The immortalized corneal (HCLE) and conjunctival (HCjE) cell lines exhibit the mucin gene expression repertoire of their native epithelia. These cell lines will be useful in determining regulation of ocular surface mucin gene expression and, potentially, goblet cell differentiation.

Antisera that recognize the a6 and (34 subunits of integrins were found by immunoelectron microscopy to localize to hemidesmosomes in the basal cells of mouse corneal epithelium. Immunoprecipitation experiments using extracts of metabolically labeled corneal epithelial cells indicate that the primary a6-subunit-containing integrin heterodimer present is a434 and not a6431. Here we extend previous studies to report that by immunofluorescence microscopy the a6 integrin subunit colocalizes with bullous pemphigoid antigen and type V1I collagen in newly forming hemidesmosomes in the developing 17-day fetal rabbit eye. Neither the composition of the anchoring filaments, which span the region between the hemidesmosomal plaque and the lamina densa of basement membrane where the globular domain of type VII collagen is located, nor the extracellular ligand of a6.84 is known. Once anchoring filament proteins are identified, it will be of interest to determine whether any bind to a484.Hemidesmosomes are cell-substrate adhesionjunctions present along the basal cell-basement membrane junction of stratified squamous epithelia (1,2). By comparison to desmosomes, biochemical characterization of the basic components of the hemidesmosome has been slow, due in part to difficulty in obtaining hemidesmosome-enriched cellular fractions. The only characterized component of the hemidesmosome is the bullous pemphigoid antigen (BPA), a 230-kDa protein present in the cytoplasmic plaque of the hemidesmosome (3-5). BPA is found within the electron-dense cytoplasmic plaque region of the hemidesmosome. The sequence of cDNA clones encoding the BPA molecule, although not entirely complete, shows no integral membrane segment (J. Stanley, personal communication). Several other monoclonal antibodies specific to hemidesmosomes have been reported, and their antigens await characterization (6, 7). Clearly, other molecules are present in both the cytoplasmic and the extracellular aspect of the hemidesmosome.The molecule(s) that traverses the cell membrane in hemidesmosomes has yet to be identified; candidates include members of the integrin superfamily of extracellular matrix receptors (8-12). The integrins are integral membrane glycoproteins that function as cell-cell and cell-substrate adhesion molecules. As a result of our studies to localize and characterize integrin subunits present in corneal epithelium, we noted that antibodies to both the a6 and (4 integrin subunits bound to the basal cell-basement membrane zone in stationary epithelia. This prompted us to determine whether the heterodimer is associated with hemidesmosomes. The present study reports that a6 and P4 integrins colocalize with BPA and type VII collagen in hemidesmosomes of developing epithelia, that both a6 and (3 antibodies bind to hemidesmosomes as shown by immunoelectron microscopy, and that both a434 and a43l heterodimers can be identified in epithelia by immunoprecipitation. MATERIALS AND METHODSTissue Preparation. For adult corneal epithelium, New Zealand White (NZW) ...

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

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.

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 is 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.

This study demonstrates that the membrane-associated mucin MUC16 is expressed by the human ocular surface epithelia and that MUC16 carries the H185 carbohydrate epitope. Future studies on the expression of MUC16 and the characterization of the molecular structure of the H185 carbohydrate epitope will determine their biological significance on the healthy ocular surface and in dry eye syndrome.

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.

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.