Differentiation-dependent expression of keratins in human oral epithelia.

The polypeptide composition of epithelial keratins varies with the state of differentiation. The epithelia lining the human oral cavity show regional variations in their histology. In the present study, paired samples of nonkeratinized buccal epithelium and keratinized hard palate epithelium were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoblots with monoclonal antibodies AE1, AE2, and AE3, and results were correlated with immunofluorescence staining of tissue sections of the same samples. Keratins from hard palate (Mr 67K, 63-65K, 58K, 56.5K, 56K, 50K, 48K) and epidermis (Mr 67K, 63-65K, 58K, 56.5K, 50K) were similar to each other but distinctly different from those of buccal epithelium (major bands of Mr 52K and 59K, minor bands of 50K and 58K). The immunoblot analysis further indicated the similarity of hard palate and epidermal keratins, in contrast to those of buccal epithelium. Each oral tissue expressed keratins of the type I (AE1, acidic) subfamily and type II (AE3, basic) subfamily. In tissue sections, the predominant staining pattern for nonkeratinized buccal epithelium was: AE1, positive in the basal layer; AE2, negative; AE3, positive in all layers. In contrast, the staining pattern for keratinized palatal epithelium was: AE1 and AE2, positive in the suprabasal layers; AE3, positive in all layers. Strong suprabasal AE1 staining in palate may be related to the presence of the 48K keratin. Some buccal samples showed an alternate staining pattern of spotty suprabasal staining with AE1 and AE2 which was correlated with the expression of the 56.5K and 63-67K keratins, as well as filaggrin. These results suggest differentiation-specific expression of the keratins and show immunologically detectable variation in the apparently normal differentiation pattern of nonkeratinized buccal epithelium.

Immunologic localization of filaggrin in human oral epithelia and correlation with keratinization.

Localization of filaggrin, a human epithelial structural protein, was investigated by indirect-immunofluorescence microscopy of oral mucosa. Thirty specimens were tested, 10 each of palate, gingiva, and buccal mucosa. Orthokeratinized and parakeratinized specimens displayed immunofluorescence within the stratum corneum, stratum granulosum, and upper stratum spinosum. Within the stratum corneum, the reaction was diffuse. Within the stratum granulosum and spinosum, the reaction was in a granular pattern, in a perinuclear position. Several of the nonkeratinized specimens had a negative reaction; however, most displayed a very weak, scattered reaction in a granular pattern within the most superficial cells. The presence of filaggrin in keratinized palate was confirmed by immunoblot studies with the same antibody. Profilaggrin was detectable in representative nonkeratinized and parakeratinized oral tissues, as well as in keratinized palatal epithelium. The localization of filaggrin is consistent with its possible function as the interfilamentous matrix protein within cells of the stratum corneum, and with its derivation from a cross-reactive precursor protein stored in keratohyaline granules. A strong positive correlation was found between the degree of keratinization and the amount of immunofluorescence; therefore, filaggrin and related antigens may be useful and sensitive marker proteins for epithelial keratinization.

Localization of basement membrane components in mucous membrane pemphigoid.

We examined the distribution of laminin, type IV collagen, and fibronectin in subepithelial vesicles of oral mucous membrane pemphigoid (MMP). Indirect immunofluorescence staining of these macromolecules was performed on 10 frozen biopsy specimens of oral MMP. We found type IV collagen in the connective tissue floor and laminin in the epithelial roof of these lesions. Our results suggest that the inflammatory injury in oral MMP may disrupt the interaction of laminin with type IV collagen in the basement membrane zone.

Use of exfoliated cells to study tissue-specific levels of beta-carotene in humans.

This study was designed to explore the feasibility of using exfoliated cells to study beta-carotene incorporation into different epithelial tissues in humans. Exfoliated cells were collected from the oral cavities (by brushing the oral mucosa) and from the urogenital tracts (by centrifuging urine samples) of 36 females and basal levels of beta-carotene (without oral supplementation) were determined. Beta-carotene levels in cells from the two sites differed significantly, although a weak correlation was observed. As a second aspect of the study, 10 of these females were given oral supplementation with beta-carotene (90 mg twice weekly for 4 weeks). Beta-carotene levels increased significantly in both exfoliated urogenital tract (6.8-fold) and oral mucosa (5-fold) cells. However, the supplemented levels remained significantly different for the two types of cells. Beta-carotene levels did not change in individuals receiving a placebo treatment (n = 7). These studies suggest that exfoliated cells collected from different sites may be of value in quantifying tissue levels of beta-carotene during cancer intervention trials.

Beta-carotene levels in exfoliated human mucosa cells following its oral administration.

Beta-carotene levels of exfoliated oral mucosa cells can be increased severalfold by the oral administration of this provitamin. Beta-carotene was estimated by HPLC analysis in pronase-treated exfoliated cells obtained by brushing the entire oral mucosa with a toothbrush. A small percentage of individuals did not respond with an increase of beta-carotene in their mucosa cell in spite of a relatively large intake of the provitamin (360 mg in 4 days, or 2880 mg in 16 weeks, respectively). Levels of beta-carotene in the mucosa cells are affected by the concurrent administration of vitamin A: 0.27 ng beta-carotene per 10(6) cells in the placebo group, 1.79 ng following the intake of beta-carotene (180 mg/week for 16 weeks), and 4.29 ng after beta-carotene (180 mg/week for 16 weeks) plus vitamin A (100,000 IU/week for 16 weeks) consumption. The considerable variations in tissue levels of beta-carotene following its oral administration must be taken into account when cancer intervention trials using this agent are designed and evaluated.

Ultrastructural morphometry of collagen from lamina propria during experimental oral carcinogenesis and chronic inflammation.

Stereological point-counting methods were used to determine the volumetric alterations in collagen from the lamina propria immediately beneath the epithelial-connective tissue junction in hamster check-pouch mucosa treated with the chemical carcinogen DMBA. In addition, a non-neoplastic inflammatory control was evaluated in which a delayed hypersensitivity reaction was induced by the contact-sensitising agent DNCB. DMBA-treated tissues were assigned to histopathologically defined hyperplasia, dysplasia and carcinoma stages. The volume densities of collagen present in unit volume of extracellular lamina propria were found to decrease progressively and significantly in DMBA-treated tissues when compared with values obtained from normal untreated mucosa. Values from the inflammatory control were comparable with those from the dysplasia stage of carcinogenesis. The mechanisms responsible for these decreases in collagen volume density are unknown, but contributory factors might include collagen destruction by enzymes originating in either the epithelium or the cells of the inflammatory infiltrate, dilution of collagen produced by inflammatory oedema or alterations in the synthetic capabilities of fibroblasts.

Histochemical determination of copper, zinc, and iron in some benign and malignant tissues.

Histochemical tests for copper, iron, and zinc revealed their presence in the stratum germinativum of normal skin and skin from which an early melanoma and basal cell and squamous cell carcinomas originated. However, only copper was seen in invasive cells of basal and squamous cell carcinomas originating from skin. The normal oral mucous membrane was free of copper, iron, and zinc, but cells of invasive squamous carcinoma originating from the oral mucous membrane contained copper. The fluorescent brightening agent, applied as a counterstain, aided in the location of the specimen.

The substance P content of peripheral tissues in several mammals.

Levels of substance P immunoreactivity (SPI) were determined in several skin and mucosal areas, in parts of the sympathetic nervous system, the urinary, biliary and respiratory systems of cats, rabbits and guinea-pigs, and in various skin and mucosal areas of humans by radioimmunoassay. Salient findings are (1) The general distribution pattern of SPI in rabbits was similar to that in rodents. (2) The highest SPI tissue levels were found in the sympathetic nervous system, notably in guinea-pigs. (3) The guinea-pig also had the highest SPI levels in ureter, urinary bladder and bile duct. (4) The aorta, pulmonary artery and portal vein of the rabbit contained very low amounts of SPI, the concentration in the carotid sinus being several fold higher. (5) Skin SPI content was generally highest in the cat, especially in the hindpaw-pad, and lowest in abdominal and back skin. (6) SPI levels found in postmortem human skin and mucosal samples are comparable to those found in other mammals. The observations are discussed in view of the sensory innervation of the various tissues.

Presence and coexistence of chromogranin A and multiple neuropeptides in Merkel cells of mammalian oral mucosa.

By the use of light microscopic (LM) immunohistochemistry, Merkel cells of the mammalian oral mucosa have been examined for the presence and coexistence of some neuropeptides and of the neuroendocrine marker chromogranin A (CG-A). Peptide and CG-A immunophenotypes of oral Merkel cells were found to vary between species and to depend on the developmental stage, as exemplarily revealed in the pig. Oral Merkel cells of adult cat, mouse and pig but not those of adult guinea pig stained for calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI). Pairs of adjacent sections alternately stained for SP, CGRP, VIP, PHI or for CG-A revealed mutual coexistence of these peptides and of CG-A (if expressed) in individual Merkel cells of hard palate, gingiva and buccal mucosa. CG-A immunoreactivity was restricted to Merkel cells of cat and pig. In adult pig and cat, a much lower number of Merkel cells stained for CG-A and peptide expression was inverse. These results indicate that the chemical coding of Merkel cells in mammalian oral mucosa is much more complex than previously described and depends on the developmental stage.

The antiperinuclear factor. I. Clinical and serologic associations.

The antiperinuclear factor (APF) test raises two main problems: the unpredictability of the cells used as substrate and the difficulty in expressing the results. We propose that 10% of the cells have to be stained by a given serum in order for it to be considered positive. APF were found to be present in 76% of rheumatoid arthritis (RA) patients, 3% of healthy controls and occasionally in disease controls. The production of APF was significantly (p less than 0.01) related to the presence of rheumatoid factor in RA, and IgG antibody was predominant in the APF test.

The antiperinuclear factor. II. Variability of the perinuclear antigen.

Since the antiperinuclear factor (APF) test on human buccal cells is rather unpredictable, we have investigated the possible factors determining the expression of appropriate antigens by the cells. We failed to find any relationship of the expression of perinuclear antigens to the donor's smoking habits, the degree of contamination with saprophytic bacteria, the presence or absence of blood group substances in saliva, or the titers of serum antibodies to Epstein-Barr virus. Family studies were also performed to further elucidate a genetic predisposition to the expression of the APF antigen.

Trauma affects cyclic AMP and DNA levels at injured and non-injured distal oral mucosal sites.

Experiments in rats were conducted to test the hypothesis that gingival trauma affects cyclic AMP and DNA levels at the gingival wound, and non-injured distal (gingival, palatal) sites. Cyclic AMP and DNA levels rose and fell in a cyclic fashion during the time (0.5-24 h) periods analyzed. Significant increases in cAMP levels occurred at 8 and 20 h and at 8 and 16 h, respectively, at the wound and non-injured palatal site, peripheral to the wound. Similar increases (not significant) in cAMP levels were also noted at the non-injured gingival contralateral site at the same time intervals. DNA distributions were found to be significantly greater 10 and 16 h after injury at the gingival wound, and distal non-injured gingival and palatal sites.

Comparative investigation of keratin-filaments in normal tissues and tumours of skin, oral mucosa, salivary glands and thymus.

Antibodies against different fractions of keratins can be helpful in various fields of special pathology. Antibodies against "small" and "large" keratins permit to evaluate epithelial maturation in skin and oral mucosa. In addition, disturbances of keratinization during inflammatory processes and malignant transformation can be analyzed. The main application of antibodies against the entire fractions of keratins is the detection of the epithelial nature of a neoplasm. By this tool, particular problems in surgical pathology concerning differential diagnosis can be handled in an easier way. Among the different tissues and their neoplasms, examples of the analysis of thymus tumours and salivary gland tumours are presented. Immunoreactivity with keratin antibodies depends crucially on tissue processing. In the normal diagnostic procedure, good results are regularly obtained if cryostat or Bouin-fixed paraffin-embedded sections are used.

A biochemical study of glycosaminoglycans in the palatal rugae of the monkey (Macaca fascicularis).

Glycosaminoglycans (GAG) were extracted from the connective tissue of the palatal rugae, separated by electrophoresis and compared with the results obtained for the remaining palatal mucosal and gingival connective tissues. The GAG content of the rugae (3.01 mg/g defatted dry weight) was higher than in the remaining palatal mucosa (2.33 mg/g defatted dry weight) or gingiva (1.68 mg/g defatted dry weight). Dermatan sulphate was the predominant GAG in both the palatal rugae (48% of total GAG) and the remaining palatal mucosa (50%) followed by hyaluronic acid (33 and 31% respectively). The results do not support previous histochemical observations in which the rugae appeared to be rich in hyaluronic acid.

The lipid composition of porcine epidermis and oral epithelium.

The permeability barrier of mammalian skin has been analysed in terms of its lipid composition whereas that of the oral mucosa is uncharacterized. As there are differences in permeability between different regions of the oral mucosa, and between the oral mucosa and skin, there may be corresponding differences in the nature of the barrier layer. Lipids were identified by thin-layer chromatography after solvent extraction of separated epithelium; their localization was determined in histological sections using standard histochemical stains. Keratinized oral epithelium and epidermis showed a similar pattern of lipid distribution, the majority of neutral lipids being ceramides, localized around the cells of the stratum corneum. The non-keratinized epithelium contained few neutral lipids but polar lipids, particularly cholesterol sulphate and glucosylceramides, were clearly evident. Histochemical staining suggested that these were localized in intercellular regions throughout the epithelium. The differences in the types and distribution of lipids accord well with known permeability differences, which show that keratinized oral epithelium and epidermis have a similar impermeability to water and that non-keratinized regions have greater permeability.

Histological localization of myxoid tissue in normal human palatal mucosa and its glycosaminoglycans.

Eighteen specimens of palatal mucosa were taken from 17 human subjects. Paraffin-wax sections were stained by routine methods and with various techniques to demonstrate glycosaminoglycans (GAG). In some sections, GAG were removed by selective degradative procedures before staining. Beneath all rugae, there were myxoid areas varying in size and marginal definition. Collagen fibres were few; elastic and reticulin fibres were numerous in a minority of sections. Alcianophilia at pH 2.5, preventable by streptomyces hyaluronidase digestion, suggested the presence of hyaluronic acid beneath the rugae. Alcian-blue staining at pH 1.0 and with the critical electrolyte concentration method using 0.5 M MgCl2 did not distinguish the myxoid tissue from the surrounding connective tissue and could be prevented by digestion with testicular hyaluronidase or chondroitinase ABC. Chondroitin sulphate and, or dermatan sulphate thus may be present but were not localized to the myxoid tissue. This unusual zone of loose connective tissue may act as a physical buffer resisting the local effects of high loads by allowing reversible extrusion of the water.

Physico-chemical characteristics of mucus glycoproteins and lipids of the human oral mucosal mucus coat in relation to caries susceptibility.

Mucus coat was isolated from oral epithelial surfaces of caries-resistant and caries-susceptible subjects, analysed for content and composition of lipids and mucus glycoproteins, and evaluated for physico-chemical characteristics. The mucus coat from caries-resistant subjects had a protein content similar to that of the caries-susceptible group but a higher content of carbohydrate and a lower content of lipids and covalently bound fatty acid. The carbohydrate component was mainly mucus glycoprotein, which accounted for 28.4% of the dry weight of caries-resistant mucus and 25.3% of caries-susceptible mucus. By chromatographic analysis on Bio-Gel A-50, both types of preparations had high (Mr approximately 2000 kdalton) and low (Mr approximately 300 kdalton) molecular-weight mucus glycoproteins. In the caries-susceptible mucus coat these two glycoproteins were in similar proportions, whereas the low molecular-weight glycoprotein predominated in caries-resistant mucus. In both preparations, the high molecular-weight glycoprotein was characterized by a high content of carbohydrates, associated lipids and covalently bound fatty acids, whereas the low molecular-weight glycoprotein was richer in protein and contained lesser amounts of associated and covalently bound lipids. Although the low molecular-weight glycoprotein showed only minor compositional differences with caries status, the high molecular-weight glycoprotein of the caries-resistant group had a 2.5 times lower content of covalently bound fatty acid, a 1.3 times lower content of associated lipids and contained 1.2 times more sulphate and sialic acid then that of the caries-susceptible group.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative biochemical and immunological analysis of cytokeratin patterns in the oral epithelium of the miniature pig and man.

In man, cytokeratin constitutes a family of 19 polypeptides that show different but distinct distribution patterns in the various epithelia. Changes in these patterns may occur during epithelial development and differentiation. The cytokeratin patterns in the oral mucosa of the miniature pig, an animal used in studies of wound healing, were investigated. Surgical biopsies were obtained from the gingiva, hard palate and alveolar mucosa of both man and pig. The cytokeratins were analysed by immunofluorescence, two-dimensional gel electrophoresis and by immunoblotting. Nine monoclonal antibodies were used to identify the different cytokeratin polypeptides in cryostat sections. Two-dimensional gel electrophoresis showed that pig oral mucosa contains at least 10 different polypeptides, five of the acidic type I and five of the basic type II cytokeratins. These were different from the human cytokeratin polypeptides and accordingly were designated P1-P10, according to their molecular weight and isoelectric mobility. Their molecular weight varied between 48 and 69 kdalton and the pHi varied between 5 and 7.3. Immunoblotting showed the monoclonal antibody Ks 13.1 (anticytokeratins Nos 13 and 14) to cross-react with the pig polypeptides P10 and P8. Immunolocalization showed that all the antibodies cross-reacted with the pig tissue except Ks 19.1 (anticytokeratin No. 19). It was possible to differentiate between pig alveolar mucosa, which expressed only P3, P4, P5, P8 and P10, and the gingival and hard palatal mucosae, which expressed all 10 polypeptides except P5. This distinction was made by antibody 6B10 (anticytokeratin No. 4), which reacted only with alveolar mucosa; antibody Ks 13.1, which strongly reacted with uncornified mucosa but weakly with cornified mucosa (gingiva and palate); and any of RKSE60, Kk 8.60 or EE21.6 (anticytokeratin No. 10, anticytokeratins Nos 10 and 11 and anticytokeratins Nos 1, 2, 10 and 11, respectively), which reacted strongly with cornified mucosa but weakly, if at all, with uncornified mucosa. These findings provide a baseline for studies on epithelial differentiation in the miniature pig such as in wound healing.

Biochemistry of glycosaminoglycans in the skin and oral mucosa of the rat.

Glycosaminoglycans (GAG) were extracted by digestion with papain followed by ultrafiltration and separated by cellulose-acetate electrophoresis and by chromatography of their cetyl-pyridinium complexes on cellulose microcolumns. The uronic-acid content of the tissues ranged from 0.8 to 2.4 mg/g of dry defatted tissue. Hyaluronic acid and dermatan sulphate were found in all tissues with chondroitin-4-sulphate also in skin, palatal mucosa and gingiva. There was 3-fold more hyaluronic acid in palatal mucosa than in any other tissue; it was concentrated in the antemolar rugae. A substance of presumptive salivary origin staining with alcian blue was found in cheek and floor of mouth mucosa. It migrated differently from reference GAG by electrophoresis and was not degraded by testicular hyaluronidase.

The immunohistochemical distribution of collagens type IV, V, VI and of laminin in the human oral mucosa.

The distribution of collagens type V (form AB2) and VI was investigated on cryostat sections of normal human oral mucosa by indirect immunofluorescence. For comparison, antibodies to fragments of type IV collagen and laminin were also used to delineate basement membrane containing structures. All antibodies used were raised against human proteins. Type V collagen appeared as a microfibrillar structure throughout the interstitium, apparently touching but not being present within epithelial or vascular basement membranes. Microfibrils in blood vessel walls were limited to the intimal layer. Pericellular areas were not specifically stained. Type VI collagen appeared as an almost amorphous stromal structure becoming more prominent and more fibrillar in the upper connective tissue papillae. Intense staining was observed in the media of blood vessels and around smooth muscle cells. A possible role of type VI collagen in tissue stabilization may be expected from this ubiquitous and abundant distribution. The findings identify types V and VI collagen as important structures in the oral mucosa and serve as a basis for understanding morbid changes.