12 with SS-KCS were obtained at baseline and after 6 months' therapy with CsA or vehicle alone. Conjunctival biopsy specimens were also obtained from 11 normal subjects. Periodic acid--Schiff staining determined the number of goblet cells present. Immunofluorescence microscopy for Ki-67 localization was used to evaluate the number of actively cycling cells.Periodic acid--Schiff staining showed fewer goblet cells at baseline in both dry eye populations when compared with normal subjects (P.001). After 6 months of CsA treatment, conjunctival biopsy specimens of both NSS-KCS and SS-KCS groups revealed an increase in goblet cells compared with baseline (P.05). More Ki-67--positive cells were observed in NSS-KCS conjunctiva at baseline than in normal conjunctiva (P.05) whereas numbers of these cells in SS-KCS conjunctiva were similar to normal at baseline. After 6 months of CsA treatment, conjunctival biopsy specimens of NSS-KCS revealed a decrease in Ki-67--labeled cells compared with baseline (P.001). In contrast, no substantial change was observed for CsA treatment in patients with SS-KCS.Treatment of dry eye syndrome for 6 months with topical CsA resulted in an increase in goblet cell numbers in patients with NSS-KCS and SS-KCS and a decrease in epithelial turnover in those with NSS-KCS. Reducing ocular surface inflammation might have an effect on the proliferative activity of the epithelium. more...

Three lectins, Wheat germ agglutinin, succinyl Concanavalin A and Ricinus communis agglutinin were used to block specific sugar moieties in the basal lamina. Corneal epithelial basal cells were plated onto freshly denuded basal lamina. Attachment was studied by quantifying the adherence of prelabeled cells and by examining attachment sites using transmission electron microscopy. Spreading was examined using scanning electron microscopy. Attachment of the cells occurred within 15 min and spreading was apparent after 45 min. Both Wheat germ agglutinin and beta-N-acetylglucosaminidase inhibited cellular attachment. Succinyl Concanavalin A and Ricinus Communis agglutinin permitted attachment, but inhibited extensive cellular spreading. The results indicate that the attachment of basal cells is dependent on N-acetylglucosamine residues, and spreading is mediated by alpha methylmannoside, glucose, and galactose residues. more...