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Eur J Oral Sci. 2000 Oct;108(5):432-41.
Bovine dental papilla-derived cells immortalized with HPV 18 E6/E7.

Thonemann B, Schmalz G.

Department of Operative Dentistry and Periodontology, University of Regensburg, Germany. birger.thonemanlinik.uni-regensburg.de

In vitro investigations of cell-specific metabolism and cell interactions as well as biocompatibility studies are often hampered by the limited lifespan of primary cells originating from target tissues like the oral mucosa, gingiva or pulp. Pulp cells, as do other primary cells, undergo senescence after several passages in vitro. However, senescence can be overcome by transfection of primary cells with oncogenes like the HPV 18 (human papillomavirus 18) E6/E7 oncogene, resulting in immortalized cell lines. The purpose of our study was to establish and preliminary characterize an immortalized bovine dental papilla-derived cell line by transfection with HPV 18 E6/E7 for future use in biocompatibility testing of dental materials. First passage dental papilla-derived cells from molar tooth germs of 6-month-old calves were transfected by electroporation with pUC18 LCR E6/E7 coding for the HPV 18 E6/E7 oncogene. Cells underwent crisis after 5 wk in culture. Distinct cell colonies arose after about 9 wk. Cells were cloned by single cell dilution in passage 15 and 17. Out of three cell clones maintained in culture, two cell clones showed cell death after 28 and 30 wk, respectively. One cell clone overcame a second crisis after 38 wk and was maintained in culture until passage 75. Stable gene expression of HPV 18 E6/E7 oncogenes was verified by polymerase chain reaction (PCR) and immunohistochemistry. Reverse transcription (RT)-PCR revealed that the established cell line (passage 50) expresses procollagen type I, alkaline phosphatase and osteocalcin. This suggests that the immortalization with HPV 18 E6/E7 results in a cell line, which maintained phenotypic characteristics of odontoblast-like cells.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11037760&dopt=Abstract

osu.edu

Healthy, adult, male and female goat temporomandibular retrodiscal tissues were characterized to determine if biochemical differences existed between the genders. RNA concentrations were not different between male and female retrodiscal tissues; however, the DNA concentration in female retrodiscal tissues was 82% greater than in male retrodiscal tissues. Collagen concentrations were significantly greater in male retrodiscal tissues, and this was reflected in significant gender differences of type I and III collagen concentrations. More specifically, male temporomandibular retrodiscal tissues contained 70% more type I collagen and 119% more type III collagen when compared to female retrodiscal tissues. These differences in collagens and DNA reflect a gender difference in temporomandibular retrodiscal tissue composition that underlies divergent biomechanical and neurophysiological properties.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11037764&dopt=Abstract

Laryngoscope. 2000 Oct;110(10 Pt 1):1739-44.
Influenza A virus infection of human middle ear cells in vitro.

Buchman CA, Fregien N.

Department of Otolaryngology, University of Miami School of Medicine, Florida, USA. cbuchmaed.miami.edu

HYPOTHESIS: Human-derived normal middle ear mucosal cells can be harvested and cultured and will support influenza A virus (INF A) infection. STUDY DESIGN: Protocols for the collection and in vitro culture of middle ear mucosal cells were developed and used to investigate the effects of INF A infection as it relates to the pathogenesis of otitis media. MATERIALS AND METHODS: Middle ear mucosa was harvested during surgeries that opened the normal middle ear. Middle ear mucosal cells were plated and grown in collagen-coated dishes. Cells were characterized before and after INF A exposure using phase-contrast and immunofluorescence microscopy as well as reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokeratin 18 gene expression and INF A. RESULTS: Primary cultures of human middle ear epithelial cells were established. Prolonged growth of middle ear cells yielded a second cell type that failed to stain for cytokeratin on immunofluorescence but continued to produce positive RT-PCR results on cytokeratin 18 analysis. After INF A exposure, cytological changes and immunofluorescence staining showed cellular infection. RT-PCR analysis using INF A-specific primers showed positive results for up to 72 hours after viral exposure. CONCLUSIONS: Primary cultures of human middle ear mucosal cells have been established. Two distinctly different cell culture systems have been developed: 1) middle ear epithelial cells and 2) either dedifferentiated epithelial cells or fibroblasts. Exposure of both cell types to INF A demonstrates that each can support cellular infection and viral replication. These models should be useful for studies of the pathogenesis of virus-mediated otitis media.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11037837&dopt=Abstract

Arthritis Rheum. 2000 Oct;43(10):2178-88.
Decreased cellular activity and replicative capacity of osteoblastic cells isolated from the periarticular bone of rheumatoid arthritis patients compared with osteoarthritis patients.

Yudoh K, Matsuno H, Osada R, Nakazawa F, Katayama R, Kimura T.

Toyama Medical and Pharmaceutical University, Japan.

OBJECTIVE: Periarticular osteopenia is frequently observed in rheumatoid arthritis (RA). Bone loss has been considered to be at least partly due to inadequate bone formation, which in turn, is largely dependent on the number of osteoblasts and the osteoblastic activity. Normal human somatic cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that the telomere, the terminal sequence of chromosomes, is the mitotic clock that triggers senescence. In the present study, we sought to clarify the relationship between periarticular osteopenia and osteoblast replicative senescence in RA. METHODS: We examined age-related changes in cellular activity (alkaline phosphatase activity, osteocalcin and C-terminal type I procollagen secretion, and cAMP response to parathyroid hormone), replicative capacity, and senescent cell expression in osteoblasts from periarticular bone samples obtained from 15 patients with RA and 15 age-matched patients with osteoarthritis (OA). Cellular replicative capacity was analyzed by the mean telomere length and in vitro remaining replicative lifespan of the cells. RESULTS: In both OA and RA groups, the cell proliferation rate, the levels of osteoblastic markers, mean telomere length, and replicative lifespan in osteoblastic cells gradually decreased with the increasing age of the donor. The percentage of senescent osteoblastic cells in the periarticular bone increased with age in both groups, and the rate of expression of senescent cells was higher in RA patients than in age-matched OA patients. The osteoblastic activities and replicative capacity of osteoblastic cells from RA patients were lower than those from OA patients at any donor age. The age-related decreases in the osteoblastic activity and replicative capacity of osteoblastic cells from periarticular bone were greater in RA patients than in OA patients. CONCLUSION: Our results suggest that osteoblast replicative senescence in periarticular bones occurs more rapidly with aging in RA than in OA patients and contributes to periarticular osteopenia in RA.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11037877&dopt=Abstract

Arthritis Rheum. 2000 Oct;43(10):2189-201.
Immortalized human adult articular chondrocytes maintain cartilage-specific phenotype and responses to interleukin-1beta.

Robbins JR, Thomas B, Tan L, Choy B, Arbiser JL, Berenbaum F, Goldring MB.

Beth Israel Deaconess Medical Center, and New England Baptist Bone & Joint Institute, Boston, Massachusetts, USA.

OBJECTIVE: To develop a reproducible immortalized human chondrocyte culture model for studying the regulation of chondrocyte functions relevant to arthritic diseases in adult humans. METHODS: Primary adult articular chondrocytes were immortalized with a retrovirus expressing a temperature-sensitive mutant of SV40-large T antigen (tsTAg). The established tsT/AC62 chondrocyte cell line was examined in monolayer and alginate culture systems. The levels of messenger RNA (mRNA) encoding cartilage matrix proteins and interleukin-1beta (IL-1beta)-inducible mRNA were analyzed by reverse transcriptase-polymerase chain reaction. Matrix protein synthesis was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-sulfate-labeled proteoglycans and Western blotting of type II collagen and aggrecan. Type II collagen (COL2A1)-luciferase reporter gene expression was analyzed by transient transfection. Phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38 mitogen-activated protein kinase (p38 MAPK), and activating transcription factor 2 (ATF-2) were detected by Western blotting. RESULTS: The tsT/AC62 cells expressed TAg at the permissive temperature (32degrees C), and the loss of TAg at 37 degrees C and 39 degrees C correlated with decreased cell proliferation. Cells in alginate culture deposited abundant alcian blue-stainable matrix and continued to proliferate at 32 degrees C. Preferential retention of aggrecan was observed in the cell-associated matrix, while biglycan and decorin were secreted into the medium of monolayer and alginate cultures. The levels of COL2A1 and aggrecan mRNA were increased after transfer from monolayer to alginate culture at 32 degrees C. Treatment with IL-1beta decreased COL2A1 and aggrecan mRNA levels and increased the levels of matrix metalloproteinases 1, 3, and 13 mRNA, as well as those of cyclooxygenase 2, type I collagen, and secretory phospholipase A2 type IIA mRNA, but not those of inducible nitric oxide synthase mRNA. IL-1beta also stimulated phosphorylation of p38 MAPK, SAPK/JNK, and ATF-2. The p38 MAPK-selective inhibitor, SB203580, partially reversed IL-1beta-induced inhibition of COL2A1 mRNA levels and COL2A1-luciferase reporter gene expression. CONCLUSION: The tsT/AC62 cells provide a reproducible model that mimics the adult articular chondrocyte phenotype, particularly in alginate culture, and demonstrates characteristic responses to IL-1beta. These studies also show, for the first time, that p38 MAPK is one of the signals required for IL-1beta-induced inhibition of COL2A1 gene expression. Availability of this model will permit identification of signals that regulate cytokine responses, and will also provide rational strategies for targeting these pathways.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11037878&dopt=Abstract

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