Surfactant-induced alterations of permeability of rabbit oral mucosa in vitro

The permeability of rabbit oral mucosa to eight nonelectrolytes was measured in vitro in the absence and in the presence of 0.025, 0.1, and 1.0% concentrations of anionic, cationic, and nonionic surfactants. The anionic surfactant sodium lauryl sulfate and the cationic surfactants cetyltrimethylammonium bromide and cetylpyridinium chloride caused greater increases in permeability than polysorbate 80, a nonionic surfactant. The increases in permeability brought about by the surfactants were concentration dependent.     

Immunobiology of the oral mucosa in the mouse.

The incidence of immunoglobulin (Ig)-synthesizing cells, Thy 1-positive cells and macrophages in the murine oral mucosa was investigated. Immunofluorescence studies of frozen tissue sections showed that IgA-, IgM- and IgG-containing cells and Thy 1-bearing cells were closely associated with the minor salivary glands. A quantitative analysis was then undertaken using single cell suspensions of the tissue. After mechanical disruption or enzymatic digestion of the mucosa, lymphoid cells were recovered almost exclusively from the mucosa of the posterior soft palate where we observed a dense accumulation of minor salivary glands. Thy 1-bearing cells were found at a higher frequency (25% of recovered cells) than membrane Ig-positive B lymphocytes (6-7%) in these suspensions. Cytoplasmic Ig+ cells accounted for about 6% of recovered cells, whereas plaque-forming cells (Ig-secreting cells) occurred at the same frequency as in the spleen (0.1%). Plasma cells of the IgA and IgM isotypes predominated over IgG-secreting cells (A:M:G ratio = 1:1:0.2); this distribution did not directly correlate with the isotype distribution of salivary Igs (A:M:G ratio = 1:0.003:0.07). In addition, about 10-14% of the cells in our preparations were esterase-positive mononuclear cells. Present data indicate that the murine oral mucosa contains both effector and regulatory cells required for the development and expression of local antibody responses.     

Merkel cells in the oral mucosa

Ninety-eight consecutive surgical biopsies of oral mucosa from 96 patients were evaluated immunohistochemically with an anti-cytokeratin 20 (CK 20) anti-body to evidence Merkel cells (MC). Fifteen cases, showing the highest number of MC, were additionally studied with chromogranin A, S-100 protein, neuro filaments, epithelial membrane antigen, and double immunostaining for CK 20 and Ki67 antibodies to evaluate MC proliferation. Electron microscopy was performed in 2 cases. MC were observed in 58 cases. The highest number of MC was found in the gingival, buccal, and palate mucosa, especially in chronically damaged oral mucosa (lichen and chronic aspecific inflammation) as well as in the mucosa overlying tumors rather than in normal or acute inflammation. MC were not observed in dysplastic or neoplastic epithelium. MC showed evidence of proliferation, as demonstrated by Ki67 positivity, in 3 cases. In conclusion, MC appear to play a role in the reparative processes of oral mucosa.     

Canine ganglioneuroblastoma in the oral mucosa

A ganglioneuroblastoma of the oral cavity in a dog was examined histologically and immunohistochemically. This rare neoplasm was considered to be derived from ectopic neural crest cells. This is the first report of a canine ectopic ganglioneuroblastoma located in the oral mucosa.     

Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue.

Two alternative integrins involved in mucosal homing (alpha 4 beta 7) or epithelial retention (alpha E beta 7) of lymphocytes were examined in the human gut. The distribution of the beta 7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by alpha E beta 7 + cells but weakly by alpha 4 beta 7 + cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T-cell receptor (TCR) gamma/delta +, and CD3- cells, expressed alpha E beta 7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B-lineage cells (CD19+) and macrophages (CD68+) were negative. In gut-associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (< 5%) cells were positive for alpha E beta 7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30-50%) expressed alpha 4 beta 7 (mAb Act-1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of alpha 4 beta 7 as in the lamina propria whereas relatively few B lymphocytes (< 50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for alpha 4 and the former three subsets as well as appendix CD8+ cells also for beta 7 while only lamina propria CD8+ cells had mRNA for alpha E. Together, the results suggested that alpha E beta 7 and alpha 4 beta 7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly alpha 4 beta 7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because alpha E beta 7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of beta 7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle-associated epithelium of GALT expressed alpha E beta 7, perhaps reflecting epithelial interaction during primary immune responses.     

Properties of high-threshold mechanoreceptors in the goat oral mucosa. II. Dynamic and static reactivity in carrageenan-inflamed mucosa.

1. We have previously described two classes of high-threshold mechanoreceptors (HTMs) of the oral mucosa of the goat. Mechanonociceptors (MNs) had very high thresholds (16-300 g) and were poor transducers of pressure. Intense pressure receptors (IPRs) had thresholds from 2 to 16 g and were good transducers of pressure. After carrageenan inflammation (CI) we observed mechanical sensitization in both classes of HTMs. The characteristics were as follows. 2. Sensitization of MNs was manifested as qualitative shifts in the capacity to encode intense pressure. MNs that were unable to code intensity before CI acquired pressure coding properties after treatment with carrageenan. Qualitative shifts in coding capacity were suggested by the greater proportion of MNs coding in preinflamed (10 of 14) compared with noninflamed tissue (8 of 25 cases). Improved afferent reactivity was directly observed, in additional experiments, in which MNs were characterized in normal tissue before the injection of carrageenan into the mucosa. In five of six cases, either qualitative or quantitative improvements were observed. In control experiments, improved reactivity was observed in one of six cases. 3. Sensitization in IPRs was manifested as both qualitative and quantitative improvement in intensity coding properties. Power functions fit to individual IPRs indicated sensitization for most mucosal afferents after inflammation. Carrageenan induced decreases in the mean response interval, pressure-frequency threshold (PFT), and pressure-frequency asymptote (PFA), and decreases in variability of functions fit to units sampled from preinflamed and noninflamed tissue (n = 23). Experiments were also conducted in which units were characterized before and after carrageenan treatment. In 7 of 10 cases, injection of carrageenan into the oral mucosal led to improved dynamic and/or static reactivity. Injection of vehicle led to changes in reactivity in one of five cases. 4. Carrageenan induced decreases in activation thresholds only when afferent receptive fields fell into restricted tissue zones. Large shifts in activation thresholds were observed for five of five (3 MNs and 2 IPRs) units in the sulcal zone of the incisal papilla (IP). In contrast, activation thresholds increased or remained the same in 11 of 12 units (10 IPRs and 3 MNs) with receptive fields in the medial or ventral tissue zones. Changes in activation threshold evolved slowly after carrageenan injection and required 1-2 h to develop. Rapid changes in activation threshold were also observed in a limited number of cases (3). These threshold shifts were unrelated to carrageenan inflammation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructures of mechanoreceptors in the oral mucosa

The present review describes the fine structures of lamellated mechanoreceptive corpuscles, Merkel cell-neurite complexes and free nerve endings in the oral mucosae of mammals, with special attention to axon terminals and lamellar cells. The mechanoreceptive nerve endings of the oral mucosa were studied using histochemistry, immunohistochemistry and transmission electron microscopy techniques. The organized mechanoreceptive corpuscles are present in the mucosae of gingiva, cheek, tongue and soft and hard palate. They are elongated or globular in shape, being located in the connective tissue papillae. The capsule is composed of several layers of cytoplasmic extensions of perineural cells. Numerous bundles of collagen fibers are noted at the periphery of the corpuscle. The lamellated corpuscles are surrounded by several layers of superimposed flattened capsular cell processes. The interlamellar spaces are 0.2-0.4 micron in width and filled with thin fibrillar collagen fibers embedded in the amorphous substance. The lamellar cells contain rich microtubules and are characterized by the presence of caveolae on the surface plasma membrane. The terminal axon contains an abundance of mitochondria and small clear vesicles (20-50 nm in diameter). There are neurofilaments in the center of the axon terminal. Intermediate-type junctions are seen between the adjacent lamellar cells and between the axon and adjacent lamellae. The free nerve endings are found in the subepithelial regions, very close to the basal laminae of mucosal epithelium. They are surrounded by a thin cytoplasm of Schwann cells. Sometimes Schwann cell basal larinae become multilayered. Merkel cells are present within the basal layer of mucosal epithelium and contain characteristic electron-dense granules that are located almost exclusively at the side of cytoplasm in contact with axon terminals. Intermediate-type junctions are noted between axon terminals and Merkel cells.     

Oxygenation of frog gastric mucosa in vitro.

We have recently shown that 5% CO2/95% O2 in the serosal bathing solution, with 100% O2 in the mucosal solution, results in CO2-diffusion limitation of acid secretion in bullfrog gastric mucosa. Changing to 10% CO2/90% 02 on both surfaces doubles the acid secretory rate. We calculate that, were the rate of oxygen consumption to increase significantly as a result of secretory stimulation, the tissue would now be oxygen limited. This prediction is tested by raising the P02 by increasing the total pressure in a hyperbaric chamber. Since no change in acid secretory rate or potential difference was observed upon changing from PO2 = 0.9 to PO2 = 1.9 atm, we conclude that the tissue is not O2 limited at normal pressure. Decreasing PO2 below 0.9 atm, by contrast, decreases the acid secretory rate and raises both PD and resistance. We infer that the rate of oxygen consumption did not rise significantly when acid secretion was increased by supplying sufficient CO2.     

Growth of crypt cell nodules in duodenal mucosa in man during organ culture in vitro.

Culture of peroral biopsy specimens from duodenal mucosa in vitro for 22 hours using a basic culture medium resulted in the formation of crypt cell nodules. The addition of collagen and serotonin to the culture medium increased the occurrence of the nodules and, invariably, their size. The nodules were situated on the pericryptal basement membrane and contained cells that resembled columnar cells, goblet cells, and endocrine cells. The overall nodular structure suggested that they had been formed by the stimulation of stem cell division at the crypt base, but the factors responsible for this have not as yet been identified. The growth of these nodules may offer opportunities for studying stem cell division and differentiation in small intestinal mucosa in man.     

Dendritic cells of the oral mucosa and the induction of oral tolerance. A local affair.

The oral mucosa is an important site to induce immunological tolerance to protein antigens. Previously we have established that oral contacts to allergen can lead to systemic tolerance in both humans and experimental animals. Because of the importance of tolerance induction as a possible way to modulate allergic reactivity, we wished to study the mechanisms involved in efficient tolerance induction via the oral mucosa. Dendritic Langerhans' cells in both skin and oral epithelium are the first cells to encounter antigen. Therefore, possible functional differences between Langerhans' cells from skin and oral mucosa were studied by migration and transfer experiments. It was found that dendritic cells derived from the oral mucosa were not able to transfer tolerance, but that they acted as antigen-presenting cells in sensu stricto irrespective of the source and route of antigen administration.     

Dendritic cells of the oral mucosa

The oral cavity contains distinct mucosal surfaces, each with its own unique distribution of dendritic cell (DC) subsets. In addition to tissue-specific properties, such organization might confer differential immune outcomes guided by tissue-resident DCs, which translate in the lymph node into an overall immune response. This process is further complicated by continual exposure and colonization of the oral cavity with enormous numbers of diverse microbes, some of which might induce destructive immunity. As a central cell type constantly monitoring changes in oral microbiota and orchestrating T-cell function, oral DCs are of major importance in deciding whether to induce immunity or tolerance. In this review, an overview of the phenotype and distribution of DCs in the oral mucosa is provided. In addition, the role of the various oral DC subsets in inducing immunity vs. tolerance, as well as their involvement in several oral pathologies is discussed.     

Expression of extracellular acid proteinase by proteolytic Candida spp. during experimental infection of oral mucosa.

We traced an acid proteinase from Candida spp. in the initial stages of the pathogenesis of the mycosis. On infection of human buccal mucosa, proteinase antigens were detected by immuno-scanning electron microscopy on the surface of adhering blastoconidia and invading filamentous cells of C. albicans serotype A. Proteinase antigens were also present on blastoconidia of C. albicans serotype B, but were missing on filamentous cells of this serotype. Proteolytic isolates of C. tropicalis behaved like C. albicans serotype A. An isolate of C. parapsilosis did not express the proteinase antigen under conditions of this study. After infection of mucosa, culture medium of C. albicans or C. tropicalis showed a time-dependent accumulation of acid proteolytic activity, indicating that the visualized antigens represent active proteinase. No such activity was detected in the medium of C. parapsilosis. Preliminary experiments with the proteinase inhibitor pepstatin A revealed an 89% reduction of mucosal adherence of C. albicans (serotype A). These results suggest that Candida proteinase is involved in fungal attachment. The pattern of adherence reflects the differential expression of secretory proteinase by different candidal strains.     

Alterations in expression of basement membrane proteins during tumour progression in oral mucosa

Expression of three basement membrane proteins—collagen IV, laminin and fibronectin—was studied in normal, hyperplastic, dysplastic and neoplastic conditions of the oral mucosa using immunohistochemistry. Collagen IV and laminin exhibited similar staining patterns, while fibronectin showed a different pattern of expression. The expression of collagen IV and laminin also demonstrated an inverse correlation between staining intensity, thickness and basement membrane continuity in various stages of tumour progression. In contrast to the continuous and intense staining of basement membrane in normal oral mucosa with collagen IV and laminin antibodies, severe dysplasia and carcinoma exhibited discontinuous, thin and weakly stained basement membrane. The expression of fibronectin showed a direct correlation with extent of tumour progression. In normal mucosa, expression of fibronectin was almost absent, whereas in carcinoma intense expression of fibronectin was evident in the basment membrane and basal cells. These results emphasize the value of basement membrane proteins as biological markers for assessing oral carcinogenesis.     

Racial variation in the O-acetylation phenotype of human colonic mucosa

O-acetylated and non-O-acetylated sialoglycoproteins can be distinguished by the mPAS (mild periodic acid-Schiff) histochemical technique. Individual adults show one of three different patterns of staining of large intestinal mucosa: uniformly mPAS-positive, uniformly mPAS-negative, or mPAS-negative with scattered mPAS-positive crypts. To test our hypothesis that these variations are the results of a single autosomal gene (oat) polymorphism, we have studied the frequency of the three patterns of staining in a total of 435 adult colon specimens from six geographically separate populations: British, South African blacks, Icelanders, Japanese, Hong Kong Chinese, and Bahrainis. The distribution of the three types of staining fell into two groups. In Japanese and Chinese, uniformly mPAS-positive cases were much more frequent than uniformly mPAS-negative cases; this distribution differed significantly (X², P<0·001) from that in non-Sino-Japanese, where the uniformly mPAS-positive phenotype was much less frequently found than the uniformly mPAS-negative phenotype. In neither of the groups did the frequency of the three phenotypes differ significantly from that predicted for a single gene polymorphism by the Hardy-Weinberg law. The variation in staining patterns between populations is consistent with variation in frequency of a single polymorphic autosomal gene (oat) controlling O-acetylation of sialic acid, probably by an O-acetyl transferase enzyme. Loss of function mutation in the high acetylator gene (oat^a) in a colonic crypt stem cell in heterozygous individuals would account for the scattered discordant crypts. Gene frequencies for a variety of enzymes differ between the Sino-Japanese and non-Sino-Japanese races. This newly described gene polymorphism may be related to differential susceptibility to organisms binding specifically to either O-acetylated or non-O-acetylated sialoglycoproteins, or to differential enteric colonization by bacterial flora that vary in their relative secretion of sialidases and sialate O-acetyl esterase.     

Role of integrins during fertilization in mammals

Fertilization includes sperm-oocyte recognition, adhesion, binding, fusion and egg activation. Integrin receptors, which are adhesion molecules, are expressed on sea urchin, mouse, hamster and human unfertilized oocytes. Potential sperm ligands have been identified. A role for integrins during fertilization is supported by inhibition of sperm-egg adhesion and/or fusion by means of anti-integrin monoclonal antibodies, Arg-Gly-Asp (RGD) or disintegrin-like peptides. In several cell-cell interactions, such as lymphocyte activation, viral fusion, bacterial infection or macrophage phagocytosis, integrins act as co-receptors after activation and, by clustering in a multimolecular complex, are able to transduce signals through cytoskeletal proteins and adaptor kinases. Experimental data suggest that they may act in a similar way during fertilization and may participate to initiation and/or propagation of the calcium signal via stimulation of phospholipase C gamma and inositol trisphosphate production.     

Interepithelial cells of the oral mucosa in mice

Summary Non-epithelial mesenchymal and neuroectodermal cells occur between the keratinocytes in the stratified squamous epithelium of the oral mucosa. These cells cannot be classified adequately by light microscopy. In the present study the oral mucosa of the lip, cheek and tongue of 50 mice were studied by light and electron microscopy. 3,025 mononuclear interepithelial cells were documented and analysed.Monocytogenic macrophages, plasma cells and mast cells were not found interepithelially and cannot be regarded as a regular constituent of the epithelium. Only a few neuroectodermal cells — in mice these are exclusively Merkel cells, with no melanocytes — were localized in the epithelium. The majority of the interepithelial cell population is made up of lymphocytes (22.8%) and Langerhans cells (56.8%). They are an integral constituent of the epithelium. Lymphocytes with rounded and indented nuclei can be identified. The larger and dendritic Langerhans cells are a specific cell of squamous epithelium and also occur in the oral mucosa. Not all cells which feature the cytological characteristics of Langerhans cells contain Langerhans or Birbeck granules. Accordingly these granules cannot be considered an exclusive identification characteristic. Two types of Langerhans cells can be differentiated. 80.9% have the more or less typical appearance known from the epidermis and were termed macrophagocytoid Langerhans cells. The nuclei are irregularly indented and moderately heterochromatic. 19.1% possessed conspicuous large, spherical, euchromatic nuclei and an electron-lucent cytoplasm. These were termed reticuloid Langerhans cells. About 20% of the interepithelial cell population could not be identified, neither as typical lymphocytes nor as Langerhans cells. These were small to medium sized cells with deeply indented cerebriform strongly heterochromatic nuclei. They are similar to the Sézary cells or mycosis fungoides cells of epidermotropic human T-cell lymphomas. The lymphocytic nature of these cells has been confirmed. It seems likely that differentiation of lymphocytes to cerebriform cells occurs within the epithelium. It is further discussed whether cerebriform cells are precursors of Langerhans cells, a conclusion suggested morphologically by transitional forms. This would imply that Langerhans cells originate from lymphocytes, and that the cerebriform cell is an intermediate step of differentiation. The microenvironment of the squamous epithelium may play a role in the process of differentiation, which could explain the epitheliotropy of lymphocytes. The possibility is considered that Langerhans cells and interdigitating reticulum cells of the T-cell area of lymph nodes are identical. The close functional cooperation of Langerhans cells, lymphocytes, and interdigitating reticulum cells in immunological defenses against external antigens is discussed.The authors wish to express their sincere appreciation to Miss P. Starck and Miss I. Brandt for invaluable technical assistance in this project.     

Increased levels of alpha6 integrins are associated with the metastatic phenotype of human breast cancer cells.

Integrins play an important role in interactions between cells and the extracellular matrix, and thus have a potential role in metastasis. Expression levels of alpha6, beta1 and beta4 integrin sub-units were measured in a panel of human breast cancer cell lines by RT/PCR, immunoprecipitation and flow cytometry. All the lines expressed alpha6, with the highest levels in the MDA-MB-231 and MDA-MB-435 cells. These grew the most aggressively and were metastastic in nude mice. Low levels of alpha6 protein were measured in breast cancer cells that were poorly tumorigenic and non-metastatic in nude mice, and there was an inverse relationship between ER and alpha6 expression. RT/PCR revealed that all lines expressed the 2 isoforms of alpha6, with the alpha6A isoform generally more abundant than alpha6B isoform. Clones of MDA-MB-435 were isolated by sterile sorting for cells with high or low alpha6 expression, and two variants established from metastases in nude mice were found to differ in alpha6 expression. When injected into nude mice. the alpha6-high variants produced significantly more lung metastases than the alpha6-low variants. beta1was abundant in all lines, while beta4 was not detected in MDA-MB- 134 cells, and in the MDA-MB-435 cells an alternately spliced variant of beta4 was identified. Sequencing of the alternate variant revealed a novel sequence from a splicing event in the cytoplasmic tail of beta4. None of the cells with this variant mRNA expressed detectable levels of beta4 protein. Our results suggest that high alpha6 expression in human breast cancer cells is associated with tumorigenicity and metastatic potential.