Generation and characterization of a monoclonal antibody to the cytoplasmic tail of MUC16.

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.

Elevated Neutrophil Elastase in Tears of Ocular Graft-Versus-Host Disease Patients.

PURPOSE: 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). DESIGN: Case-control study. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Epidemic Keratoconjunctivitis-Causing Adenoviruses Induce MUC16 Ectodomain Release To Infect Ocular Surface Epithelial Cells.

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.

Comparison of the transmembrane mucins MUC1 and MUC16 in epithelial barrier function.

Membrane-anchored mucins are present in the apical surface glycocalyx of mucosal epithelial cells, each mucosal epithelium having at least two of the mucins. The mucins have been ascribed barrier functions, but direct comparisons of their functions within the same epithelium have not been done. In an epithelial cell line that expresses the membrane-anchored mucins, MUC1 and MUC16, the mucins were independently and stably knocked down using shRNA. Barrier functions tested included dye penetrance, bacterial adherence and invasion, transepithelial resistance, tight junction formation, and apical surface size. Knockdown of MUC16 decreased all barrier functions tested, causing increased dye penetrance and bacterial invasion, decreased transepithelial resistance, surprisingly, disruption of tight junctions, and greater apical surface cell area. Knockdown of MUC1 did not decrease barrier function, in fact, barrier to dye penetrance and bacterial invasion increased significantly. These data suggest that barrier functions of membrane-anchored mucins vary in the context of other membrane mucins, and MUC16 provides a major barrier when present.

Stability and in vitro toxicity of an infliximab eye drop formulation.

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 (P > 0.05). 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.

Methods for culture of human corneal and conjunctival epithelia.

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.

Gene expression in human accessory lacrimal glands of Wolfring.

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.

A metalloproteinase secreted by Streptococcus pneumoniae removes membrane mucin MUC16 from the epithelial glycocalyx barrier.

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.

Comparison of mucin levels at the ocular surface of postmenopausal women with and without a history of dry eye.

PURPOSE: 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. METHODS: 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. RESULTS: Postmenopausal women with a history of dry eye displayed significantly increased MUC1 mRNA expression and cellular protein compared with normal subjects (P < 0.001 and P < 0.01, respectively). Similarly, cellular MUC16 protein levels were significantly higher (P < 0.001). 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. CONCLUSIONS: 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.

Effect of pro-inflammatory mediators on membrane-associated mucins expressed by human ocular surface epithelial cells.

Membrane-associated mucins are altered on the ocular surface in non-Sjögren's dry eye. This study sought to determine if inflammatory mediators, present in tears of dry eye patients, regulate membrane-associated mucins MUC1 and -16 at the level of gene expression, protein biosynthesis and/or ectodomain release. A human corneal limbal epithelial cell line (HCLE), which produces membrane-associated mucins, was used. Cells were treated with interleukin (IL)-6, -8, or -17, tumor necrosis factor-alpha (TNF-alpha), and Interferon-gamma (IFN-gamma), or a combination of TNF-alpha and IFN-gamma, or IFN-gamma and IL-17, for 1, 6, 24, or 48 h. Presence of receptors for these mediators was verified by RT-PCR. Effects of the cytokines on expression levels of MUC1 and -16 were determined by real-time PCR, and on mucin protein biosynthesis and ectodomain release in cell lysates and culture media, respectively, by immunoblot analysis. TNF-alpha and IFN-gamma each significantly induced MUC1 expression, cellular protein content and ectodomain release over time. Combined treatment with the two cytokines was not additive. By comparison, one of the inflammatory mediators, IFN-gamma, affected all three parameters-gene expression, cellular protein, and ectodomain release-for MUC16. Combined treatment with TNF-alpha and IFN-gamma showed effects similar to IFN-gamma alone, except that ectodomain release followed that of TNF-alpha, which induced MUC16 ectodomain release. In conclusion, inflammatory mediators present in tears of dry eye patients can affect MUC1 and -16 on corneal epithelial cells and may be responsible for alterations of surface mucins in dry eye.

The Boston Keratoprosthesis: comparing corneal epithelial cell compatibility with titanium and PMMA.

PURPOSE: To determine in vitro whether titanium is superior in corneal cell compatibility to standard polymethyl-methacrylate (PMMA) for the Boston Keratoprosthesis (KPro). METHODS: 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. P < 0.05 was statistically significant. RESULTS: 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). CONCLUSION: 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.

Release of membrane-associated mucins from ocular surface epithelia.

PURPOSE: Three membrane-associated mucins (MAMs)--MUC1, MUC4, and MUC16--are expressed at the ocular surface epithelium. Soluble forms of MAMs are detected in human tears, but the mechanisms of their release from the apical cells are unknown. The purpose of this study was to identify physiologic agents that induce ocular surface MAM release. METHODS: An immortalized human corneal-limbal epithelial cell line (HCLE) expressing the same MAMs as native tissue was used. An antibody specific to the MUC16 cytoplasmic tail was developed to confirm that only the extracellular domain is released into the tear fluid or culture media. Effects of agents that have been shown to be present in tears or are implicated in the release or shedding of MAMs in other epithelia (neutrophil elastase, tumor necrosis factor [TNF]), TNF-alpha-converting enzyme, and matrix metalloproteinase-7 and -9) were assessed on HCLE cells. HCLE cell surface proteins were biotinylated to measure the efficiency of induced MAM release and surface restoration. Effects of induced release on surface barrier function were measured by rose bengal dye penetrance. RESULTS: MUC16 in tears and in HCLE-conditioned medium lacked the cytoplasmic tail. TNF induced the release of MUC1, MUC4, and MUC16 from the HCLE surface. Matrix metalloproteinase-7 and neutrophil elastase induced the release of MUC16 but not of MUC1 or MUC4. Neutrophil elastase removed 68% of MUC16, 78% of which was restored to the HCLE cell surface 24 hours after release. Neutrophil elastase-treated HCLE cells showed significantly reduced rose bengal dye exclusion. CONCLUSIONS: Results suggest that the extracellular domains of MUC1, MUC4, and MUC16 can be released from the ocular surface by agents in tears. Neutrophil elastase and TNF, present in higher amounts in the tears of patients with dry eye, may cause MAM release, allowing rose bengal staining.

Photodistribution of blue-gray hyperpigmentation after amiodarone treatment: molecular characterization of amiodarone in the skin.

BACKGROUND: For decades, the photodistributed blue-gray skin hyperpigmentation observed after amiodarone therapy was presumably attributed to dermal lipofuscinosis. Using electron microscopy and high-performance liquid chromatography, we identified amiodarone deposits in the hyperpigmented skin sample from a patient treated with this antiarrhythmic agent. Our findings therefore indicate that the hypothesis relating the blue-gray hyperpigmentation to lipofuscin should be challenged. OBSERVATIONS: A 64-year-old man, skin phototype III, presented with asymptomatic skin hyperpigmentation that had been slowly developing on sun-exposed areas since April 2004. He had been taking amiodarone for 4 years (cumulative dose, 277 g). Electron microscopy did not show lipofuscin pigments in his skin. Conversely, abundant electron-dense membrane-bound granule deposits were observed in most of the dermal cells (fibroblasts, macrophages, pericytes, Schwann cells, and endothelial cells), especially in photoexposed skin. High-performance liquid chromatography confirmed that the skin deposits were composed of amiodarone. These results demonstrate that amiodarone hyperpigmentation is related to drug deposition on photoexposed skin. CONCLUSION: Amiodarone-related hyperpigmentation should be considered a skin storage disease that is secondary to drug deposition.

MUC16 is lost from the uterodome (pinopode) surface of the receptive human endometrium: in vitro evidence that MUC16 is a barrier to trophoblast adherence.

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 LH+0 to LH+13 show that another membrane-spanning mucin, MUC16, was lost from uterodome surfaces in all samples taken during the receptive phase, LH+6 to LH+8 (n = 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.

Functions of MUC16 in corneal epithelial cells.

PURPOSE: The membrane-associated mucin MUC16, a heavily O-glycosylated transmembrane protein, is expressed by the ocular surface epithelia and localized on the tips of the surface microplicae. Although its functions in the ocular surface glycocalyx are unknown, it is thought that MUC16 provides a disadhesive barrier to the epithelial membrane. Two other membrane-associated mucins expressed by ocular surface epithelia, MUC1 and MUC4, are multifunctional and have signaling capabilities through their cytoplasmic tails and EGF-like domains, respectively. The MUC16 cytoplasmic tail has not been characterized, but, because it contains a polybasic amino acid sequence, it potentially interacts with the actin cytoskeleton through ezrin/radixin/moesin (ERM) actin-binding proteins. METHODS: The interaction of MUC16 with the actin cytoskeleton through ERMs was investigated using cytoplasmic tail peptides and ERM pull-down experiments. MUC16 functions were determined using RNA interference in immortalized human corneal-limbal epithelial (HCLE) cells. The effect of MUC16 knockdown on microplicae structure in HCLE cells was determined using scanning and immunoelectron microscopy. HCLE cells were incubated with rose bengal dye to measure the role of MUC16 in ocular surface barrier function. Binding of fluorescently labeled Staphylococcus aureus to HCLE cells was measured to determine the role of MUC16 in the protection of pathogen adherence on the ocular surface epithelium. RESULTS: MUC16 cytoplasmic tail peptides bound the N-terminus of ERMs, with no detectable binding of MUC1 and MUC4 peptides. No effect on surface membrane projections could be detected in HCLE cells after MUC16 suppression; however, HCLE cells incubated with rose bengal showed that exclusion of the dye was significantly reduced in cells with MUC16 suppression. In addition, S. aureus binding to HCLE cells was significantly increased with MUC16 suppression. CONCLUSIONS: These results suggest that MUC16 is a multifunctional molecule linked to the actin cytoskeleton. The expression of MUC16 in the ocular surface glycocalyx helps provide a disadhesive protective barrier for the epithelial surface.

Assay of mucins in human tear fluid.

Mucin genes, both secreted (MUC2, MUC5AC, MUC5B, MUC7) and membrane associated (MUC1, MUC4, MUC16), have been reported to be expressed by ocular surface epithelia. The purpose of this study was to comprehensively assay the mucin content of human tear fluid using multiple antibodies for each mucin and to develop a sensitive, semi-quantitative method for the assay of mucins in tears. Tear washes were obtained by instillation of saline onto the ocular surface, followed by collection from the inferior fornix. Tear proteins were separated in 1% agarose gels, transferred to nitrocellulose membrane by vacuum blotting and probed with multiple antibodies recognizing MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 and MUC16. Binding was detected using chemiluminescence, and quantity was determined by densitometry. Serial dilutions of pooled tears from normal individuals were assayed to determine the linear range of detectability. MUC1, MUC4, MUC16, MUC5AC and low levels of MUC2 were consistently detected in human tear fluid, while MUC5B and MUC7 were not. Use of several antibodies recognizing different epitopes on the same mucin confirmed these findings. The antibodies to mucins bound to serial dilutions of tears in a linear fashion (r2 > 0.9), indicating the feasibility of semi-quantitation. MUC5AC in tear fluid had an increased electrophoretic mobility compared to MUC5AC isolated from conjunctival tissue. This study provides clear evidence that the mucin component of tears is a mixture of secreted and shed membrane-associated mucins, and for the first time demonstrates MUC16 in tear fluid. Immunoblots of tears using agarose gel electrophoresis and chemiluminescence detection provide a semi-quantitative assay for mucin protein that will be useful for comparisons with tears from diseased eyes or after pharmacological intervention.

Mucin gene expression in human male urogenital tract epithelia.

BACKGROUND: Mucins are large, hydrophilic glycoproteins that protect wet-surfaced epithelia from pathogen invasion as well as provide lubrication. At least 17 mucin genes have been cloned to date. This study sought to determine the mucin gene expression profile of the human male urogenital tract epithelia, to determine if mucins are present in seminal fluid and to assess the effect of androgens on mucin expression. METHODS AND RESULTS: Testis, epididymis, vas deferens, seminal vesicle, prostate, bladder, urethra and foreskin were assessed for mucin expression by RT-PCR (for 14 mucin genes) and immunohistochemistry (nine antibodies for five mucins). Epithelia of the vas deferens, prostate and urethra expressed the greatest number of mucins, each with mRNA for between 5 and 8 mucins. Except for MUC20 in epididymis, mRNA for MUC1 and MUC20, both membrane-associated mucins, was detected in all tissues analysed. By comparison, MUC6 was more restricted in expression, being primarily detected in seminal vesicle. MUC1, MUC5B and MUC6 were detected in seminal fluid samples by immunoblot analysis. Androgens had no effect on mucin expression in cultured human prostatic epithelial cells. CONCLUSIONS: Each region of urogenital tract epithelium expressed a unique mucin gene repertoire. Secretory mucins are present in seminal fluid, and androgens do not appear to regulate mucin gene expression in prostatic epithelial cells in culture.

Mucins and contact lens wear.

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.

Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye.

PURPOSE: Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins. METHODS: HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay. RESULTS: The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake. CONCLUSIONS: These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia.

Effect of retinoic acid on gene expression in human conjunctival epithelium: secretory phospholipase A2 mediates retinoic acid induction of MUC16.

PURPOSE: How vitamin A contributes to the maintenance of the wet-surfaced phenotype at the ocular surface is not well understood. This study sought to identify vitamin A-responsive genes in ocular surface epithelia using gene microarray analysis of cultures of a human conjunctival epithelial (HCjE) cell line grown with all-trans-retinoic acid (RA). The analysis showed that secretory phospholipase A(2) group IIA (sPLA(2)-IIA) was the gene most upregulated by RA, followed by the membrane-associated mucin MUC16 at a later time point. Since eicosanoids, the product of arachidonic acid generated by the PLA(2) family, have been shown to increase mucin production, this study sought to determine whether sPLA(2) mediates the RA induction of MUC16. METHODS: HCjE cells were cultured with or without RA for 3, 6, 24, and 48 hours. Complementary RNA prepared from RNA of the HCjE cells was hybridized to human gene chips and analyzed using commercial software. Microarray data on mucin expression were validated by real-time PCR. To investigate whether sPLA(2) is associated with RA-induced MUC16 upregulation, HCjE cells were incubated with RA and the broad-spectrum PLA(2) inhibitor aristolochic acid (ArA) or the specific sPLA(2)-IIA inhibitor LY315920, followed by analysis of MUC16 mRNA and protein by real-time PCR and Western blot analysis. RESULTS: After RA addition, 28 transcripts were upregulated and 6 downregulated by more than twofold (P < 0.01) at both 3 and 6 hours (early phase). Eighty gene transcripts were upregulated and 45 downregulated at both 24 and 48 hours (late phase). Group IIA sPLA(2), significantly upregulated by 24 hours, and MUC16 were the most upregulated RNAs by RA at 48 hours. sPLA(2) upregulation by RA was confirmed by Western blot analysis. When HCjE cells were incubated with RA plus ArA or specific inhibitor of sPLA(2)-IIA, LY315920, the RA-induced MUC16 mRNA was significantly reduced (P < 0.01). CONCLUSIONS: The RA-associated upregulation of membrane-associated mucin MUC16 at late phase appears to be through sPLA(2)-IIA. Upregulation of this hydrophilic membrane-associated mucin may be one of the important mechanisms by which vitamin A facilitates maintenance of the wet-surfaced phenotype on the ocular surface.