Reconstituted human oral and esophageal mucosa in culture

Summary We have successfully established monolayer and organotypic culture techniques for growing human oral and esophageal epithelial cells. Cells in monolayer culture were grown in serum-free medium, modified from techniques previously reported by our group. The organotypic cultures were grown in a defined medium supplemented with 10% fetal calf serum. Oral and esophageal cells were maintained in keratinocyte basal medium with pituitary extract and other supplements, and 0.05 mM calcium for 7–9 and 9–11 passages, respectively. Both cell types had similar morphology by phase contrast microscopy. When confluent, the cells were predominantly small, basaloid, and uniform and interspersed with larger, differentiated cells. By immunohistochemistry, both cell types in monolayer were positive to AE1, AE3, and 34BE12 antibodies to keratins of stratified epithelia. Oral epithelial cells in monolayer also were positive to 35BH11, representative of simple epithelial keratins, while esophageal cells were not. The esophageal cells were focally positive to K13, while the oral cells were negative. Both were negative for K19. When comparing monolayer to organotypic cultures and to in vivo specimens, there was a significant difference in the expression of keratins. Using organotypic cultures, AE1, AE3, and 34BE12 were strongly positive in both oral and esophageal cells, similar to in vivo tissues. In contrast to monolayers, both were also focally positive for K19. Esophageal cells were strongly positive for K13, while the oral cells were middly but uniformly positive. Both were negative for keratins of simple epithelia. These two cell culture techniques offer unique opportunities to study the pathobiology, including carcinogenesis, of stable cell systems from the oral and esophageal epithelia.     

Functional anatomy of human esophageal mucosa in postnatal ontogenesis

Morphological and morphometric analysis of human oesophagus mucous membrane layers in the norm in postnatal period show that all membranes have definite stages of development. Among them we can single out: first: from birth to 12 months--stage of secondary differentiation; second: from 12 months to 3 years--stage of combined development; third: 3 to 12 years--stage of intensive development; from 30 to 50 years--stage of before involutive development and sixth: from 50 to old age--stage of involutive development. Each of the stages is characterized by a combination of correlative morphological and morphometric features, which form individual ontogenesis of organ membrane.     

Cross-circulation and cell distribution kinetics in parabiotic mice

Blood‐borne nucleated cells participate not only in inflammation, but in tissue repair and regeneration. Because progenitor and stem cell populations have a low concentration in the blood, the circulation kinetics and tissue distribution of these cells is largely unknown. An important approach to tracking cell lineage is the use of fluorescent tracers and parabiotic models of cross‐circulation. Here, we investigated the cross‐circulation and cell distribution kinetics of C57/B6 GFP⁺/wild‐type parabionts. Flow cytometry analysis of the peripheral blood after parabiosis demonstrated no evidence for a “parabiotic barrier” based on cell size or surface characterstics; all peripheral blood cell subpopulations in this study reached equilibrium within 14 days. Whole blood fluorescence analysis indicated that the mean exchange flow rate was 16 µl/h or 0.66% of the circulating blood volume per hour. Studies of peripheral lymphoid organs indicated differential cell distribution kinetics. Some subpopulations, such as CD8⁺ and CD11c⁺, equilibrated in both lymph nodes and spleen indicating a residence time <28 days; in contrast, other lymphocyte subpopulations, such as B220⁺ and CD4⁺ cells, had not yet reached equilibrium at 28 days. We conclude that parabiosis can provide important insights into defining tissue distribution, residence times, and recirculating pools using fluorochrome markers of cell lineage. J. Cell. Physiol. 227: 821–828, 2012. © 2011 Wiley Periodicals, Inc.     

Epithelial cell kinetics of the gastric mucosa during Helicobacter pylori infection

Helicobacter pylori is an important pathogen in major gastroduodenal diseases, including inflammation with ulceration and gastric malignancies. Alterations in H. pylori associated cell turnover in gastric epithelial cells are examined in relation to inflammatory activity, bacteria load and cytokines which may improve knowledge concerning the outcome of gastric diseases caused by H. pylori. Antral biopsies from 42 dyspeptic patients including 27 H. pylori-positive and 15 H. pylori-negative patients were tested for apoptotic activity by the TUNEL assay, and immuno-histochemically for p53 and the proliferative marker Ki-67. H. pylori infection, bacteria load and inflammatory activity were associated with increased cell turnover as judged by enhanced activities of TUNEL, p53 and Ki-67. Only p53 was significantly correlated to IFN-gamma, IL-8 and IL-10. The H. pylori-positive state was furthermore accompanied by varying degrees of altered distribution pattern of the markers studied, with occasional presence of apoptosis in the deeper pit zones, upward extension of Ki-67 and to a lesser degree of p53. Given a similar pattern of change in proliferation and apoptosis in some neoplastic lesions in other parts of the gastrointestinal tract, such studies in cell turnover may provide insights valuable in the investigations of potential precursors of gastric malignancies.     

Lectin binding pattern and proteoglycan distribution in human eccrine sweat glands

The distribution pattern of glycoconjugates in human eccrine sweat glands has been studied by the binding of newly discovered lectins and by antibodies against a chondroitin sulphate proteoglycan and chondroitin sulphate glycosaminoglycans. Mannose-specific lectins labelled large intracellular granules, part of which could be extended cisternae of the endoplasmic reticulum or Golgi apparatus. In contrast, lectins specific for terminal mannose/glucose residues predominantly labelled basement membranes and the glycocalyx. Lectins recognizing terminal N-acetylgalactosamine groups left most parts of the glands unstained, but stained some dark cells intensely. These last cells were also intensively labelled by N-acetylglucosamine-specific and by fucose-specific lectins. Sialic acid residues were preferentially located in luminal borders of secretory coils. No terminal galactose residues were detected. All antibodies against chondroitin glycoconjugates stained large granules similar to those revealed by the mannose-specific lectins in the secretory cells. The basement membrane is only stained by the proteoglycan antibody and the chondroitin-6-sulphate antibody.Thus, a complex composition of glycoconjugates exists not only in matrix elements but also in the cells of eccrine glands of the human skin. A possible secretion of glycoconjugates is discussed.     

Histochemical studies on irradiated and obstructive human submandibular glands. Protein distribution and lectin-binding in the degenerative glands.

Immunohistochemical protein distribution of alpha-amylase (Am), lysozyme (Ly), cytokeratinin (CK), S-100 protein (S-100) and secretory component (SC), and lectin-binding (SBA and UEA-I) profiles were studied in 10 obstructive and 20 irradiated human submandibular glands which were surgically extirpated. Degenerative intensity of the glands was graded as I, II and III based on the order of severity. All proteins generally existed in serous acinic cells of the intact glands. The proteins immunoreactivities became weak even in mildly inflamed glands (grade I), and nearly disappeared from the moderately damaged glands (grade II). Duct cells had clear CK and some cells reacted with the anti-SC antibody, but other proteins were not observed on the ducts. Mucous cells possessed none of the proteins, and their lectin-binding was only traceable in some glands. Compared with immunoreactivities in the proteins, lectin-binding profiles were different. SBA and UEA-I bound somewhat similarly to both acinic and duct cells, and the binding was hardly affected even by severe degeneration (grade III). Between obstructive and irradiated glands, no obvious difference was observed in either protein distribution or lectin-binding. From the above, it seems that some proteins are more affective to the degeneration and that lectin-binding sugar residues are non-affective against the degenerative changes of the tissues.     

Endocrine cells of the esophageal glands]

Distribution of endocrine cells in the human oesophagus was studied histochemically. Large amount of endocrine cells was discovered in terminal parts and excretory ducts of the cardial glands. Endocrine cells of the oesophageal cardial glands are represented, at least, by three types: argentaffine, argyrophil in the reactions of Grimelius and Sevier, Munger and argyrophil in the reaction of Grimelius only. The amount of argentaffine cells in the oesophageal cardial glands was observed to be 5 times as large as that of in the gastric cardial glands. The reason is evidently in functional difference of the oesophageal and gastric cardial glands. The endocrine cells were absent in the mucous glands of the oesophagus.     

Glycoconjugate distribution in the human fundic mucosa revealed by lectin- and glycoprotein-gold cytochemistry

Summary The glycoconjugates of the human fundic mucosa were characterized at the ultrastructural level by means of direct (Helix pomatia agglutinin-gold complex) and indirect lectin techniques (Concanavalin A and horseradish peroxidase-gold complex; wheat germ agglutinin and ovomucoid-gold complex). Surface mucous cells and mucous neck cells secreted O-glycoproteins with N-acetylgalactosamine and N-acetylglucosamine residues at the non reducing terminus of the saccharidic chain. The secretory granules of the mucous neck cells showed condensed areas slightly reactive to ConA. The results obtained in the chief cells suggest that these cells secrete N-glycoproteins rich in mannose and/or glucose residues. Transitional cells, presenting both morphological characteristics and lectin binding pattern intermediate to the mucous neck and chief cells have been observed. The surface of the intracellular canaliculi of the parietal cell was labelled by HPA, WGA and ConA. In the neck region of the gastric glands, immature parietal cells containing abundant mucous granules reactive to HPA, WGA and ConA were observed. The present results further corroborate the existence of a common cell precursor for surface mucous, mucous neck and parietal cells. In a further step, mucous neck cells gradually differentiate into chief cells the transitional cells being an intermediate stage.     

Glycoconjugate distribution in the human fundic mucosa revealed by lectin- and glycoprotein-gold cytochemistry

The glycoconjugates of the human fundic mucosa were characterized at the ultrastructural level by means of direct (Helix pomatia agglutinin-gold complex) and indirect lectin techniques (Concanavalin A and horseradish peroxidase-gold complex; wheat germ agglutinin and ovomucoid-gold complex). Surface mucous cells and mucous neck cells secreted O-glycoproteins with N-acetylgalactosamine and N-acetylglucosamine residues at the non reducing terminus of the saccharidic chain. The secretory granules of the mucous neck cells showed condensed areas slightly reactive to ConA. The results obtained in the chief cells suggest that these cells secrete N-glycoproteins rich in mannose and/or glucose residues. "Transitional cells", presenting both morphological characteristics and lectin binding pattern intermediate to the mucous neck and chief cells have been observed. The surface of the intracellular canaliculi of the parietal cell was labelled by HPA, WGA and ConA. In the neck region of the gastric glands, immature parietal cells containing abundant mucous granules reactive to HPA, WGA and ConA were observed. The present results further corroborate the existence of a common cell precursor for surface mucous, mucous neck and parietal cells. In a further step, mucous neck cells gradually differentiate into chief cells the transitional cells being an intermediate stage.     

Spatial distribution of lead in the roots of human primary teeth

Lead remains one of the most hazardous metals in our environment. The concentrations of lead in coronal dentine and enamel have previously been reported but limited information is available regarding lead levels in radicular dentine and cementum. This study reports the distribution of lead in 26 roots of 16 human maxillary primary teeth from seven individuals. In addition, calcium and phosphorous concentrations were also measured to detect any variations in the degree of mineralization in different regions of the roots. The mean lead concentration in these roots was 1.67 +/- 1.43 microg/g, which is comparable to other studies. In all cases there were higher lead concentrations in the apices of non-resorbed roots of primary maxillary teeth relative to middle and cervical regions. The findings reported here are of potential significance during the process of physiological root resorption whence periapical tissue may be exposed to higher levels of lead.     

Lymphokine-activated and natural killer cell activity in human intestinal mucosa

The activity of natural effector (NE) cells was studied in lamina propria lymphocytes (LPL) obtained from 61 histologically normal specimens of human intestine, which included 45 resected for colon carcinoma and 16 resected for nonmalignant conditions. The mean spontaneous natural killer (NK) cell activity in LPL (1.7 X 10(2) cytotoxic units (C.U.)/10(5) cells) was very low in contrast to that found in peripheral blood mononuclear cells (PBMC) (38.5 X 10(2) C.U./10(5) cells). Significant NK activity was detected in only 16 (47%) of the tissues resected for carcinoma, and in five (38%) of those removed for nonmalignant conditions. Exposure to human leucocyte interferon resulted in only minimal increases in cytotoxicity for K562 target cells. Consistent with these findings, large granular lymphocytes represented less than 0.5% of freshly isolated LPL. Cultures of LPL from both carcinoma and nonmalignant conditions in MLA144-conditioned medium (CM), a source of interleukin 2 (IL 2), generated marked increases in cytotoxicity to levels comparable with or exceeding those found in PBMC. (Mean cytotoxicities were 90.4 X 10(2) and 49 X 10(2) C.U./10(5) cells, respectively.) Cytotoxicity induced by culture in MLA144-CM could be blocked by pretreatment of LPL with the monoclonal antibody anti-Tac directed against the IL 2 receptor. In addition, LPL cultured in recombinant human IL 2 were induced to levels of cytotoxicity that were similar to those induced by MLA144-CM. These data indicate that IL 2 is the factor in MLA144-CM responsible for generating lymphokine-activated killer (LAK) cells in LPL. The IL 2-activated LPL killer cells were OKT11+, OKT3-, Leu-7-, Leu-11b-, as determined by antibody and complement-mediated lysis, and the precursor cells in the lamina propria necessary for generation of killer cells by IL 2 were also OKT11+, OKT3-, Leu-7-, Leu-11b-. These studies indicate that LAK cells may be an important potential source of nonspecific cytotoxicity in the intestinal mucosa.     

Mathematical modeling the kinetics of cell distribution in the process of ligand-receptor binding

A statistical approach is presented to model the kinetics of cell distribution in the process of ligand-receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with fluorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand-receptor complexes was observed during the reaction process by real-time measuring of the fluorescence and light-scattering traces of individual cells with the scanning flow cytometer. The experimental data were fitted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors.     

Repopulation kinetics in irradiated mouse lip mucosa: the relative importance of treatment protraction and time distribution of irradiations

The repopulation kinetics of the irradiated lip mucosa of mice has been investigated. Split-dose experiments showed that, in this tissue, repopulation starts within 3 days after the first irradiation and increases exponentially within 10 days. To assess the relative importance of protraction and distribution of irradiations as a function of time, 10 fractions were given in (1) 3 days (three irradiations per day with a 4-hr interval), (2) 11 days (daily fractions), or (3) two short courses, each consisting of five fractions given in 1.5 days separated by a rest period of 8 days, with an overall time of 11 days. The results show that by protracting the treatment from 3 to 11 days (with daily irradiations) repopulation accounts for recovery of approximately 13 Gy. Delivering the radiation in two short courses separated by a rest period leads to an additional recovery of approximately 5 Gy. The most plausible explanation for this observation is that repopulation is much more efficient during the rest period between the two courses than during continuous daily irradiation. Although the regimen of two short courses with a rest period spares the acute reaction, it will not enhance the late tolerance. Before thorough knowledge about the repopulation kinetics of the tumors can be gained, caution should be observed for indiscriminate use of split-course multiple-fraction-per-day (MFD) regimens for treating various tumors.     

HCO3- secretion in the esophageal submucosal glands

The mammalian esophagus has the capacity to secrete a HCO(3)(-) and mucin-rich fluid in the esophageal lumen. These secretions originate from the submucosal glands (SMG) and can contribute to esophageal protection against refluxed gastric acid. The cellular mechanisms by which glandular cells achieve these secretions are largely unknown. To study this phenomenon, we used the pH-stat technique to measure luminal alkali secretion in an isolated, perfused pig esophagus preparation. Immunohistochemistry was used to localize receptors and transporters involved in HCO(3)(-) transport. The SMG-bearing esophagus was found to have significant basal alkali secretion, predominantly HCO(3)(-), which averaged 0.21 +/- 0.04 microeq.h(-1).cm(-2). This basal secretion was doubled when stimulated by carbachol but abolished by HCO(3)(-) or Cl(-) removal. Basal- and carbachol-stimulated secretions were also blocked by serosal application of atropine, pirenzipine, DIDS, methazolamide, and ethoxzolamide. The membrane-impermeable carbonic anhydrase inhibitor benzolamide, applied to the serosal bath, partially inhibited basal HCO(3)(-) secretion and blocked the stimulation by carbachol. Immunohistochemistry using antibodies to M(1) cholinergic receptor or carbonic anhydrase-II enzyme showed intense labeling of duct cells and serous demilunes but no labeling of mucous cells. Labeling with an antibody to Na(+)-(HCO(3)(-))(n) (rat kidney NBC) was positive in ducts and serous cells, whereas labeling for Cl(-)/HCO(3)(-) exchanger (AE2) was positive in duct cells but less pronounced in serous cells. These data indicate that duct cells and serous demilunes of SMG play a role in HCO(3)(-) secretion, a process that involves M(1) cholinergic receptor stimulation. HCO(3)(-) transport in these cells is dependent on cytosolic and serosal membrane-bound carbonic anhydrase. HCO(3)(-) secretion is also dependent on serosal Cl(-) and is mediated by DIDS-sensitive transporters, possibly NBC and AE2.