Establishment and first characterization of a sublingual epithelial and immune cell co-culture model.

We describe here the establishment and first characterization of a co-culture model of human epithelial sublingual cells (HO-1-u-1 cell line) and human dendritic cells derived from human peripheral blood monocytes (PBMC). Cell culture conditions for HO-1-u-1 cells were optimized. First characterization of phenotypic features by electron microscopy and fluorescence imaging revealed resemblance to sublingual tissue specimen from healthy donors. Successful co-culturing of epithelial and dendritic cells (DCs) was confirmed by confocal laser scanning microscopy. Stimulation of HO-1-u1 cells alone and the epithelial/DC co-culture by incubation with liposomes, virosomes and influenza virus lead reproducibly to the release of inflammatory cytokine GM-CSF. This co-culture model may be suitable for elucidation of mechanisms involved in the immune response at the sublingual epithelium as well as for the evaluation of novel topical vaccines, potentially replacing cumbersome ex vivo and in vivo methods currently in place.

Mapping of the lingual immune system reveals the presence of both regulatory and effector CD4+ T cells.

BACKGROUND: Sublingual immunotherapy (SLIT) is safe and reduces both symptoms and medication requirements in patients with type I respiratory allergies. Nonetheless, immune mechanisms underlying SLIT need to be further documented. OBJECTIVE: A detailed characterization of the lingual immune system was undertaken in mice, to investigate the presence of tolerogenic and pro-inflammatory mechanisms. METHODS: Immune cells were characterized in lingual tissues from BALB/c mice using immunohistology and flow cytometry. Resident CD4(+) T cells were sorted and toll-like receptor (TLR) expression profiles as well as functional characterization were assessed by RT-PCR, T cell suppressive assays and cytokine gene expression, respectively. RESULTS: Eosinophils and mast cells were only detected in submucosal tissues. No NK, NK-T, gamma/delta, CD8(+) T cells, nor B-lymphocytes were detected. Potential antigen presenting cells include various subsets of dendritic cells (CD207(+) Langerhans cells, CD11b(+)CD11c(+) myeloid cells and 120G8(+) plasmacytoid DCs) together with F4/80(+) macrophages. Noteworthy, both CD103(-) and CD103(+) CD4(+) T cells expressing TLR2 and TLR4 receptors are present along the lamina propria, in vicinity of myeloid CD11b(+)CD11c(+/-) dendritic cells. Such resident lingual CD4(+) T lymphocytes comprise both suppressive T cells as well as cells with memory/effector functions (i.e. expressing IFN gamma, IL4, IL10 and IL17 genes following stimulation), irrespective of the presence of the mucosal addressing marker CD103. CONCLUSION: The sublingual route is pertinent to induce antigen-specific tolerance, due to (i) limited numbers of pro-inflammatory cells, rather located in submucosal tissues, (ii) co-localization of APCs and resident CD4(+) T cells with regulatory functions. Since the oral immune system can also elicit pro-inflammatory effector responses, the cytokine milieu in which allergens are presented by sublingual APCs needs to be controlled during immunotherapy (e.g. with adjuvants) in order to favour tolerance over inflammation.

Development of cellular magnesium nano-analysis in treatment of clinical magnesium deficiency.

A novel technique, using energy dispersive X-ray microanalysis (EXAtm), for noninvasive intracellular (i.c.) measurement of magnesium [Mg2+]i has now been accomplished and proven to be a valuable tool in multiple aspects of normal as well as pathological magnesium metabolism. Since only 1% of total body Mg2+ is found in the intravascular space, serum levels of Mg2+ give little information about a patient's overall Mg2+ status with respect to this essential mineral. Using the EXAtm analysis it has shown been determined that Mg2+ levels are significantly reduced in many physiological states which may lead to serious pathological conditions [15]. Description of the methodology and examples of data as well as potential applications will focus on intracellular (i.c.) [Mg2+]i determinations obtained in cells from subjects with cardiovascular disease (CVD) syndromes related to Mg2+ deficiency. Examples of the application of EXAtm evaluation include examination of intracellular magnesium and other minerals in a wide spectrum of conditions which include cardiovascular conditions, arrhythmias, heart failure, myocardial infarction, and bypass surgery. Standardization of control values were performed at NASA.

Evaluation of the nucleolar organizer region associated proteins in exfoliative cytology of normal buccal mucosa. Effect of smoking.

An evaluation of cellular alterations in the smoker's oral mucosal cells was performed. The Exfoliative Cytology technique was applied and the cytological smears stained with silver for the enumeration of Argyrophilic nucleolar organizer regions (AgNORs). Cytological smears were collected from two anatomic sites: floor of the mouth and tongue border, in both groups, smokers and non smokers, with a purpose of correlating the smoking habit to possible cellular alterations. The enumeration of the AgNORs showed that the average number of AgNORs is higher in smokers. There is a significant difference (P=0.0001) between smears from the floor of the mouth and from tongue border in the smoking group. In this study, no correlation between number of cigarettes, age and gender was found, but the results suggest that there might be a correlation between the smoking habit and an increased rate of cellular proliferation in the oral mucosal cells.

Localization, morphology and ultrastructure of taste buds in the domestic duck (Cairina moschata domestica L.) oral cavity.

The Authors report on localization of the taste buds in epithelium of the palate (70%), the floor of the oral cavity (28%) and the tongue (2%) in Domestic Duck. Each taste bud is oval-shaped, measuring roughly 130 x 60 microm, and communicates with the oral cavity by a short duct. There is topographical correspondence between the buds and the parietal salivary glands. Ultrastructural examination showed the following 4 cell types: 1) light cells--characterized by cytoplasm containing sparse perinuclear filaments, numerous light vesicles, a uniformly granular nucleus. 2) dark cells--with electron-dense cytoplasm, numerous perinuclear filaments, occasional light vesicles, an irregular and densely granular nucleus. 3) intermediate cells--with characteristics common to the two previous cell types. 4) basal cells--located at the ventral part of the button.

Immunoperoxidase detection of polycyclic aromatic hydrocarbon-DNA adducts in mouth floor and buccal mucosa cells of smokers and nonsmokers.

Tobacco smoking is a major risk factor for oral cancer; mouth floor and buccal mucosa are among the most and least cancer-prone subsites, respectively, in the oral cavity. We investigated the applicability of immunohistochemistry of smoking-induced DNA adducts in oral cells for assessing the exposure to carcinogens, and estimating the risk for oral cancer. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were measured in mouth floor and buccal mucosa cells of smokers (n = 26) and nonsmokers (n = 22) by means of a semiquantitative immunoperoxidase assay. Smokers had elevated levels of PAH-DNA adducts compared to nonsmokers in their mouth floor cells (0.045 +/- 0.022 versus 0.022 +/- 0.016, P = 0.0008 arbitrary units of immunohistochemistry) as well as in their buccal mucosa cells (0.058 +/- 0.028 versus 0.028 +/- 0.012, P = 0.001). Also, there was a correlation between the levels of PAH-DNA adducts in mouth floor cells and those in buccal mucosa cells (r = 0.4, P = 0.01). Furthermore, PAH-DNA adduct levels in both mouth floor and buccal mucosa cells were significantly related to current smoking indices (amount of tar and number of cigarettes consumed per day). Expectedly, the levels of PAH-DNA adducts neither in mouth floor cells nor in buccal mucosa cells, both being short-lived cells, were related to smoking history index (pack years). The levels of PAH-DNA adducts, however, in mouth floor cells as the cancer prone cells were lower than those in buccal mucosa cells (0.037 +/- 0.023 versus 0.044 +/- 0.026, P = 0.04). We conclude that immunohistochemistry of PAH-DNA adducts in oral cells can be used for exposure assessment of tobacco-related carcinogens, however, it cannot be used for oral cancer risk estimation.

Noninvasive measurement of tissue magnesium and correlation with cardiac levels.

BACKGROUND: Intracellular magnesium ([Mg]i) plays an important role in the regulation of myocardial metabolism, contractility, and the maintenance of transsarcolemmal and intracellular ionic gradients. An understanding of the role of magnesium in the clinical setting, however, is hampered by the lack of an assay of intracellular tissue magnesium levels. METHODS AND RESULTS: We used energy-dispersive x-ray analysis to measure [Mg]i in sublingual epithelial cells and to correlate the level with those in atrial biopsy specimens from the same patients during cardiopulmonary bypass. Levels were also measured in acute myocardial infarction (AMI) patients before and after intravenous magnesium sulfate administration and compared with those from intensive care unit (ICU) patients and healthy individuals. A strong correlation between sublingual epithelial cell (mean, 32.1 +/- 0.3 mEq/L) and atrial tissue (mean, 32.1 +/- 0.3 mEq/L) [Mg]i was present in 18 cardiac surgery patients (r = .68, P < .002). Epithelial and atrial [Mg]i levels were lower than in healthy individuals (33.7 +/- 0.5 mEq/L, P < .01) studied at that time and correlated poorly with serum magnesium. Mean [Mg]i in 22 AMI patients was 30.7 +/- 0.4 mEq/L, which was significantly lower than in 21 ICU patients and 15 healthy individuals (35.0 +/- 0.5 mEq/L and 34.5 +/- 0.7 mEq/L, respectively, P < .001). Intravenous magnesium sulfate was administered to most of the AMI patients (mean dose, 36 +/- 6 mmol). [Mg]i rose significantly in the AMI patients over the first 24 hours, and the magnitude of the increase was greater in those who received higher doses of intravenous magnesium sulfate. CONCLUSIONS: Sublingual epithelial cell [Mg]i correlates well with atrial [Mg]i but not with serum magnesium. [Mg]i levels are low in patients undergoing cardiac surgery and those with AMI. Intravenous magnesium sulfate corrects low [Mg]i levels in AMI patients. Energy-dispersive x-ray analysis determination of sublingual cell [Mg]i may expedite the investigation of the role of magnesium deficiency in heart disease.

Regional variation in Langerhans cell distribution and density in normal human oral mucosa determined using monoclonal antibodies against CD1, HLADR, HLADQ and HLADP.

The distribution, density and activation of Langerhans cells (LC) has been established in biopsies of normal human buccal mucosa, hard palate, lateral border and dorsum of tongue, floor of mouth and lip taken from sudden death post mortems. LC were identified in cryostat sections with monoclonal antibodies against CD1, HLADR, HLADQ and HLADP using an immunoalkaline phosphatase technique. The use of post mortem material was validated by comparison with biopsies taken from volunteers. LC were predominantly situated in the basal and immediately suprabasal layers of the epithelium. In floor of mouth, lip, lateral border and dorsum of tongue the cells were found along the length of the epithelium. In buccal mucosa, although LC showed fundamentally a similar distribution, a tendency to cluster around the connective tissue papillae was also noted. In hard palate LC were found parallel to the surface in the mid zone of the epithelium. No evidence of LC free areas was found. Dorsum of tongue had the highest density of LC per mm epithelial surface length (28.3 cells per mm) which was significantly greater (P less than 0.05) than buccal mucosa (25.2) which in turn had significantly more cells (P less than 0.05) than lip (22.4). The lowest density of LC was found in lateral border of tongue (17.6), hard palate (17.6) and floor of mouth (16.7). These sites had significantly fewer cells than lip, buccal mucosa and dorsum of tongue (P less than 0.05). Class II MHC molecules are necessary for antigen presentation and in all sites except buccal mucosa there was no significant difference between the number of cells expressing CD1, HLADR, HLADQ and HLADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochemical study of exfoliated cells of oral mucosa. I. The glycogen deposition and keratinization.

Cytochemical studies of glycogen of oral mucosa cells have been made on the smears by freeze-drying and PAS staining. The specimens were obtained from different areas of oral cavity of 77 human subjects and an attempt was made to find some interrelation amoung glycogen deposition, keratinization and inflammation. The largest glycogen deposition was found in the mucosa cells from mouth floor and cheek, a little in those from gingiva and quite a small or no glycogen in those from mucosa of hard palate and tongue. In gingiva the cells showing much more keratinization were less in glycogen contents, and vice versa. In inflammation some increase in glycogen contents were found in the gingivitis and the highest glycogen content in the cases of denture irritation of the palate as far as the present observation is concerned.