Suppression of Toll-like receptor-mediated innate immune responses at the ocular surface by the membrane-associated mucins MUC1 and MUC16.

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.

Alteration in goblet cell numbers and mucin gene expression in a mouse model of allergic conjunctivitis.

PURPOSE: To determine whether the number of filled conjunctival goblet cells and mucin gene expression are altered in a mouse model of allergic conjunctivitis. METHODS: A/J mice were sensitized intraperitoneally with cat dander or the peptide P3-1 from the protein Fel d1. Two weeks later, the mice were challenged for 7 consecutive days with eye drops containing the allergens. Conjunctival tissue was harvested at 0, 6, 24, or 48 hours after final antigen challenge. Control samples were naïve animals and mice sensitized with cat dander and challenged with OVA-peptide or PBS. The mean number of filled goblet cells per square millimeter in three forniceal fields for each group was determined in wholemounts of conjunctiva prepared using rhodamine-phalloidin labeling followed by confocal microscopy. RNA was isolated from conjunctiva of the contralateral eye and taken for relative quantitation of mRNA of the goblet cell mucin Muc5AC and the epithelial membrane-spanning mucin Muc4, by real-time RT-PCR. RESULTS: The number of filled goblet cells was significantly decreased with both cat dander and P3-1, after final ocular challenge (P < 0.001). The most significant decrease over naïve mice was seen at 6 hours after final challenge with both allergens. The number of filled goblet cells was still decreased but was returning toward naïve levels at 24 hours (P < 0.05), and at 48 hours no significant difference was seen compared with naïve, PBS-treated, and OVA-peptide-treated control samples. For both cat dander and P3-1, Muc5AC and Muc4 mRNA was found to be decreased at the time of final ocular challenge. The level of Muc5AC mRNA from goblet cells rebounded from the decrease to show an increase over control by 24 hours after final challenge, and by 48 hours, the mRNA level had returned to naïve control range. In contrast, significant increases in Muc5AC mRNA were evident after final control challenge with PBS or OVA-peptide, indicating a potential irritant effect of drop application. The Muc4 mRNA level was significantly reduced at all time points except 24 hours after the last challenge. By comparison with allergen-challenged eyes, no change in Muc4 message levels was noted at any time point in OVA-peptide- or PBS-treated control eyes. CONCLUSIONS: These findings demonstrate that, in the conjunctiva of mice, repetitive application of allergens induces a reduction in the number of filled goblet cells and a decrease in Muc5AC and Muc4 mRNAs. After a period of 24 to 48 hours, the goblet cell number return to naïve levels, and goblet cell mucin mRNA levels return to above or within normal range, indicating a rapid recovery in the mucus secretion system.

The Amount of MUC5B mucin in cervical mucus peaks at midcycle.

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.

Vitamin A deficiency alters the expression of mucin genes by the rat ocular surface epithelium.

PURPOSE: To study effects of depletion of retinoic acid on expression of the mucins ASGP (rMuc4), rMuc5AC, and rMuc1, by the corneal and conjunctival epithelia of the rat. METHODS: Nineteen-day-old Sprague-Dawley male rats were fed a casein-based vitamin A- deficient diet or casein-based diet with vitamin A as control. Rats from both groups were killed at 1, 3, 5, 13, 15, 18, and 20 weeks after initiation of feeding. Expression of the three mucin genes by the ocular surface epithelium was assayed by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: In vitamin A-deficient rats, ASGP mRNA was not detected by RT-PCR after 15 weeks of feeding. rMuc5AC mRNA was detected by RT-PCR at 15 weeks, but by 18 and 20 weeks was no longer detectable. By in situ hybridization, ASGP mRNA was localized in the entire ocular surface epithelium after 1 week of feeding, was diminished but detectable above background by 13 weeks, and was not detectable at 20 weeks. rMuc5AC mRNA was detected in the goblet cells of vitamin A- deficient rats by in situ hybridization at 13 weeks, but was lost by 20 weeks, as were identifiable goblet cells. rMuc1 mRNA were detected by RT-PCR through all time points of 1 to 20 weeks in both vitamin A-deficient and control rats, indicating no significant change in rMuc1 mRNA expression with vitamin A deficiency. CONCLUSIONS: Both the membrane-spanning mucin ASGP (rMuc4) and the secretory mucin rMuc5AC are directly or indirectly regulated by vitamin A in the ocular surface epithelium, whereas the membrane-spanning mucin rMuc1 is not.

MUC4 and MUC5B transcripts are the prevalent mucin messenger ribonucleic acids of the human endocervix.

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.

Cloning of rat Muc5AC mucin gene: comparison of its structure and tissue distribution to that of human and mouse homologues.

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.

Mucin genes expressed by human female reproductive tract epithelia.

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.

Expression of secretory mucin genes by human conjunctival epithelia.

PURPOSE: To determine whether human conjunctival epithelium expresses any of the human mucin genes designated MUC2 through MUC7. METHOD: Northern blot analysis was performed using total RNA isolated from surgically removed conjunctival tissues. Complementary DNA or oligonucleotides to the tandem repeat region of each mucin gene were labeled and hybridized to conjunctival RNA. In situ hybridization also was performed to determine the distribution of mucin mRNA. RESULTS: Only MUC4 and MUC5 probes hybridized to conjunctival RNA by Northern blot analysis. Both probes bound in a polydispersed pattern, which is characteristic of mucin genes. Using in situ hybridization, MUC4 mRNA was detected in the cells of the stratified conjunctival epithelium, whereas MUC5 mRNA expression was limited to goblet cells MUC4 or MUC5 probes did not hybridize to sections of corneal epithelium. CONCLUSIONS: The mucins MUC4 and MUC5 are expressed by the human conjunctiva. These mucins may play an important role in forming the tear-film layer at the air and ocular surface epithelium interface.

Stratified squamous epithelia produce mucin-like glycoproteins.

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.

Human corneal and conjunctival epithelia express MUC1 mucin.

PURPOSE: To determine if human corneal and conjunctival epithelial synthesize MUC1 mucin, a membrane-spanning mucin present in a variety of simple epithelia. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was carried out to examine the expression of MUC1 mRNA by epithelial cells, using total cellular RNA prepared from cultured corneal epithelial cells and conjunctival epithelial cells stripped from the ocular surface with nitrocellulose filter paper. Northern blot analysis was performed to examine the transcription of MUC1 gene in cultured corneal epithelial cells. In situ hybridization histochemistry was performed to determine distribution of MUC1 mRNA in ocular surface epithelium. Human milk fat globule antibody (HMFG-1) and monoclonal antibody 139H2, which are specific for MUC1 core protein, were used in immunoblot analysis, immunohistochemistry, or both, to determine the presence and distribution of MUC1. RESULTS: MUC1 mRNA was detected in cultured corneal and ex vivo conjunctival epithelial cells by RT-PCR. Northern blot analysis showed production of two different sizes of transcripts in the cultured corneal epithelium. Expression of MUC1 mRNA was observed in all layers of corneal epithelium. By immunoblot analysis, HMFG-1 binding (> 200 kd) was detected in human cultured corneal epithelium, and its binding was enhanced by neuraminidase pretreatment. Immunohistochemically, HMFG-1 binding was observed along the apical membranes of corneal epithelium after neuraminidase pretreatment. In the conjunctiva, HMFG-1 and 139H2 binding were detected in the basal region and sporadically on the apical surface, but not in the goblet cells. CONCLUSIONS: The stratified epithelia of the cornea and conjunctiva synthesize MUC1 mucin. This transmembrane mucin may have a role in tear film spread and may prevent adhesion of foreign debris, cells, or pathogens to the ocular surface.

Human corneal and conjunctival epithelia produce a mucin-like glycoprotein for the apical surface.

PURPOSE: The authors have determined that the corneal and conjunctival epithelia of the rat produce a mucin-like glycoprotein at the apical surface of the epithelium. The purpose of this study was to determine if human ocular surface epithelium produces similar glycoproteins. METHODS: Because our initial attempts at production of monoclonal antibodies yielded blood type A-specific antibodies, corneal epithelia from blood type O donor eyes were used for the production of monoclonal antibodies. Screening of hybridoma products was accomplished by immunofluorescence microscopy of cryostat sections of blood type O donor eyes. The monoclonal antibody produced was used for immunofluorescence microscopy and immunoelectron microscopy to determine tissue and cellular distribution, respectively. Immunoblot analysis of SDS-PAGE-separated proteins from corneal epithelial tissue and from cultured human corneal epithelium was used to determine molecular weight range and epitope binding after periodate oxidation, N-glycanase, and O-glycanase treatment. RESULTS: A monoclonal antibody, designated H185, that binds to apical cell layers of human corneal, conjunctival, laryngeal, and vaginal epithelium was produced. The antibody binds to apical membranes of apical cells, particularly at the tips of microplicae. In subapical cells, the antibody binds to small cytoplasmic vesicles. Cultured human corneal epithelium produces H185 antigen. By immunoblot analysis, H185 binds a high molecular weight protein, > 205 kD, from corneal epithelium and cultured corneal epithelium. The protein band visualized by immunoblot analysis cannot be stained by Coomassie or silver stain on SDS-PAGE, but it does stain with Alcian blue followed by silver stain, indicating that the protein is highly glycosylated. H185 binding to blotted proteins is destroyed by sodium periodate treatment and O-glycanase incubation, suggesting that the epitope of H185 is an O-linked carbohydrate. CONCLUSION: Human corneal and conjunctival epithelia produce a molecule, similar in size, cellular localization, and distribution to the mucin-like glycoprotein of rat ocular surface epithelium. These data suggest that the entire ocular surface epithelium produces mucins for the tear film.

Redistribution of the hemidesmosome components alpha 6 beta 4 integrin and bullous pemphigoid antigens during epithelial wound healing.

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.

Integrins in the wounded and unwounded stratified squamous epithelium of the cornea.

PURPOSE: The authors determined the synthesis, cell surface expression, and localization of integrins in the rat corneal epithelium to detect whether any changes in integrins occur during epithelial migration in response to simple debridement wounding. METHODS: Immunoprecipitation analysis of extracts from either metabolically or surface-labeled rat epithelia was done to assess the synthesis and cell surface expression of integrins in the normal cornea. The localization of integrins was determined by indirect immunofluorescence of frozen sections obtained from control corneas and from those after debridement wounding. Immunoblotting of extracts from time course experiments was done on organ cultures of rat corneas after debridement to determine if any changes in the amounts of integrins occurred. The cell adhesion function of integrins on control and migrating epithelial cells was evaluated by cell adhesion assays. RESULTS: The data indicated that the corneal epithelium has a variety of distinct integrin subunits including beta 1, beta 4, beta 5, alpha 2, alpha 3, alpha 5, alpha 6, and alpha v. Although beta 1, beta 5, alpha 2, alpha 3, and alpha v were localized to sites of apparent cell-cell contact, alpha 5, alpha 6, and beta 4 were localized specifically to the basal membrane of the basal cells. Little change occurred in the localization of integrins in the migrating epithelial sheets. At 3, 6, 9, 12, 18, and 24 hr after wounding, the amount of the beta 1, beta 4, alpha 3, alpha 5, and alpha 6 integrin subunits (as measured by immunoblots) was not altered relative to that of the control corneas. Adhesion assays also showed no differences in adhesion of stationary versus migrating corneal epithelial cells to fibronectin and laminin. CONCLUSIONS: Integrin localization, production, and cell adhesion function in the stratified squamous epithelium of the cornea are not dramatically altered during epithelial cell migration over simple debridement wounds. Integrins in the cell membrane at sites of cell-cell interaction and as components of the hemidesmosomes in stationary epithelia may be available for rapid recruitment as epithelial cell migration proceeds.

Altered epithelial-basement membrane interactions in diabetic corneas.

Morphologic alterations of the extracellular matrix in diabetes include thickening of the basement membrane and, as evidenced in the corneal epithelium, a decrease in the area of the basal cell membrane occupied by hemidesmosomes. To determine if the abnormal basement membrane in diabetes has altered epithelial recognition of sites for hemidesmosome formation and if diabetic epithelium can recognize sites for hemidesmosome formation on normal basement membrane, we recombined epithelial sheets and corneal stromas with denuded basement membranes in vitro using diabetic and control corneas. In recombinations of diabetic epithelium on normal stromas, the area of the basal cell membrane occupied by hemidesmosomes was 10.7%, significantly less than control recombinations (19.5%). Similarly, in recombinations of normal epithelium on diabetic stromas, the value (8.6%) was significantly decreased from control. These data may indicate altered cell-extracellular matrix interactions in diabetes.

Characteristics of a glycoprotein in the ocular surface glycocalyx.

A monoclonal antibody has been produced that binds to the apical squames (flattened cells) of the rat ocular surface epithelium and to the goblet cells of the conjunctiva. Immunoelectron microscopic localization of the antigen indicates that in apical cells it is present along the apical-microplical membrane in the region of the glycocalyx. In subapical squames, the antigen is in cytoplasmic vesicles. In some goblet cells, the antigen is in the Golgi network, and in others, it is located primarily in the membrane of the mucous granules. SDS-PAGE and immunoblot analysis demonstrate that the molecular weight of the antigen is greater than 205 kD, and the electrophoretic band stains with Alcian blue followed by silver stain. Periodate oxidation of immunoblots and cryostat sections removes antibody binding. Neuraminidase treatment of cryostat sections does not remove antibody binding, whereas N-glycanase does. Taken together, these data indicate that the antigen recognized by the monoclonal antibody is a carbohydrate epitope on a high-molecular-weight, highly glycosylated glycoprotein in the glycocalyx of the ocular surface epithelium and goblet cell mucin granule membrane. The antigen appears to be stored within cytoplasmic vesicles and reaches the glycocalyx when cells differentiate to the apical-most position where the glycocalyx interfaces with the mucin layer of the tear film.

Reassembly of the corneal epithelial adhesion structures following human epikeratoplasty.

Ten epikeratoplasty lenticules removed after surgery were obtained for immunohistochemical and electron microscopic analysis to determine the pattern of re-formation of corneal epithelial adhesion structures. Antibodies to laminin and type VII collagen were used to determine the presence of basement membrane and anchoring fibrils, respectively. Electron micrographs were used to determine the percentage of basal cell membrane occupied by hemidesmosomes, the area of basal lamina per 100 microns of basal cell membrane, and the average maximum depth of penetration of anchoring fibrils into the stoma. Nine normal corneas served as controls. Compared with normal corneas (24.5% of basal cell membrane occupied by hemidesmosomes; 32.0 microns 2 basal lamina per 100 microns of basal cell membrane), lenticules removed for optical reasons had near-normal hemidemosomes as early as 10 weeks following surgery (mean, 20.3%). The area of basement membrane was reduced (16 microns 2 basal lamina per 100 microns of basement cell membrane). During the course of 2 to 3 years, irregularities and duplications of the basement membrane were noted. Compared with normal corneas, the two lenticules removed for persistent defects had a marked reduction of hemidesmosomes and basement membrane present under epithelium at 3 and 4 weeks (9.6% of basal cell membrane occupied by hemidesmosomes and 13.6 microns 2 basal lamina per 100 microns of basal cell membrane, and 5.4% of basal cell membrane occupied by hemidesmosomes and 7.2 microns 2 basal lamina per 100 microns of basal cell membrane, respectively.

Alpha 6 beta 4 integrin heterodimer is a component of hemidesmosomes.

Antisera that recognize the alpha 6 and beta 4 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 alpha 6-subunit-containing integrin heterodimer present is alpha 6 beta 4 and not alpha 6 beta 1. Here we extend previous studies to report that by immunofluorescence microscopy the alpha 6 integrin subunit colocalizes with bullous pemphigoid antigen and type VII 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 alpha 6 beta 4 is known. Once anchoring filament proteins are identified, it will be of interest to determine whether any bind to alpha 6 beta 4.

Localization of smooth muscle myosin-containing cells in the aqueous outflow pathway.

Cells containing smooth muscle myosin were localized in the human aqueous outflow pathway by immunohistochemical techniques. In the majority of eyes, immunoreactive cells were observed adjacent to the collector channels and slightly distal to the outer wall of Schlemm's canal. In a few eyes, smooth muscle myosin was localized to cells in the juxtacanalicular tissue and the trabecular meshwork. The immunoreactive cells from these regions may be true smooth muscle cells or pericytes, which can contain smooth muscle myosin. No obvious differences were observed in the pattern of distribution of smooth muscle myosin-containing cells in a comparison of age groups. In the majority of eyes, we observed an apparent direct insertion of the longitudinal portion of the ciliary muscle in the corneoscleral meshwork far internal to the scleral spur.

Decreased penetration of anchoring fibrils into the diabetic stroma. A morphometric analysis.

Morphometric measurements of epithelial adhesion structures were performed on electron micrographs of corneas of diabetic patients (ages 35 to 77 years) and controls of similar age (ages 21 to 79 years). Penetration of the anchoring fibrils from the deepest layer of the basal lamina into the stroma was significantly decreased in diabetics (0.41 +/- 0.02 microns) compared with controls of similar ages (0.65 +/- 0.03 microns). Significant thickening of the basal lamina in diabetics (50.1 +/- 7.6 microns 2/100 microns basal cell membrane) was noted compared with controls (32.0 +/- 6.3 microns 2/100 microns), but no significant differences were noted in the percentage of the basal cell membrane occupied by hemidesmosomes. Decreased penetration of the anchoring fibrils into the stroma may cause the loose adhesion between the stroma and basement membrane observed in diabetes.