Serum immunoglobulin G antibody titer to Fusobacterium nucleatum is associated with unfavorable outcome after stroke.

Stroke can be a cause of death, while in non-fatal cases it is a common cause of various disabilities resulting from associated brain damage. However, whether a specific periodontal pathogen is associated with increased risk of unfavorable outcome after stroke remains unknown. We examined risk factors for unfavorable outcome following stroke occurrence, including serum antibody titers to periodontal pathogens. The enrolled cohort included 534 patients who had experienced an acute stroke, who were divided into favorable (n = 337) and unfavorable (n = 197) outcome groups according to modified ranking scale (mRS) score determined at 3 months after onset (favorable = score 0 or 1; unfavorable = score 2-6). The associations of risk factors with unfavorable outcome, including serum titers of IgG antibodies to 16 periodontal pathogens, were examined. Logistic regression analysis showed that the initial National Institutes of Health stroke scale score [odds ratio (OR) = 1·24, 95% confidence interval (CI) = 1·18-1·31, P < 0·001] and C-reactive protein (OR = 1·29, 95% CI = 1·10-1·51, P = 0·002) were independently associated with unfavorable outcome after stroke. Following adjustment with those, detection of the antibody for Fusobacterium nucleatum ATCC 10953 in serum remained an independent predictor of unfavorable outcome (OR = 3·12, 95% CI = 1·55-6·29, P = 0·002). Determination of the antibody titer to F. nucleatum ATCC 10953 in serum may be useful as a predictor of unfavorable outcome after stroke.

TNF-α-induced IL-6 and MMP-9 expression in immortalized ameloblastoma cell line established by hTERT.

OBJECTIVE: Ameloblastoma (AM) shows locally invasive behaviour. However, biological investigations regarding regulation of gene expression associated with AM pathological features are difficult to perform, because AM cells can be passaged for a few generations due to senescence. We report a newly established immortalized AM cell line, AMB cells, by transfection with human telomerase reverse transcriptase (hTERT). Furthermore, we examined whether TNF-α modulates bone resorption-related genes, IL-6 and MMP-9 in cooperation with TGF-ß or IFN-γ. MATERIALS AND METHODS: Following transfection of an hTERT expression vector into AM cells using a non-viral method, the effects of cytokines on the expressions of IL-6 and MMP-9 mRNA were examined using real-time PCR. TNF-α-induced NF-κB activity was examined by western blotting and transcription factor assays. RESULTS: AMB cells continued to grow for more than 100 population doublings. Stimulation with TNF-α increased IL-6 and MMP-9 mRNA expressions, as well as NF-κB activation. Furthermore, TGF-ß and IFN-γ dramatically increased TNF-α-mediated expressions of MMP-9 and IL-6 mRNA, respectively, while those responses were suppressed by NF-κB inhibitor. CONCLUSION: We established an immortalized AM cell line by hTERT transfection. TNF-α-mediated regulation of MMP-9 and IL-6 via NF-κB may play an important role in the pathological behaviour of AMs, such as bone resorption.

Establishment of mesenchymal cell line derived from human developing odontoma.

OBJECTIVES: An odontoma, which shows proliferating odontogenic epithelium and mesenchymal tissue, is one of the most common odontogenic tumors encountered. These are commonly found in tooth-bearing regions, although the etiology remains unknown. There are no previous reports of an established line of immortalized human odontoma cells. METHODS: Using odontoma fragments obtained from a girl treated at our department, we established an immortalized human odontoma cell line and investigated cell morphology, dynamic proliferation, the presence of contamination, and karyotype. Moreover, cell characterization was examined using osteogenic and odontogenic markers. RESULTS: We successfully established a mesenchymal odontoma cell (mOd cells). The cells were found to be fibroblastic and had a high level of telomerase activity. Cell growth was confirmed after more than 200 population doublings without significant growth retardation. mOd cells expressed mRNA for differentiation markers, including collagen type I (COLI), alkaline phosphatase, bone sialoprotein, osteopontin, osteocalcin, cementum-derived protein (CP-23), dentin sialophosphoprotein (DSPP), and distal-less homeobox 3 (DLX3), as well as bone morphogenetic proteins (BMPs). In addition, they showed a high level of calcified nodule formation activity in vitro. CONCLUSIONS: We successfully established a cell line that may be useful for investigating the mechanisms of normal odontogenesis as well as characteristics of odontoma tumors.

Influence of factors related to implant stability detected by wireless resonance frequency analysis device.

Resonance frequency analysis (RFA) was introduced as a method for measuring implant stability more than a decade ago. Implant stability quotient (ISQ) values obtained using a recently introduced wireless RFA device have made it possible to evaluate stability in a non-invasive technique; however, there are few studies of the factors that affect ISQ values determined using this device. The aim of the present study was to evaluate the association between ISQ values determined by wireless RFA and various factors related to dental implant stability using a pig cortical bone model. Dental implants (Replace) Select Tapered implants) with a length of 10 mm were placed into pig cortical bone samples, then, ISQ values were determined using wireless RFA under various conditions (probe orientation, diameter of implant, insertion torque and peri-implant bone loss). The results of this study showed that ISQ values were not affected by the direction of the probe from parallel to perpendicular to the long axis of the pig bone or to the smart peg. In addition, the diameter of the implant did not have a significant effect on the measured ISQ values. Statistically significant correlations were found between insertion torque and ISQ values (Spearman's test, P < 0.05), and lower ISQ values were observed for deeper peri-implant vertical defects (Mann-Whitney U-test, P < 0.05). A wireless RFA device appears to be useful for measuring implant stability within the limits of the present in vitro study.

Amphiregulin induces proliferative activities in osseous dysplasia.

Human osseous dysplasia (OD) is a benign fibro-osseous neoplasm of periodontal ligament origin in which normal bone is replaced with fibrous connective tissue containing abnormal bone or cementum. However, cellular differentiation and proliferation in OD have not been fully elucidated. In vitro culture systems have distinct advantages for analytical studies. Therefore, we established immortalized cell lines (OD-1) from OD lesions of the jaw from an individual with gnathodiaphyseal dysplasia (GDD). We hypothesized that OD-1 had a characteristic growth mechanism different from that of mineralized-associated cells such as osteoblasts. To clarify the difference of gene expression patterns between OD-1 and osteoblasts, we compared the profiles of genes expressed in the 2 cell types by microarray analysis. We identified amphiregulin to be highly expressed in OD-1 compared with osteoblasts and gingival fibroblasts. OD-1 showed proliferative activities regulated in an autocrine manner by amphiregulin, and amphiregulin may play a significant role in the proliferation of OD.

Regulation of CXCL9/10/11 in oral keratinocytes and fibroblasts.

Th1 and Th2 cytokines such as interferon-gamma (IFN-gamma ) , tumor necrosis factor- alpha (TNF-alpha ), and IL-4 are expressed in T-cell-mediated inflammation in the oral cavity. We tested the hypothesis that those cytokines may act on CXCR3-agonistic chemokines, T-cell recruiting factors, and on neighboring cells, including oral keratinocytes and fibroblasts. Human immortalized oral keratinocytes (RT7) and fibroblasts (GT1) after 24-hour stimulation with IFN-gamma showed increased mRNA levels of CXCL9 (600- and 700-fold), CXCL10 (10,000- and 150-fold), and CXCL11 (5000- and 300-fold), respectively. In contrast, TNF-alpha caused an increase in CXCL9 (300-fold), CXCL10 (2000-fold), and CXCL11 (2000-fold) mRNA levels in GT1, but not RT7 cells, at 24 hrs. IL-4 reinforced the promotion of CXCL9, CXCL10, and CXCL11 expression by IFN-gamma in RT7 cells, whereas IL-4 inhibited the increased levels by IFN-gamma and TNF-alpha in GT1 cells. Thus, IFN-gamma , TNF-alpha , and IL-4 appear cooperatively to regulate CXCR3-agonistic chemokines in oral keratinocytes and fibroblasts in T-cell-mediated oral inflammation sites.

Expression of Bub1 gene correlates with tumor proliferating activity in human gastric carcinomas.

Bub1 plays an important role at the spindle assembly checkpoint to prevent cell cycle progression following spindle damage. We examined the expression of human Bub1 mRNA in 20 gastric carcinoma tissues and corresponding nonneoplastic mucosas by reverse transcriptase-polymerase chain reaction and analyzed the relation with proliferative activity monitored by the expression of proliferating cell nuclear antigen (PCNA) on Western blotting as well as Ki-67 labeling index by immunohistochemistry. Increased expression of Bub1 mRNA was detected in 8 (40%) of the gastric carcinomas in comparison with their nonneoplastic counterparts, while 4 (20%) expressed Bub1 at lower levels. The expression of Bub1 mRNA was confirmed by in situ hybridization. The expression levels of Bub1 mRNA were well correlated with the levels of PCNA protein in 16 (80%) gastric carcinoma cases. The examination of Ki-67 labeling indices proved the close correlation between the expression levels of Bub1 and proliferating activity. These findings suggest that mRNA expression of human Bub1 gene is closely associated with the tumor-proliferating activity. Since genetic alterations of human Bub1 rarely occur in gastrointestinal cancers, the functional machinery of Bub1 to prevent cell cycle progression into anaphase might be well preserved in gastric carcinomas even with high proliferative activity.

Promoter hypermethylation of MGMT is associated with protein loss in gastric carcinoma.

Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumor suppressor genes in neoplasms. Recently, O(6)-methylguanine-DNA methyltransferase, MGMT, was shown to be hypermethylated in certain carcinomas, resulting in loss of MGMT protein. We studied DNA methylation of CpG islands of the MGMT gene by methylation specific PCR in 26 gastric carcinoma tissues and 8 gastric carcinoma cell lines for comparison with levels of MGMT protein expression. In addition, we examined p53 mutation status in the same tissues by PCR-SSCP analysis for comparison with MGMT protein expression levels. In total, promoter hypermethylation of the MGMT gene was found in 8 (31%) of the 26 gastric carcinomas with reduced expression of MGMT protein, whereas the hypermethylation was not detected in the 18 carcinomas with non-reduced MGMT expression. MGMT protein expression levels were associated with promoter hypermethylation of MGMT (p = 0.0001; Mann-Whitney test); however, MGMT expression was not associated with p53 mutation status (p = 0.461; Mann-Whitney test). Among in gastric carcinoma cell lines, the TMK-1 cell line showed loss of the MGMT protein association with promoter hypermethylation and this loss was rectified by treatment with a demethylating agent, 5-Aza-2'-deoxycytidine. Our results suggest that transcriptional inactivation of MGMT by aberrant methylation of the promoter region may participate in carcinogenesis in the stomach.

No mutations of the Bub1 gene in human gastric carcinomas.

Chromosomal instability in colorectal cancers is associated with functional loss of a mitotic check point partly due to mutations of the Bub1, one of the mitotic check point genes. However, mutation of coding sequences of human Bub1 gene has not been fully elucidated in gastric carcinomas. We performed sequencing analysis on reverse transcriptase-polymerase chain reaction (RT-PCR) product of the Bub1 cDNA (entire coding sequence) from 5 human gastric carcinomas as well as on genomic PCR products of Bub1 kinase domain from 7 gastric carcinoma tissues. Although sequencing analysis of the Bub1 cDNA revealed several point mutations in 2 gastric carcinoma cases, we could not confirm the mutations by analyzing genomic DNA. Furthermore, genomic DNA sequencing revealed no mutations in the kinase domain of the Bub1 gene in any gastric carcinoma examined. These results suggest that mutational inactivation of the Bub1 gene might not play a key role in human stomach carcinogenesis.

Prospective study of Ki-67 labeling index in the mucosa adjacent to cancer as a marker for colorectal cancer metastasis.

Multivariate regression analysis has shown that Ki-67 labeling index in the mucosa adjacent to cancer was the most significant marker for colorectal cancer metastasis among several metastasis-related parameters according to our previous retrospective data base (7). We have performed a prospective study to ascertain whether Ki-67 labeling index in the mucosa adjacent to cancer is a useful preoperative diagnostic marker for colorectal cancer metastasis. In 182 registered cases colonoscopically biopsied, we performed surgical resection of the cancer in 37 adenocarcinoma cases, which were registered in the study. In 31 out of the 37 cases except for 6 cases with an insufficient amount of non-neoplastic mucosa, preoperative diagnosis for metastasis was performed using the Ki-67 cutoff line. The cutoff line was set at 15% according to our previous retrospective database. The preoperative diagnosis for metastasis was compared to the pathological findings of the resected specimen. The incidences of correct diagnosis for metastasis was 81% (25/31). There were 3 false positive cases and 3 false negative cases in Dukes' A-B and Dukes' C, respectively. The mean Ki-67 labeling index in the mucosa adjacent to cancer of Dukes' A-B and Dukes' C-D cases, except for the 6 misdiagnosed cases, was 7.4+/-5.0% and 29.9+9.8%, being significant at p<0.0001 by unpaired Mann-Whitney U test. These results suggest that Ki-67 labeling index in the mucosa adjacent to cancer might be a good marker for metastasis in colorectal cancer.

Pleomorphic adenoma of the parotid gland with extensive bone formation.

We report an unusual case of pleomorphic adenoma with extensive bone formation, occurring in the parotid gland of a 58-year-old Japanese man. The tumor was a well-circumscribed mass that measured 20 x 20 x 15 mm and contained extensive bone formation. Histologically, most of the tumor was composed of bone and chondroid tissues. The bone formation with a marrow-like structure occupied half the volume of the tumor. The chondroid tissues merged continuously into bone tissues. The bone tissue seemed to be formed within areas of chondral tissue by a process of enchondral ossification. Except for the unusual amount of large bone formation, the tumor showed histology of pleomorphic adenoma, particularly at the periphery of the tumor. These histological findings suggest the possibility of extensive enchondral ossification in pleomorphic adenoma.

Atmospheric halocarbons, hydrocarbons, and sulfur hexafluoride: global distributions, sources, and sinks.

The global distribution of fluorocarbon-12 and fluorocarbon-11 is used to establish a relatively fast interhemispheric exchange rate of 1 to 1.2 years. Atmospheric residence times of 65 to 70 years for fluorocarbon-12 and 40 to 45 years for fluorocarbon-l1 best fit the observational data. These residence times rule out the possibility of any significant missing sinks that may prevent these fluorocarbons from entering the stratosphere. Atmospheric measurements of methyl chloroform support an 8-to 10-year residence time and suggest global average hydroxyl radical (HO) concentrations of 3 x 10(5) to 4 x 10(5) molecules per cubic centimeter. These are a factor of 5 lower than predicted by models. Additionally, methyl chloroform global distribution supports Southern Hemispheric HO levels that are a factor of 1.5 or more larger than the Northern Hemispheric values. The long residence time and the rapid growth of methyl chloroform cause it to be a potentially significant depleter of stratospheric ozone. The oceanic sink for atmospheric carbon tetrachloride is about half as important as the stratospheric sink. A major source of methyl chloride (3 x 10(12)grams per year), sufficient to account for nearly all the atmospheric methyl chloride, has been identified in the ocean.