Dolphine Oda

Human oral epithelial cell culture. I, improved conditions for reproducible culture in serum-free medium

SummaryGingival tissue from healthy adult human donors was used as a source of epithelial cells for culture. An overnight incubation of this tissue with dispase facilitated the mechanical separation of the surface epithelium from the underlying fibrous connective tissue. This step minimized culture contamination with fibroblasts. The epithelium was then trypsinized to prepare a single cell suspension. The cell pellets were collected by centrifugation and resuspended in keratinocyte growth medium, incubated at 37° C and 5% CO2 in a humid atmosphere. Primary cultures grew in small islands that coalesced at confluency. Immunohistochemistry demonstrated uniform staining of the cells with antibodies to keratins of stratified squamous epithelium. Ultrastructurally, the cells contained distinct intermediate filaments. When cells were grown in media with low calcium (0.15 mM), cell-to-cell contacts were via interlacing papillary projections with no desmosomes. However, when cells were grown under physiologic calcium (1.2 mM), desmosomes were prominent and well developed. Cells were maintained in culture for over 100 d (7 passages).

Head and neck osteosarcoma at the University of Washington

Head and neck osteosarcoma is a comparatively rare and aggressive malignancy. Our goal was to examine the experience of head and neck osteosarcoma patients seen over a 15‐year period at the University of Washington Medical Center and compare this with the published experience of other centers in terms of demographics, histology, treatment, and survival rate.

Human papilloma viruses and p53 mutations in normal pre-malignant and malignant oral epithelia.

HPV infections have been previously observed in oral cancers, and inactivation of the p53 gene has been shown to be one of the most common genetic alterations in human tumors. We examined 179 oral specimens from 70 individuals with histologic findings of either normal mucosa (n = 6) or oral disease that ranged from mild dysplasia to invasive squamous‐cell carcinoma (n = 64) to determine the occurrence of both HPV infection and p53 mutations and their relationship with several clinical factors. HPV infection was detected by PCR amplification of viral DNA, and the presence of p53 mutations was assayed using the single‐strand conformation polymorphism (SSCP)‐PCR technique. HPV infection was found in 31% of individuals with oral disease and was not seen in healthy individuals. Mutations in exons 5, 6, 7 or 8 of the p53 gene were detected in 37.5% of patients with oral lesions and in a biopsy from 1 healthy individual who was a heavy smoker. Approximately one‐third of lesions classified as pre‐malignant (dysplasia and carcinoma in situ) and 42% of invasive carcinomas contained p53 mutations. The majority of these mutations were G:T transversions located within exons 7 and 8. Tumor tissues from 6 patients with oral lesions were found both to be HPV‐16‐positive and to contain p53 mutations; of these, 4 were poorly differentiated carcinomas that were diagnosed as late‐stage disease. In this study, p53 mutations were detected in the early stages of cancer development.

Model Bile and Bile Salts Accelerate Mucin Secretion by Cultured Dog Gallbladder Epithelial Cells

BACKGROUND & AIMS Hypersecretion of gallbladder mucin has been proposed as a pathogenic factor in gallstone formation. We investigated whether mucin secretion is modulated by biliary constituents using normal, well-differentiated dog gallbladder epithelial cells. METHODS Model biles or bile salts were applied to monolayers of epithelial cells. Mucin secretion was studied by measuring the secretion of [3H]N-acetyl-D-glucosamine-labeled glycoproteins. RESULTS Model biles with different cholesterol saturation indices increased mucin secretion by the cells to an average 251% after 5 hours of incubation (P < 0.01). Mucin secretion remained elevated during a 24-hour period, suggesting a sustained effect on mucin secretion. There was no relation between the cholesterol or phospholipid concentration and the extent of stimulation of mucin secretion. Taurocholate caused a dose-dependent increase in mucin secretion, suggesting that bile salt was the bile component responsible for the stimulatory effect. At a concentration of 0.5 mmol/L, only the more hydrophobic bile salts taurochenodeoxycholate and taurodeoxycholate, but not the hydrophylic bile salts taurocholate and tauroursodeoxycholate, stimulated mucin secretion (P < 0.01). CONCLUSIONS Bile salts play an important role in the regulation of mucin secretion. A shift in the bile salt composition of bile towards the more hydrophobic bile salts may cause mucin hypersecretion, thereby initiating cholesterol gallstone formation.

The fibroblast-like nature of myofibroblast

Abstract The myofibroblast is found in normal tissue as well as in a wide variety of pathological processes. We have cultured myofibroblasts and dermal fibroblasts and have found that they secrete similar type-specific procollagens into the culture media. These were primarily type I and III procollagens with a predominance of type I procollagen. These patterns are distinctly different from those of smooth muscle cells, which synthesize predominantly type III procollagen. Cultured fixed cells were also examined by immunohistochemistry. Both myofibroblasts and fibroblasts stained positively with antibodies to type I and III procollagens. Reaction to type V procollagen antibodies was prominent only in the myofibroblast, as was immunostaining with anti-muscle actin antibodies. Immunostaining with desmin antibodies was negative in both cell types. By electron microscopy, the myofibroblast had well-developed dense microfilament fibers of 40–80° that were prominent in the long axes of the cells near the cellular margins. Although the myofibroblast has properties of both smooth muscle cells and fibroblasts, it appears to be most likely a modified fibroblast that has undergone differentiation, probably in response to specific signals from the extracellular matrix.

RECONSTITUTED HUMAN ORAL AND ESOPHAGEAL MUCOSA IN CULTURE

SummaryWe 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.

Proliferating oral epithelial cells in culture are capable of both extracellular and intracellular degradation of interstitial collagen.

The potential of epithelial cells to degrade interstitial collagen was studied by culturing human masticatory mucosa on decalcified dentin matrix. Morphological changes were observed in the underlying collagen substratum and in the connective tissue of the explant. Degradation of the substratum was initiated two days after the first contact with epithelial cells exhibiting basal cell markers. Electron microscopic studies confirmed extensive collagen degradation in the vicinity of these cells. No collagen degradation was observed underneath the connective tissue portion of the explant. Experiments in which the explant was partially separated from the underlying substratum by a filter further showed that connective tissue was apparently not involved in the collagen degradation by the epithelial cells. Lysis of connective tissue of the explant was observed in association with epithelial cells that showed a disrupted basal lamina and release of vesicular material from the exposed cell membrane. Collagen fibers were visible inside some epithelial cells suggesting intracellular collagenolysis. Primary cultures of human gingival epithelial cells and porcine periodontal ligament epithelial cells (epithelial cell rests of Malassez) that expressed similar basal cell cytokeratins as the active cells of the mucosal explants secreted collagenase, gelatinase and TIMP to the culture medium. They also contained acid collagenolytic proteinases. When cultured on a porous polycarbonate membrane the epithelial cells secreted collagenolytic enzymes from the pores at cell membrane sites lacking basal lamina. These results provide evidence that proliferating basal epithelial cells have a strong capacity for collagen degradation. It seems that the absence of basement membrane is the signal for these cells to secrete matrix degrading enzymes.

Oral histoplasmosis as a presenting disease in acquired immunodeficiency syndrome.

A 43-year-old homosexual man visited his dentist with painful, nodular, ulcerated lesions on the soft palate, right buccal mucosa, and right posterior maxillary gingiva. Serologic studies for exposure to human immunodeficiency virus, performed before biopsy, were positive. Biopsy of the maxillary gingiva demonstrated sheets of histiocytes containing small intracellular yeasts, which on culture were identified as Histoplasma capsulatum. Bilateral leukoplakic lesions with some vertical furrowing involving the lateral borders of the tongue were also noted. Histologically, hyperkeratosis and fungal hyphae were identified. The patient was treated for histoplasmosis with amphotericin B, which resulted in significant improvement of the oral lesions. He was subsequently hospitalized for fatigue and dyspnea and was found to have Pneumocystis carinii pneumonia. Pulmonary status deteriorated within a 3-week period, and the patient died. Autopsy findings were negative for histoplasmosis but positive for necrotizing and cavitary P. carinii pneumonia, pulmonary and hepatic herpes simplex infections, and pulmonary and intestinal cytomegalovirus infection.

Human oral epithelial cell culture ii. keratin expression in fetal and adult gingival cells

SummaryEpithelial cells from human fetal and adult gingiva were cultured in keratinocyte growth medium (KGM), a serum-free medium. The expression of keratin proteins in these cells was evaluated using immunohistochemistry and SDS-PAGE-immunoblot analysis and compared with expression in the tissue. Keratins 5, 6, 14, 16, and 19 were identified in cells cultured from both fetal and adult tissues. K19 was localized in basal cells of fetal oral tissue but was not seen in adult gingiva (except for scattered Merkel cells). K1 and K10 were expressed in tissue, but not in cultured cells. The keratin profiles of cultured epithelial cells from several adult donors were similar and were identical in cultures from primary through Passage 5. K13, a differentiation-specific keratin, was expressed in all suprabasal cells of fetal oral epithelium, but shows only spotty expression in adult gingival tissue. K13 was expressed in cultures of fetal cells, but very weakly or not at all in cultures of adult cells. K13 expression was greater in cultures grown with physiologic calcium concentrations (1.2 mM) than in those grown at 0.15 mM or less. Our findings are consistent with basal-like characters of these cells in 0.15 mM calcium growth conditions. Differentiation of fetal oral cells in culture to the suprabasal basal cell stage in 1.2 mM Ca2+ is shown by the expressionof K13.

Instability of the myofibroblast phenotype in culture

Myofibroblasts are cells that have features of both smooth muscle cells and fibroblasts. Myofibroblasts from rat granulation tissue were studied using a coordinated biochemical and morphological approach. These myofibroblasts were maintained in culture for more than 17 passages. After establishing that these cells were indeed myofibroblasts by immunohistochemical and ultrastructural criteria, their biochemical parameters and phenotypic stability were studied. Rat dermal fibroblasts grown under similar conditions served as controls. Biochemically, it was found that both cells produced similar types of procollagens, namely collagens I and III, in similar proportions at or near a 1:1 ratio. However, myofibroblasts at early passage produced threefold more procollagens than did fibroblasts at the same stage of passage, while similar quantities were produced in each at late passage. These observations for procollagens were confirmed by separate studies for collagens, as measured by hydroxyproline determinations. Such differences were also reflected ultrastructurally. Secretory vesicles were identified in myofibroblasts at early stage, but not at late stage of passaging. No such vesicles were seen in fibroblasts at any stage of passage. In conclusion, myofibroblasts are active secretory variants of fibroblasts which have an unstable phenotype in culture and become indistinguishable from fibroblasts at late passage.