Effect of high glucose, Porphyromonas gingivalis lipopolysaccharide and advanced glycation end-products on production of interleukin-6/-8 by gingival fibroblasts.

BACKGROUND AND OBJECTIVE: It is known that chronic periodontal infection can magnify the cytokine responses in patients with diabetes. Hyperglycemia increases the proinflammatory status, including the levels of advanced glycation end-products (AGEs), in patients with periodontitis. However, whether AGEs have additional effects on the production of those proinflammatory cytokines in diabetic patients with periodontitis is still unknown. To examine in vitro the effect of hyperglycemia and AGEs on the amounts of interleukin (IL)-6 and IL-8 produced in periodontally infected gingiva, human gingival fibroblasts (HGFs) were stimulated with glucose, AGE-modified bovine serum albumin (AGE-BSA) and Porphyromonas gingivalis LPS in the present study. MATERIAL AND METHODS: Primary culture of HGFs was incubated with various concentrations of AGE-BSA (0, 50, 100 and 200 µg/mL) and LPS (0, 10, 100 or 1000 ng/mL) at two different glucose concentrations - normal glucose (5 mm) and high glucose (25 mm). The amounts of IL-6 and IL-8 produced by HGFs were evaluated using ELISA. Expression of the AGE receptor on HGFs was determined by flow cytometry. RESULTS: High glucose stimulated a significant increase in the production of IL-6 and IL-8 by HGFs compared with normal glucose. This enhanced production of IL-6 and IL-8 could also be observed in the presence of LPS and/or AGE-BSA. When both LPS and AGE-BSA were present, especially at high concentrations (≥ 500 µg/mL of LPS and ≥ 25 µg/mL of AGE-BSA), a synergistic effect on IL-8 production was found in the high-glucose condition. CONCLUSIONS: A synergistic effect of the production of IL-8 could be induced in HGFs with the combination of high glucose, LPS and AGEs.

An estimation of the incidence of tuberous sclerosis complex in a nationwide retrospective cohort study (1997-2010).

BACKGROUND: Tuberous sclerosis complex (TSC) is caused by mutations in TSC1 and TSC2, leading to mammalian target of rapamycin hyperactivation. Patients with TSC develop hamartomas in brain, lungs, liver and skin. Two epidemiological studies, performed in Minnesota, U.S.A., have estimated the incidence of TSC to be 0·28-0·56 per 100 000 person-years (PY), based on < 12 patients. Furthermore, whether common comorbidities are associated with this rare disease is not known. OBJECTIVES: To estimate the incidence of TSC and investigate the associations of TSC with other comorbidities, including diabetes, peptic ulcers, stroke and myocardial infarction. METHODS: We estimated the incidence and prevalence of TSC and its comorbidities from 1997 to 2010, based on the Catastrophic Illness Certificate disease database and a beneficiary cohort of 1 million people. RESULTS: The incidence of TSC in Taiwan is 0·153 per 100 000 PY. The number of patients identified with TSC in Taiwan doubled from 206 in 2006 to 471 in 2010. In 2010, the prevalence of TSC in Taiwan was estimated to be 1·58 in 100 000. We confirmed that female patients with TSC are more likely to develop renal tumours than male patients. Surprisingly, patients with TSC have a significantly decreased risk of developing peptic ulcers compared with controls. CONCLUSIONS: This is the first large-scale and longitudinal incidence study of TSC. This study provides compelling evidence that TSC mutations in humans are associated with a decreased risk of peptic ulcers.

The effects of diallyl sulfide upon Porphyromonas gingivalis lipopolysaccharide stimulated proinflammatory cytokine expressions and nuclear factor-kappa B activation in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Diallyl sulfide (DAS), a flavor compound from garlic, has varied potential therapeutic activities. Periodontitis is a disease that develops because of host-mediated inflammation to periodontal pathogens. In this study, the effects of DAS on the common proinflammatory cytokines and nuclear factor-kappa B (NF-κB) in human gingival fibroblasts (HGFs) being stimulated with lipopolysaccharide from Porphyromonas gingivalis, a potent periodontal pathogen, were evaluated. MATERIAL AND METHODS: Cytotoxicities of DAS and lipopolysaccharide on HGFs were measured with MTS assay. The mRNA and protein expressions of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α, from the HGFs treated with lipopolysaccharide with and without DAS were examined with reverse transcription-polymerase chain reaction and immunocytochemistry, respectively. In addition, the activation and nuclear translocation of NF-κB with and without DAS were compared. RESULTS: DAS and lipopolysaccharide treatments within 3 mm and 10 µg/mL, respectively, did not affect the survival rate of HGFs. Lipopolysaccharide (1 µg/mL) significantly increased the mRNA expressions of IL-1ß, IL-6 and TNF-α; however, DAS (1 mm) inhibited these expressions. The protein expressions of TNF-α, IL-1ß, as well as the NF-κB nuclear translocation were increased after lipopolysaccharide treatment, but decreased when there was a DAS pretreatment. CONCLUSION: DAS diminished P. gingivalis lipopolysaccharide-stimulated cytokine expression and NF-κB activation in HGFs; we therefore suggest DAS may be beneficial on periodontal inflammation.

Up-regulation of retinoblastoma protein phosphorylation in gingiva after cyclosporine A treatment: an in vivo and in vitro study.

BACKGROUND AND OBJECTIVE: Cyclosporine A can induce gingival cell proliferation; however, the precise molecular regulation of the proliferation is uncertain. Therefore, this study was carried out to examine, in vivo and in vitro, the expression of genes and proteins associated with gingival cell proliferation after treatment with cyclosporine A. MATERIAL AND METHODS: Forty Sprague Dawley rats with right maxillary posterior edentulous gingivae were assigned to a cyclosporine A group (30 mg/kg daily of cyclosporine A, administered orally) or a control group (administered mineral oil only). The animals were killed 4 wk after treatment. The edentulous gingivae were dissected out and analyzed for the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (Rb1) mRNA and/or protein, and phosphorylated Rb1 (pRb1), by real-time RT-PCR or immunohistochemistry. In human gingival fibroblast (HGF) cultures, the expression of PCNA, CDK4, cyclin D1 and Rb1 proteins and Rb1 phosphorylation were determined by western blotting after cyclosporine A treatment (0-10(4) ng/mL). RESULTS: Proliferating cell nuclear antigen and cyclin D1 mRNAs (Pcna and Ccnd1, respectively) were expressed more strongly in the gingivae of cyclosporine A-treated animals than in the gingivae of the controls. Immunohistochemical analyses showed that a greater number of gingival cells stained positive for cyclin D1, CDK4 and pRb1 in the cyclosporine A group than in the control group. Increased expression of cyclin D1, CDK4 and PCNA proteins was observed in HGFs after cyclosporine A treatment. The phosphorylation of Rb1 was enhanced in HGFs after treatment with cyclosporine A at concentrations of 10(2)-10(3) ng/mL. CONCLUSION: The increases in cyclin D1, PCNA and CDK4, together with the enhanced phosphorylation of Rb1, suggest that cyclosporine A promotes cell-cycle progression through the G(1)/S transition in the gingiva.

Cyclosporine A enhances apoptosis in gingival keratinocytes of rats and in OECM1 cells via the mitochondrial pathway.

BACKGROUND AND OBJECTIVE: We reported previously that cyclosporine A induces a high level of expression of p21 in rat gingival keratinocytes and in OECM1 cells. In this study, the apoptosis of gingival keratinocytes after treatment with cyclosporine A was evaluated using the same models. MATERIAL AND METHODS: Forty Sprague-Dawley rats with right edentulous ridges were assigned into cyclosporine A (30 mg/kg) and control groups. Four weeks later, gingivae were screened for expression of apoptotic genes using microarray analyses and DNA fragmentation. The expression of bcl2-associated X protein (Bax), apoptosis-inducing factor (AIF) and Caspase 3 mRNAs, and the expression of Bax, AIF, Caspase 9 and Fas proteins, were analyzed using the reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. Apoptosis in OECM1 cells (keratinocytes of a gingival carcinoma cell line), after treatment with cyclosporine A, was evaluated by 4',6-diamidino-2-phenylindole (DAPI) staining and flow cytometry, whereas the expression of Bax, AIF, Caspase 3 and 8, Bcl-2 and Fas proteins were examined using western blotting. RESULTS: According to microarray analyses, the expression of certain apoptotic genes was altered in the gingiva of rats who received cyclosporine A, and increased number of DNA fragments were detected. Expression of mRNA or protein for Bax, AIF and Caspase 3 and 9 in the gingivae of rats increased after treatment with cyclosporine A. An increased number of apoptotic bodies and of OECM1 cells in the sub-G1 phase was observed after treatment with cyclosporine A. Increased expression of AIF, Bax and Caspase 3 protein, but not of bcl-2, Caspase 8 or Fas protein, was observed in cells after treatment with cyclosporine A. CONCLUSION: Based on the above findings, we suggest that cyclosporine A might enhance the apoptosis of gingival keratinocytes, mainly via the mitochondrial pathway.

Association study between keratinocyte-derived growth factor gene polymorphisms and susceptibility to vitiligo vulgaris in a Taiwanese population: potential involvement of stem cell factor.

BACKGROUND: Vitiligo vulgaris is a depigmentary disorder resulting from the disappearance of functional melanocytes. Currently, the pathogenesis of this disorder remains obscure. OBJECTIVES: Genetic analysis of patients with vitilgo may provide important clues for elucidating the complex pathomechanisms involved in the disease process. Because dysfunctional keratinocytes have recently been implicated in the pathogenesis of vitiligo vulgaris, we conducted a case-control association study to investigate this phenomenon. PATIENTS AND METHODS: Fifty-one patients with vitiligo vulgaris and 118 healthy controls from Taiwan were recruited to investigate the association between relevant keratinocyte-related genes and the occurrence of vitiligo vulgaris. This study genotyped 11 single-nucleotide polymorphisms (SNPs) in five genes including stem cell factor (SCF, also known as KITLG), basic fibroblast growth factor (bFGF, also known as NuDT6), endothelin-1 (EDN1), hepatocyte growth factor (HGF) and stem cell growth factor (SCGF, also known as CLEC11A). RESULTS: Our results revealed that the A allele for SNP rs11104947 in the SCF gene and the T allele for SNP rs13866 in the SCGF gene were, respectively, associated with a 1.95- and a 2.14-fold risk of developing vitiligo vulgaris. A higher risk was also detected among subjects who carried the SCF rs995029/rs11104947 C/A haplotype (odds ratio = 2.45). Furthermore, the at-risk alleles for SCF rs11104947 (A allele) and for SCGF SNP rs13866 (T allele) were found to display a 7.92-fold increased gene-gene combined risk. No significant relationship between polymorphic frequency for genes bFGF, EDN1 as well as HGF and occurrence of vitiligo vulgaris was observed. CONCLUSIONS: These novel genetic findings provide new insights in relation to the mechanisms that might be involved in the development of vitiligo vulgaris.

Expression of p21 and p53 in rat gingival and human oral epithelial cells after cyclosporine A treatment.

BACKGROUND AND OBJECTIVE: Expression of p21 and p53 were examined, at gene and protein levels, in edentulous gingival epithelial cells from rats and from a human oral epidermoid carcinoma cell line, OECM1, after cyclosporine A therapy. MATERIAL AND METHODS: In vivo: 20 partially edentulous SD rats were assigned into cyclosporine A feeding and control groups. After the rats were killed, p21 and p53 in gingiva were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. In vitro: after cyclosporine A treatment, p21 and p53 of OECM1 cells were evaluated by western blot and the luciferase assay. The distribution of OECM1 cells in each phase of the cell cycle was evaluated by flow cytometry. RESULTS: The mRNA expression of p21 was significantly higher in the cyclosporine A group than in the control group. A greater number of positive anti-p21-stained cells were observed in the gingival epithelium of the cyclosporine A group than in the control group. Significantly higher levels of p21 protein and activity were observed in OECM1 cells after cyclosporine A treatment than in cells without treatment. A relative increase of cells in G0/G1 phases, and a decrease of cells in G2/M phases, were observed in OECM1 cells after cyclosporine A treatment. CONCLUSION: In the present study, higher p21 mRNA and protein expressions were observed after cyclosporine A treatment. Thus, an up-regulation of p21 expression, via a p53-independent pathway, by cyclosporine A in gingival and oral epithelial cells was suggested.

Cyclosporin-A inhibits the expression of cyclooxygenase-2 in gingiva.

BACKGROUND AND OBJECTIVE: Various inflammatory mediators are involved in the development of cyclosporine A-induced gingival overgrowth. In this study, the gingival expression of cyclooxygenase-2 after cyclosporine A therapy was examined in vivo and in vitro. MATERIAL AND METHODS: After edentulous ridges on maxilla were established, 21 Sprague-Dawley rats received cyclosporine A daily for 4 wk, and a further 21 rats received solvent. After the rats were killed, the expression of cyclooxygenase-2 mRNA, interleukin-1beta mRNA, tumor necrosis factor-alpha mRNA, and interleukin-6 mRNA was examined in the edentulous gingiva. The expression of cyclooxygenase-2 protein and the production of prostaglandin E2 were also evaluated. RESULTS: In cultured human gingival fibroblasts and epithelial cells, the expression of cyclooxygenase-2 mRNA was measured after treatment with cyclosporine A. Significantly lower expression of cyclooxygenase-2 and interleukin-1beta mRNA, but higher interleukin-6 expression, were observed in gingiva from cyclosporine A-treated rats than in those from the control rats. Significantly less prostaglandin E2 production was observed in cyclosporine A-treated rats. Immunohistochemistry revealed that fewer gingival stromal cells were positively stained for cyclooxygenase-2 in cyclosporine A-treated rats. In cultured cells, significantly less cyclooxygenase-2 mRNA was detected after treatment with cyclosporine A. CONCLUSION: The expression of cyclooxygenase-2 was lower in the plaque nonretentive gingivae and the in vitro gingival cells upon treatment with cyclosporine A. Thus, we propose that cyclosporine A inhibits the expression of gingival cyclooxygenase-2.