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Purpose To evaluate the antifungal properties of topical antibiotics (already being used successfully to prevent bacterial endophthalmitis) and some promising antiseptics for antifungal prophylaxis in the setting of artificial corneal implantation. Methods Several commonly used antibiotics for antimicrobial prophylaxis after artificial corneal implantation, in addition to antiseptics [benzalkonium chloride (BAK), povidone-iodine (PI), and some ionic liquids (ILs)], were tested in vitro against Candida albicans, Fusarium solani, and Aspergillus fumigatus. The time-kill activity was determined. Toxicity was assayed in vitro on human corneal epithelial cultures using trypan blue. Adhesion and tissue invasion experiments were also carried out on porcine corneas and commonly used contact lenses, with or without gamma irradiation, and by analysis with fluorescence microscopy. Results Polymyxin B (PMB)/trimethoprim/BAK (Polytrim), PMB alone, gatifloxacin with BAK (Zymaxid), and same-concentration BAK alone exhibited antifungal activity in vitro. Moxifloxacin (MOX) or gatifloxacin without BAK-as well as trimethoprim, vancomycin, and chloramphenicol-had no effect. 1% PI and ILs had the highest efficacy/toxicity ratios (>1), and Polytrim was species dependent. Subfungicidal concentrations of Polytrim reduced adhesion of C. albicans to Kontur contact lenses. Gamma-irradiated corneas showed enhanced resistance to fungal invasion. Conclusions Of antibiotic preparations already in use for bacterial prophylaxis after KPro surgery, Polytrim is a commonly used antibiotic with antifungal effects mediated by both PMB and BAK and may be sufficient for prophylaxis. PI as a 1% solution seems to be promising as a long-term antifungal agent. Choline-undecanoate IL is effective and virtually nontoxic and warrants further development.

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.

Claes Dohlman’s early research and leadership mark the beginning of the field of corneal research and the corneal specialty within ophthalmology. Upon establishing a cornea research department at the then Retina Foundation, then a clinical cornea service at the Massachusetts Eye and Ear Infirmary, he recruited basic researchers and corneal fellows to the laboratory, and the findings that ensued contributed significantly to our knowledge of corneal function and physiology. He applied these findings to clinical disease and to development of the keratoprosthesis. He was an exceptionally productive pioneer in the field and continues to this day as the ultimate “translational” scientist.

Goblet cells within the conjunctival epithelium are specialized cells that secrete mucins onto the surface of the eye. Recent research has demonstrated new characteristics of the cells, including factors influencing their differentiation, their gene products and their functions at the ocular surface. The following review summarizes the newly discovered aspects of the role of Spdef, a member of the Ets transcription factor family in conjunctival goblet cell differentiation, the newly discovered goblet cell products including claudin2, the Wnt inhibitor Frzb, and the transmembrane mucin Muc16. The current concepts of conjunctival goblet cell function, including debris removal and immune surveillance are reviewed, as are changes in the goblet cell population in ocular surface diseases. Major remaining questions regarding conjunctival cell biology are discussed.

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.

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.

Importance The number and size of guttae increase over time in Fuchs endothelial corneal dystrophy (FECD); however, the association between these physical parameters and disease pathogenesis is unclear. Objective To determine the role of guttae in corneal endothelial cell function. Design, Settings, and Participants In an in vitro model, cells from a human corneal endothelial cell line, HCENC-21T, were seeded on decellularized normal (n = 30) and FECD (n = 70) endothelial basement (Descemet) membranes (DMs). Normal human corneas were sent to our laboratory from 3 sources. The study took place at the Schepens Eye Research Institute, Massachusetts Eye and Ear, Boston, and was performed from September 2015 to July 2017. Normal DMs were obtained from 3 different tissue banks and FECD-DMs were obtained from patients undergoing endothelial keratoplasty in 2 departments. Main Outcomes and Measures Endothelial cell shape, growth, and migration were assessed by live-cell imaging, and gene expression analysis as a function of guttae diameter was assessed by laser capture microscopy. Results Mean (SD) age of normal-DMs donors was 65.6 (4.4) years (16 women [53%]), and mean (SD) age of FECD-DMs donors was 68.9 (10.6) years (43 women [61%]). Cells covered a greater area (mean [SD], 97.7% [8.5%]) with a greater mean (SD) number of cells (2083 [153] cells/mm2) on the normal DMs compared with the FECD DMs (72.8% [11%];P = .02 and 1541 [221] cells/mm2221/mm2;P = .01, respectively). Differences in endothelial cell growth over guttae were observed on FECD DMs depending on the guttae diameter. Guttae with a mean (SD) diameter of 10.5 (2.9) μm did not impede cell growth, whereas those with a diameter of 21.1 (4.9) μm were covered only by the cell cytoplasm. Guttae with the largest mean (SD) diameter, 31.8 (3.8) μm, were not covered by cells, which instead surrounded them in a rosette pattern. Moreover, cells adjacent to large guttae upregulatedαSMA,N-cadherin,Snail1, andNOX4genes compared with ones grown on normal DMs or small guttae. Furthermore, large guttae induced TUNEL-positive apoptosis in a rosette pattern, similar to ex vivo FECD specimens. Conclusions and Relevance These findings highlight the important role of guttae in endothelial cell growth, migration, and survival. These data suggest that cell therapy procedures in FECD might be guided by the diameter of the host guttae if subsequent clinical studies confirm these laboratory findings.

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.

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.

Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef(-/-) mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef(-/-) mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef(-/-) mice revealed down-regulation of goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-alpha, Il-1beta, Tnf-alpha), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjogren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef(-/-) mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease.

To investigate the levels of neutrophil elastase (NE), matrix metalloproteinases (MMPs), and myeloperoxidase (MPO) in tear washes of patients with ocular graft-vs-host disease (oGVHD).Case-control study.Based on established criteria, oGVHD patients (n = 14; 28 eyes) and age-/sex-matched healthy controls (n = 14; 28 eyes) were enrolled. Tear washes were collected and analyzed for NE using a single-analyte enzyme-linked immunosorbent assay (ELISA). MMPs (1, 2, 3, 7, 8, 9, 12), MPO, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were analyzed using multianalyte bead-based ELISA assays. Total MMP activity was measured using a fluorimetric assay. Correlation studies were performed between NE, MMP-8, MMP-9, and MPO within study groups.NE, MMP-8, MMP-9, and MPO levels were elevated in oGVHD tears when compared with controls (P.0001). NE was the most elevated analyte. MMP activity was higher and TIMP-1 levels were lower in oGVHD than in control (P.0001). In oGVHD, NE significantly correlated with MMP-8 (r = 0.92), MMP-9 (r = 0.90), and MPO (r = 0.79) (P.0001). MMP-8 correlated with MMP-9 (r = 0.96, P.0001), and MPO (r = 0.60, P = .001). MMP-9 correlated with MPO (r = 0.55, P = .002). In controls, NE, MMP-9, and MPO significantly correlated with each other (P.0001).The marked increase in NE in oGVHD tears that correlated strongly with elevated MMP-8, MMP-9, and MPO suggests a common neutrophilic source and provides evidence of neutrophil activity on the ocular surface of oGVHD patients.

Human adenoviruses (HAdVs) are double-stranded DNA viruses that cause infections across all mucosal tissues in the body. At the ocular surface, HAdVs cause keratoconjunctivitis (E. Ford, K. E. Nelson, and D. Warren, Epidemiol Rev 9:244–261, 1987, and C. M. Robinson, D. Seto, M. S. Jones, D. W. Dyer, and J. Chodosh, Infect Genet Evol 11:1208–1217, 2011, doi:10.1016/j.meegid.2011.04.031)—a highly contagious infection that accounts for nearly 60% of conjunctivitis cases in the United States (R. P. Sambursky, N. Fram, and E. J. Cohen, Optometry 78:236–239, 2007, doi:10.1016/j.optm.2006.11.012, and A. M. Pihos, J Optom 6:69–74, 2013, doi:10.1016/j.optom.2012.08.003). The infection begins with HAdV entry within ocular surface epithelial cells; however, the mechanisms used by HAdVs to transit the otherwise protective mucosal barrier of ocular surface epithelial cells prior to entry remain unknown. Here, we report that the highly virulent keratoconjunctivitis-causing HAdV-D37 induces release of the extracellular domain (ectodomain) of MUC16, a major component of the mucosal barrier of ocular surface epithelial cells, prior to infecting underlying cells. Currently, there is no specific treatment for controlling this infection. Understanding the early steps involved in the pathogenesis of keratoconjunctivitis and using this information to intercept adenoviral entry within cells may guide the development of novel strategies for controlling the infection., Human adenoviruses (HAdV), species D in particular (HAdV-D), are frequently associated with epidemic keratoconjunctivitis (EKC). Although the infection originates at the ocular surface epithelium, the mechanisms by which HAdV-Ds bypass the membrane-associated mucin (MAM)-rich glycocalyx of the ocular surface epithelium to trigger infection and inflammation remain unknown. Here, we report that an EKC-causing adenovirus (HAdV-D37), but not a non-EKC-causing one (HAdV-D19p), induces ectodomain release of MUC16—a MAM with barrier functions at the ocular surface—from cultured human corneal and conjunctival epithelial cells. HAdV-D37, but not HAdV-D19p, is also found to decrease the glycocalyx barrier function of corneal epithelial cells, as determined by rose bengal dye penetrance assays. Furthermore, results from quantitative PCR (qPCR) amplification of viral genomic DNA using primers specific to a conserved region of the E1B gene show that, in comparison to infection by HAdV-D19p, infection by HAdV-D37 is significantly increased in corneal epithelial cells. Collectively, these results point to a MUC16 ectodomain release-dependent mechanism utilized by the EKC-causing HAdV-D37 to initiate infection at the ocular surface. These findings are important in terms of understanding the pathogenesis of adenoviral keratoconjunctivitis. Similar MAM ectodomain release mechanisms may be prevalent across other mucosal epithelia in the body (e.g., the airway epithelium) that are prone to adenoviral infection. IMPORTANCE Human adenoviruses (HAdVs) are double-stranded DNA viruses that cause infections across all mucosal tissues in the body. At the ocular surface, HAdVs cause keratoconjunctivitis (E. Ford, K. E. Nelson, and D. Warren, Epidemiol Rev 9:244–261, 1987, and C. M. Robinson, D. Seto, M. S. Jones, D. W. Dyer, and J. Chodosh, Infect Genet Evol 11:1208–1217, 2011, doi:10.1016/j.meegid.2011.04.031)—a highly contagious infection that accounts for nearly 60% of conjunctivitis cases in the United States (R. P. Sambursky, N. Fram, and E. J. Cohen, Optometry 78:236–239, 2007, doi:10.1016/j.optm.2006.11.012, and A. M. Pihos, J Optom 6:69–74, 2013, doi:10.1016/j.optom.2012.08.003). The infection begins with HAdV entry within ocular surface epithelial cells; however, the mechanisms used by HAdVs to transit the otherwise protective mucosal barrier of ocular surface epithelial cells prior to entry remain unknown. Here, we report that the highly virulent keratoconjunctivitis-causing HAdV-D37 induces release of the extracellular domain (ectodomain) of MUC16, a major component of the mucosal barrier of ocular surface epithelial cells, prior to infecting underlying cells. Currently, there is no specific treatment for controlling this infection. Understanding the early steps involved in the pathogenesis of keratoconjunctivitis and using this information to intercept adenoviral entry within cells may guide the development of novel strategies for controlling the infection.

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.

Recent studies have shown that galectin-3 (Gal-3; also known as LGALS3), a beta-galactoside-binding lectin, promotes cell migration during re-epithelialization of corneal wounds. The goal of this study was to characterize the molecular mechanism by which Gal-3 stimulates cell migration. We demonstrate here that exogenous Gal-3, but not Gal-1 or Gal-8, promotes cell scattering and formation of lamellipodia in human corneal epithelial cells in a beta-lactose-inhibitable manner. alpha3beta1 integrin was identified as the major Gal-3-binding protein in corneal epithelial cells by affinity chromatography of cell lysates on a Gal-3-Sepharose column. Preincubation of cells with anti-alpha3 integrin function-blocking antibody significantly inhibited the induction of lamellipodia by Gal-3. Furthermore, exogenous Gal-3 activated both focal adhesion kinase, a key regulator of integrin-dependent intracellular signaling, and Rac1 GTPase, a member of the family of Rho GTPases, well known for its role in the reorganization of the actin cytoskeleton and formation of lamellipodial extensions. Experiments involving knockdown of beta-1,6-N-acetylglucosaminytransferase V, an enzyme that synthesizes high-affinity glycan ligands for Gal-3, revealed that carbohydrate-mediated interaction between Gal-3 and complex N-glycans on alpha3beta1 integrin plays a key role in Gal-3-induced lamellipodia formation. We propose that Gal-3 promotes epithelial cell migration by cross-linking MGAT5-modified complex N-glycans on alpha3beta1 integrin and subsequently activating alpha3beta1-integrin-Rac1 signaling to promote lamellipodia formation.

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.

In corneal tissue from a diabetic patient 15 months after delayed epithelial healing following vitrectomy surgery, electron microscopy revealed a discontinuous basement membrane; immunohistochemically, fibronectin was present in the basement membrane zone. To determine whether alterations in repair of the basement membrane, anchoring fibrils and hemidesmo-somes occur in wounds in diabetics, we studied the pattern of histochemical localization of bullous pemphigoid antigen (the intracellular hemidesmosome plaque component), laminin and type VII collagen (the anchoring fibril collagen) in alloxan-induced diabetic rabbits following superficial keratectomy. Ultrastructural studies of basal laminae and hemidesmosomes also were performed. Epithelial wound closure was complete by 66–72 h after 7-mm superficial keratectomies. Type VII collagen localized at the base of the epithelium at the wound periphery by 66 h, appearing as a beaded line underlying the epithelium; at 1 week, as in control rabbits, the localization zone extended across the wound bed. Polyclonal antibodies to laminin and bullous pemphigoid antigen showed similar localization. Small segments of basal laminae were noted by electron microscopy of the wound periphery as early as 66 h post-kera-tectomy. By 2 weeks, an increase in hemidesmosomes associated with segments of discontinuous basal lamina was apparent. No obvious differences in the time or pattern of re-appearance of the adhesion complex in keratectomy wounds could be discerned between normoglycemic and hyperglycemic rabbits. Thus, healing defects in diabetics may be due to factors other than reassembly of adhesion structures.

The corneal epithelium adheres to the stroma through a series of linked structures termed collectively the adhesion complex. These structures include; intermediate filaments (keratin filaments) which are linked to the hemidesmosome; the hemidesmosome; the anchoring filaments which extend from the membrane at the hemidesmosome through the lamina lucida to the lamina densa region of the basement membrane; the anchoring fibrils which insert into the basement membrane from the stromal side; and the anchoring plaque on which anchoring fibrils terminate distal from their insertion on the basement membrane. Upon wounding, basal cells of the corneal epithelium disassemble their hemidesmosomes. During migration, the membranes along the wound bed exhibit a different kind of adhesion junction, the focal contact. This junction is present primarily in cells of the leading edge of migration and may be the provisional adhesion junction used by epithelial sheet moving to cover a wound.

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.

In order for the preimplantation embryo to implant into the uterus, the trophoblast cells must initially adhere to the uterine epithelial surface. In preparation, the luminal secretory cells of the epithelium lose their nonadhesive character and their surface microvilli and bulge into the lumen, forming uterodomes (pinopodes; uterodome is used instead of pinopode, since in humans the surface membrane exocytoses rather than endocytoses (Murphy, Hum Reprod 2000; 15:2451–2454). Previous research has led to the hypothesis that loss of the nonadhesive membrane-spanning mucin MUC1 from the uterodome surface allows trophoblast adherence. Immunofluorescence microscopic assay of luminal epithelia on human uterine biopsies taken from LHþ 0t o LH þ13 show that another membrane-spanning mucin, MUC16, was lost from uterodome surfaces in all samples taken during the receptive phase, LHþ 6t o LH þ 8( n¼ 12), and that MUC1 was present on uterodomes in 4 of 12 samples and on all ciliated cells of the epithelium in the receptive phase. Short interfering RNA (siRNA) knockdown of MUC16 in a uterine epithelial cell line ECC-1 that, like uterine epithelium, expresses MUC16 and MUC1 allowed increased adherence of cells of a trophoblast cell line. In parallel experiments, siRNA knockdown of MUC1 did not affect trophoblast cell adherence. These data indicate that MUC16 is a membrane component of the nonreceptive luminal uterine surface, which prevents cell adhesion, and that its removal during uterodome formation facilitates adhesion of the trophoblast. female reproductive tract, implantation, trophoblast, uterus

Members of the DEWS Research Subcommittee reviewed research into the basic mechanisms underlying dry eye disease. Evidence was evaluated concerning the tear film, lacrimal gland and accessory lacrimal glands, ocular surface epithelia (including cornea and conjunctiva), meibomian glands, lacrimal duct system and the immune system. Consideration was given to both animal and human research data. Results are presented as a series of information matrices, identifying what is known and providing supporting references. An attempt is made to identify areas for further investigation.

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.

At the ocular surface, three types of mucins are present. The large gel-forming mucin MUC5AC is expressed by conjunctival goblet cells. Some cells of the lacrimal gland acini express the small soluble mucin MUC7. The corneal and conjunctival epithelia express the membrane-associated mucins MUCs 1, 4, and 16. With the characterization of the mucin gene repertoire of the ocular surface epithelia, studies of the function of specific mucins, their gene regulation, and their alteration in ocular surface disease have begun. Current information suggests that all the mucins are hydrophilic and play a role in maintenance of water on the surface of the eye. The large secreted mucins represent the "janitorial service" that moves over the surface of the eye to wrap up and remove debris. The membrane-associated mucins form the glycocalyx, which provides a continuous barrier across the surface of the eye that prevents pathogen penetrance and has signaling capabilities that influence epithelial activity. Factors regulating mucin gene expression include retinoic acid, serum, and dexamethasone. Alteration in both secreted and membrane-associated mucins occur in drying ocular surface diseases. In Sjogren syndrome, MUC5AC expression is reduced, and in non-Sjogren dry eye, glycosylation of MUC16 appears to be altered. The pattern of expression of enzymes that glycosylate mucins is altered in ocular cicatricial pemphigoid. Therapies being evaluated for dry eye, including cyclosporine A, P2Y2 agonists, gefarnate, 15-(S)-HETE, and corticosteroids, may be efficacious due to their effect on mucin gene expression and secretion.

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.

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.

The purpose of this study was to develop a novel 10-mg/mL infliximab eye drop, to characterize its physical and biological stability under recommended storage conditions, and to assess the formulation's toxicity to ocular surface epithelium in vitro. Infliximab (10 mg/mL) was reconstituted using equal volumes of sterile water and 1% carboxymethylcellulose artificial tears. Aliquots were stored in either a 4 degrees C refrigerator or -20 degrees C freezer for up to 45 days. Physical stability was assessed through monitoring the solution's appearance, pH, ultraviolet-visible-near infrared absorbance and scattering, as well as protein gel electrophoresis. Biological stability was assayed through binding to tumor necrosis factor-alpha using an enzyme-linked immunosorbent assay. In vitro cytotoxicity to human corneal-limbal epithelial cells was examined following a 4-hour exposure to the study drug. Refrigerated and frozen infliximab eye drops remained clear and colorless for the duration of study. The formulation's pH (7.0) was comparable to that of the artificial tear vehicle alone. Low levels of ultraviolet-visible-near infrared light absorbance and scattering established the lack of protein precipitate after refrigeration or freezing. Protein gel electrophoresis performed under reducing conditions revealed the presence of two main protein bands of approximately 50 kDa and 25 kDa, representing immunoglobulin G heavy and light chains. The migration pattern of the proteins did not change under the different storage conditions and between day 10 and 45 after formulation. Infliximab binding to tumor necrosis factor-alpha remained stable for up to 45 days, with conservation of 101% and 102% of its initial binding activity when refrigerated or frozen, respectively. In vitro human corneal-limbal epithelial cultures showed no increase in cytotoxicity with infliximab treatment when compared to vehicle and culture media controls (P0.05). Infliximab can be formulated as an eye drop and remains stable when stored in accordance with current regulations regarding compounded eye drops. The demonstrated physical and biological stability as well as in vitro innocuity of this infliximab eye drop formulation may facilitate future clinical investigation targeting tumor necrosis factor-alpha as a modulator of various ocular surface diseases.

It has been hypothesized that the carbohydrate portion of mucins present in the endocervical canal plays an important role in conferring specific physicochemical properties (e.g. viscosity and hydration) to the mucus gel through the menstrual cycle. Our recent finding showing an increase in the amount of MUC5B mucin protein at midcycle has raised the question of whether the mucin O-glycan content also varies to confer specific hydrodynamic properties to secreted mucins during ovulation. Using lectins as carbohydrate probes, we have identified two common mucin oligosaccharide structures, T antigen and N-acetyllactosamine, within secretory granules in human endocervical glands during the proliferative phase of the menstrual cycle. Analysis of endocervical secretions by enzyme-linked lectin assay revealed that the amounts of T antigen and N-acetyllactosamine are maximal at midcycle. Lectin blot assay of immunoprecipitated MUC5B demonstrated that the mucin is a carrier of the T antigen and N-acetyllactosamine oligosaccharides in cervical mucus secretions. The amounts of T antigen and N-acetyllactosamine oligosaccharides on MUC5B increased during the first half of the cycle, peaked at midcycle, and dramatically dropped at the end of the cycle. The peak in MUC5B mucin protein and carbohydrate content coincides with the change in mucus character that occurs at midcycle. The role of O-glycans on mucins may be to hold water within the endocervical canal during ovulation to facilitate sperm migration.

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.

The physical character and amount of mucus secreted by the endocervix changes dramatically during the menstrual cycle to facilitate sperm migration at the time of midcycle ovulation. Mucins are highly glycosylated, high-molecular-weight proteins, which are the major structural components of the protective mucus gel covering all wet-surfaced epithelia, including that of the endocervix. We have previously demonstrated that the endocervical epithelium expresses messenger RNA (mRNA) of three of the large gel-forming mucins, designated MUC5AC, MUC5B, and MUC6, with mRNA of MUC5B predominating. Because mucin protein levels may be regulated posttranscriptionally, measurement of MUC5B protein levels with cycle are needed for correlation to mRNA levels. Measurement of specific mucin gene products within mucus secretions has been limited by availability of specific, well-characterized antibodies and by volume requirements of the isolation protocols for mucins, which include CsCl density centrifugation and fraction isolation. To measure MUC5B protein within the cervical mucus through the hormone cycle, we developed a polyclonal antibody specific to the mucin. The antibody, designated no. 799, is to a synthetic peptide mimicking a 19-amino-acid segment of an intercysteine-rich region within the D4 domain in the 3' region of the MUC5B protein. It recognizes native as well as denatured MUC5B on immunoblot, is preadsorbable with its peptide, and binds to apical secretory vesicles of epithelia expressing MUC5B. We used the MUC5B antibody along with a cervical mucin standard cervical mucin isolate in enzyme-linked immunosorbent assay to determine the relative amount of MUC5B mucin in samples of human cervical mucus taken through the menstrual cycle. We demonstrate a peak of MUC5B mucin in human cervical mucus collected at midcycle, compared with mucus from early or late in the cycle. This peak in MUC5B content coincides with the change in mucus character that occurs at midcycle, suggesting that this large mucin species may be important to sperm transit to the uterus.

Human mucin genes include membrane-bound mucins (MUC1, MUC3, MUC4) and secretory mucins (MUC2, MUC5AC, MUC5B, MUC6). Our aim was to determine mucin gene expression in human gallbladder cell lines, normal gallbladder from liver donors (N = 7) and surgical specimens with mild chronic cholecystitis (N = 29), chronic cholecystitis (N = 48), and acute and chronic cholecystitis (N = 27). MUC1 mRNA was ubiquitous; however, only rare MUC1 immunoreactivity was detected. MUC3, MUC5AC, MUC5B, and MUC6 mRNA were present in all gallbladder specimens and cell lines examined. Prominent MUC3, MUC5AC, MUC5B, and MUC6 immunoreactivity was present in 86-100% of normal gallbladders. The frequency of MUC5AC reactivity was decreased in specimens with acute cholecystitis (P < 0.05). In contrast, MUC2-reactivity was absent in normal gallbladder and present in 53.8% of acute cholecystitis specimens (P < 0.05). Surface epithelium is characterized by MUC3, MUC5AC, and MUC5B, whereas deeper mucosal folds display MUC5B and MUC6 immunoreactivity. Gallbladder epithelium demonstrates a unique and diverse pattern of mucin core proteins that becomes altered with increasing degrees of inflammation.

Mucins secreted by the endocervical epithelium protect the surfaces of the reproductive tract epithelium from pathogen penetrance and modulate sperm entry into the uterus. Three large gel-forming mucins, MUCs 5AC, 5B, and 6, are expressed by the endocervical epithelium, as is MUC4, a relatively uncharacterized mucin for which only tandem repeat sequence has been reported. We sought to determine the relative abundance of each of these mucin gene transcripts and to relate their expression to blood progesterone and estradiol. Samples were obtained from six subjects at successive stages in the menstrual cycle. Primers to nontandem repeat sequences of MUCs 4, 5AC, 5B, and 6 were used in semiquantitative reverse transcription-polymerase chain reaction to determine relative abundance of each mucin gene in relation to beta2-microglobulin message control. In order to design primers from a nontandem repeat region of MUC4 so that MUC4 message levels could be quantitated, we obtained approximately 2.7-kilobase nontandem repeat sequence 5' to the tandem repeat sequence of a MUC4 genomic clone. The sequence showed lack of cysteine-rich D-domains and was rich in serine and threonine. Semiquantitative polymerase chain reaction analyses indicated that the principal mucin transcripts of human endocervix are MUC4 and MUC5B, with MUC4 predominant in 15 of 21 samples. When correlated with plasma steroid levels, message levels of both MUC4 and MUC5B were inversely related to progesterone levels.

The human mucin gene MUC5AC codes for a large mucin which has tandem repeat units and cysteine rich regions characteristic of several members of this class of glycoproteins. Human epithelia expressing the mucin include that of stomach, bronchus/trachea, endocervix and conjunctiva. We report here a 3.8 kb partial sequence of a rat homologue for the human MUC5AC gene and compare its tandem repeat sequence and cysteine rich domains to those of the human and mouse gene. Rat and mouse have the same number of amino acids (16) in their Muc5AC tandem repeat units and share 69% sequence similarity, whereas human MUC5AC has only 8 amino acids in its tandem repeat. In rat, the tandem repeat domain is flanked at its 3' end by a non-repeat region coding for 1142 amino acids. Four cysteine rich subdomains were identified in this region; one of these has 64% similarity to a corresponding region in human MUC5AC and 80% similarity to a mouse MUC5AC cysteine rich region. Southern blot analysis revealed cross hybridization of a probe for the rat cysteine rich region, to human, mouse, rabbit, and porcine genomic DNA; the rat tandem repeat probe hybridized with mouse and rabbit only. Unlike humans, rat expressed MUC5AC message detectable by Northern blot and in situ hybridization only in stomach epithelium and conjunctival goblet cells.

Recent characterizations of mucins at the molecular level indicate that at least eight mucin genes are expressed by epithelia of mucosal surfaces. The purpose of this study was to determine whether these cloned mucins, designated MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7, are expressed by epithelia of the female reproductive tract. Northern blot analysis, in situ hybridization, and immunohistochemistry were performed using RNA and tissue from surgically removed human reproductive tract specimens including endocervix, ectocervix, vagina, endometrium, and fallopian tube. Complementary DNA to the tandem repeat regions of MUCs 1, 2, 3, 5AC, 5B, and 6; oligonucleotides to the tandem repeat regions of MUCs 4, 6, and 7; and antibodies that recognize unique mucin tandem repeats were used. The data demonstrate that the endocervical epithelium expresses five mucin genes: MUCs 1, 4, 5AC, 5B, and 6. The ectocervical and vaginal epithelia express MUCs 1 and 4, although vaginal expression of MUC4 appears patchy. Endometrial epithelium expresses MUC1 and low amounts of MUC6. MUC6 immunoreactivity was detected only in scattered endometrial glands located in the basalis region in specimens from pre- and postmenopausal women. The only mucin detected in the fallopian tube was MUC1. These data indicate that the endocervical epithelium expresses multiple mucin genes and that the stratified epithelia of the ectocervix and vagina also produce mucins that may function in reproductive processes and protection of the reproductive tract tissues.

Mucins are the glycoproteins which form the viscous, gel-like mucus layer of the tear film. Molecular characterization of mucins has been slow due to their heavy glycosylation and high molecular weight, but recent cloning of human gut and trachea, mammary gland, and salivary gland mucins has begun to shed light on the primary structure of these important protective molecules. To date nine human mucin genes, designated MUCI-8, have been cloned. The presence in the protein backbone of tandem repeats of series of amino acids rich in serine and/or threonine is a feature common to all. Numbers and sequences of amino acids in each tandem repeat varies with each mucin. Numbers of amino acids per repeat vary from 169 (MUC6) to 8 (MUC5AC). Until recently little has been known regarding the expression of these genes by the ocular surface epithelium. This review summarizes recent work from our laboratory aimed at determining the molecular character of mucins expressed by conjunctival and corneal epithelium. Using northern blot analysis and in situ hybridization techniques, we have demonstrated that the stratified epithelium of both cornea and conjunctiva express the transmembrane mucin, MUC1. This widely expressed mucin is the best characterized of the cloned mucins. Using similar methodologies we demonstrated that the conjunctival goblet cell expresses MUC5AC, a gel-forming mucin, which has a cysteine-rich domain responsible for the disulfhydryl bonding between mucin molecules. Conjunctival stratified epithelium expresses MUC4, a relatively uncharacterized mucin, whose function is not known. Are there additional and/or unique mucins expressed by the ocular surface epithelium? We summarize our recent work on characterization of a mucin isolated from rat and human corneal epithelium. Antibodies to these mucins localize to the glycocalyx of the corneal epithelial apical cells. Since MUCI appears to be the only one of the cloned mucins expressed by the corneal epithelium, it is a candidate for comparison to our mucin isolate. Current efforts are directed toward this comparison. Identification of mucin genes expressed by the ocular surface epithelium opens many new avenues for investigation into expression, regulation, and glycosylation of mucins in ocular surface pathologies and into the specific character of these molecules which enhance protection from pathogen invasion.

Aging of the ocular surface and corneal tissues, major components of the visual system, causes major eye disease and results in substantial cost in medical and social terms. These diseases include the highly prevalent dry eye disease that affects the ocular surface and its glands, leading to tear film alterations, discomfort, and decreased vision. Studies show that 14.4% of the population in the United States older than 50 years have dry eye disease and demonstrate that it is particularly prevalent among women. Annual medical costs per patient with dry eye in the United States are estimated at $783 per year, with an overall medical cost adjusted to prevalence of $3.84 billion per year. Societal costs, which include loss of productivity, are estimated per patient at $11,302 per year, with overall costs adjusted to prevalence of $55.4 billion per year. Because there are few effective treatments for the disease, more research on its etiology and mechanisms is warranted and needed. Increased public education about risk factors for the disease is also required. Another major age-related eye disease of the cornea that leads to vision impairment and potentially blindness if left untreated is Fuchs' endothelial corneal dystrophy. This disease leads to loss of the endothelial cells on the internal side of the cornea that are responsible for keeping the cornea in the proper hydration state to ensure its transparency to light. The mechanism of cell loss is unknown, and the only treatment available to date is surgical transplantation of the cornea or inner part of the cornea. These medically costly procedures require donor corneas, eye banking, and medical follow-up, with accrued costs. Fuchs' endothelial corneal dystrophy is a major cause of corneal transplantation in the United States; therefore, research support is needed to determine the mechanism of this age-related disease, to develop medical, nonsurgical methods for treatment.

The stratified squamous epithelia of the ocular surface, larynx, and vagina are mucus-coated epithelia, apices of which are subject to abrasive pressure from epithelia — epithelia interactions from eyelid, vocal cords, or vaginal folds, respectively. Mucus coats on these epithelia have generally been considered to be derived from the specialized mucin-producing cells embedded either in the epithelia or in adjacent tissues. Here we report the isolation, partial characterization, and cellular localization of a mucin-like glycoprotein produced by these stratified epithelia. In all three epithelia, the mucin-like molecule is present on cytoplasmic vesicles in subapical cells. As cells differentiate to their apical-most position adjacent to their mucus coat, the mucin-like molecule moves to the cell membrane where it is particularly prominent on microplicae folds. Lectin affinity chromatography was used to isolate the molecule from rat vaginal and corneal epithelium. Isolated material was approximately 60% carbohydrate and 40% protein. The major monosaccharide was N-acetylgalactosamine with lesser amounts of N-acetylglucosamine, galactose, mannose, xylose and fucose. Amino acid analysis demonstrated the predominant amino acids to be glycine, serine, threonine and proline. These data plus PAS and Alcian blue binding to the isolate indicate a mucin-like glycoprotein.

Healing of corneal wounds is a complex process involving epithelial, keratocyte, and endothelial interactions that are affected by their associations with wound bed matrix and by cytokine availability and activation. The spectrum of possible cellular-matrix-growth factor interactions is indeed great and growing. Several of the significant contributions made during the past year include development of an organotypic organ culture model of the cornea that allows in vitro assembly of the epithelial extracellular matrix-anchoring complex, demonstration of epithelial synthesis of Bowman's layer collagens, demonstration of transforming growth factor-beta 2's inhibition of stromal cell collagenase synthesis, and demonstration of the paracrine pathway of keratinocyte growth factor action in the cornea.

The surface of the eye is exposed to the outside world and is, thus, subject to surface abrasion, infections, and drying, cicatrizing diseases. Availability of in vitro methods for culture of the human corneal and conjunctival epithelia, which cover the ocular surface, is therefore important in understanding the biology of these epithelia and their response to disease/infections, as well as for providing human-relevant models for preclinical testing of potential therapeutic agents. The ensuing chapter describes several methods for primary culture of both corneal and conjunctival epithelia and culture of immortalized cell lines, and methods employed to induce differentiation in the cultured epithelia.

PURPOSE. The accessory lacrimal glands are assumed to contribute to the production of tear fluid, but little is known about their function. The goal of this study was to conduct an analysis of gene expression by glands of Wolfring that would provide a more complete picture of the function of these glands. METHODS. Glands of Wolfring were isolated from frozen sections of human eyelids by laser microdissection. RNA was extracted from the cells and hybridized to gene expression arrays. The expression of several of the major genes was confirmed by immunohistochemistry. RESULTS. Of the 24 most highly expressed genes, 9 were of direct relevance to lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and lacritin. The glands of Wolfring are enriched in genes related to protein synthesis, targeting, and secretion, and a large number of genes for proteins with antimicrobial activity were detected. Ion channels and transporters, carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal polypeptide, purinergic, androgen, and prolactin receptors were also expressed in gland of Wolfring. CONCLUSIONS. The data suggest that the function of glands of Wolfring is similar to that of main lacrimal glands and are consistent with secretion electrolytes, fluid, and protein under nervous and hormonal control. Since these glands secrete directly onto the ocular surface, their location may allow rapid response to exogenous stimuli and makes them readily accessible to topical drugs. (Invest Ophthalmol Vis Sci. 2012; 53:6738‐6747) DOI:10.1167/iovs.12-10750

Mucins of the corneal and conjunctival epithelia are necessary for the protection of the ocular surface against desiccation, pathogen access, and injury. Detection and quantification of mucins is important for the understanding of ocular surface diseases that cause impaired vision and, in advanced stages, blindness. Advances in the field of molecular biology have made it possible to study membrane mucins and their associated O-glycans in established cell culture models of human ocular surface epithelia. This chapter discusses procedures to detect and quantify mucin RNA and protein in biological samples, as well as methods to experimentally manipulate the epithelia in culture by shRNA, to understand the function of specific mucins. Example protocols are provided to evaluate the role of ocular surface mucins in mucosal barrier function and bacteria-host interactions.

The surface of the eye is exposed to the outside world and is, thus, subject to surface abrasion, infections, and drying, cicatrizing diseases. Availability of in vitro methods for culture of the human corneal and conjunctival epithelia, which cover the ocular surface, is therefore important in understanding the biology of these epithelia and their response to disease/infections, as well as for providing human-relevant models for preclinical testing of potential therapeutic agents. The ensuing chapter describes several methods for primary culture of both corneal and conjunctival epithelia and culture of immortalized cell lines, and methods employed to induce differentiation in the cultured epithelia.

To determine if levels of the glycocalyx membrane mucins, MUC1 and MUC16, and the secreted goblet cell mucin MUC5AC are altered in conjunctival cells and tears of postmenopausal women presenting with a history of non-Sjögren dry eye and if mucin levels correlate with dry eye clinical diagnostic data.Eighty-four postmenopausal women with a history of non-Sjögren dry eye and 30 normal subjects were recruited for this study. Impression cytology samples were collected for mucin messenger RNA (mRNA) and protein analysis. Tears were collected for mucin protein assay. Quantitative polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were used to quantitate MUC1, MUC16, and MUC5AC levels.Postmenopausal women with a history of dry eye displayed significantly increased MUC1 mRNA expression and cellular protein compared with normal subjects (P0.001 and P0.01, respectively). Similarly, cellular MUC16 protein levels were significantly higher (P0.001). Mucin levels were found to be correlated with the clinical characterization of the subjects, including staining and symptoms. Although cellular MUC5AC protein levels were increased in symptomatic subjects, the increase did not reach statistical significance.Elevation in MUC1 and MUC16 mRNA and/or protein levels in postmenopausal women with non-Sjögren dry eye with a history of dry eye may be a compensatory response to irritation and inflammation associated with the disease. Understanding the pattern of mucin expression associated with the dry eye pathology may clarify factors involved in the progression of the disease and enhance the development of targeted therapies.

The majority of bacterial infections occur across wet-surfaced mucosal epithelia, including those that cover the eye, respiratory tract, gastrointestinal tract and genitourinary tract. The apical surface of all these mucosal epithelia is covered by a heavily glycosylated glycocalyx, a major component of which are membrane-associated mucins (MAMs). MAMs form a barrier that serves as one of the first lines of defense against invading bacteria. While opportunistic bacteria rely on pre-existing defects or wounds to gain entry to epithelia, non opportunistic bacteria, especially the epidemic disease-causing ones, gain access to epithelial cells without evidence of predisposing injury. The molecular mechanisms employed by these non opportunistic pathogens to breach the MAM barrier remain unknown. To test the hypothesis that disease-causing non opportunistic bacteria gain access to the epithelium by removal of MAMs, corneal, conjunctival, and tracheobronchial epithelial cells, cultured to differentiate to express the MAMs, MUCs 1, 4, and 16, were exposed to a non encapsulated, non typeable strain of Streptococcus pneumoniae (SP168), which causes epidemic conjunctivitis. The ability of strain SP168 to induce MAM ectodomain release from epithelia was compared to that of other strains of S. pneumoniae, as well as the opportunistic pathogen Staphylococcus aureus. The experiments reported herein demonstrate that the epidemic disease-causing S. pneumoniae species secretes a metalloproteinase, ZmpC, which selectively induces ectodomain shedding of the MAM MUC16. Furthermore, ZmpC-induced removal of MUC16 from the epithelium leads to loss of the glycocalyx barrier function and enhanced internalization of the bacterium. These data suggest that removal of MAMs by bacterial enzymes may be an important virulence mechanism employed by disease-causing non opportunistic bacteria to gain access to epithelial cells to cause infection.

As basal cells of stratified squamous epithelia become migratory in response to wounding, they lose their cell-substrate adhesion junctions, the hemidesmosomes. We report here studies to determine the fate of the hemidesmosome components, alpha 6 beta 4 integrin and the bullous pemphigoid antigens (BPAGs), as recognized by bullous pemphigoid autoantisera (BPA), in migrating epithelium. In addition, we report studies to determine whether relative synthesis and amount of alpha 6 beta 4 is altered during migration. Mouse corneas with 1.5- to 2-mm-diameter central epithelial debridements were allowed to heal in vitro or in vivo for 1-18 h. In order to do preembedding immunoelectron microscopic localization of alpha 6 beta 4, sheets of stationary and migrating corneal epithelium were removed from their basal laminae after organ culture. BPA and antibodies to alpha 6 and beta 4 were used for immunofluorescence microscopy on frozen sections of intact corneas healing in vivo 1-18 h. Both alpha 6 and beta 4 were found to redistribute from their clustered location within hemidesmosomes to a more even distribution within the substrate-associated membrane of basal cells of the tip of the leading edge of migrating epithelium. Behind the tip of the leading edge, basal cells bound the integrin antibodies around their entire membrane. BPAGs moved from their location along the basal cell membrane of stationary epithelium to a diffuse location within the cytoplasm of migrating cells at the leading edge of migration. Quantitative immunoprecipitation and immunoblotting of alpha 6 beta 4 as well as beta 1 integrin from stationary and migrating epithelium were done to determine whether the synthesis or total amount of the integrins were altered during migration. The relative syntheses of alpha 6 beta 4 and beta 1 per milligram of protein or per cell do not appear to differ between stationary and migrating epithelium and the total amount of the beta 4 and beta 1 does not change despite increased rates of protein synthesis in migrating epithelium. Taken together, these studies suggest that as hemidesmosomes disassemble, their clustered integrin component distributes more evenly in the basal cell membrane, the components recognized by BPA and associated with intermediate filaments are released from the membrane, and these events occur in the absence of any measurable change in the synthesis or total amount of the alpha 6 beta 4 component.