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Aim To investigate the effects of 2-hydroxy-ethyl methacrylate (HEMA) on cytotoxicity and cyclooxygenase-2 (COX-2) protein expression in human osteoblasts. Methodology Cytotoxicity was judged using an Alamar Blue reduction assay on human osteoblast cell line U2OS. Western blot was used to evaluate the expression of COX-2 protein by HEMA. To determine whether glutathione (GSH) levels were important in cytotoxicity and COX-2 expression of HEMA, cells were pre-treated with the GSH precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. Paired Student's t-tests were applied for the statistical analysis of the results. Results HEMA demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P

Kuan Y-H, Li Y-C, Huang F-M, Chang Y-C. The upregulation of tumour necrosis factor-α and surface antigens expression on macrophages by bisphenol A-glycidyl-methacrylate. International Endodontic Journal, 45, 619–626, 2012. Abstract Aim To evaluate the expression of tumour necrosis factor-α and surface antigens by bisphenol A-glycidyl-methacrylate (BisGMA) on murine macrophage cell line RAW264.7. Methodology Cytotoxicity was measured by tetrazolium bromide reduction assay. Tumour necrosis factor (TNF)-α was analysed by enzyme-linked immunosorbent assay. Cell surface antigens were investigated by flowcytometry. Statistical analyses were performed using anova followed by the Bonferroni’s t-test for multigroup comparisons. Results BisGMA exhibited cytotoxicity to RAW264.7 in a dose-dependent manner (P 0.05). Conclusions Taken together, the ability of macrophages to induce an appropriate immune response when exposed to BisGMA has the potential to upregulate TNF-α production and expression of surface antigens.

Background Dysregulated T helper (Th) cells are considered important in the pathophysiology of chronic primary immune thrombocytopenia (ITP). The present study investigated whether levels of Th cytokines in pediatric patients with chronic ITP were different compared with healthy controls. Procedures Fifty-seven pediatric patients with chronic ITP and 28 healthy controls were enrolled. Patients were divided into three groups based on their platelet counts at the time of the study: (i) active disease 150 × 109/l (n = 11). Plasma concentration of Th1 [interferon gamma (INF-γ), interleukin 2 (IL-2)], Th2 (IL-4, IL-10), Th3 [transforming growth factor-β1 (TGF-β1)], and Th17 (IL-17) cytokines were investigated by enzyme-linked immunosorbent assay. Results IFN-γ was increased in patients with active (P

Background and Objective: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. However, little is known about how nicotine influences the expression of osteolytic mediators in cigarette smoking-associated periodontal diseases. The aim of this study was to investigate the expression of interleukin-1, interleukin-8, receptor activator of nuclear factor-κB ligand (RANKL), gelatinases and tissue-type plasminogen activator in U2OS cells (from the human osteosarcoma cell line) stimulated with nicotine. Material and Methods: Differences in the expression of interleukin-1, interleukin-8 and RANKL mRNAs, in response to exposure to various concentrations of nicotine (0, 0.125, 0.25, 0.5 and 1 mm) were evaluated in U2OS cells using the reverse transcripttion–polymerase chain reaction.In addition, the levels of interleukin-1, interleukin-8 and RANKL proteins were determined using enzyme-linked immunosorbent assays. The gelatinolytic and caseinolytic activities in nicotine treated-U2OS cells were demonstrated using gelatin and casein zymography, respectively. Results: Nicotine was found to increase the expression of interleukin-1, interleukin-8 and RANKL mRNA and protein in U2OS cells (p

Background and Objective: Cystatin C is a 13 kDa non-glycosylated, basic protein belonging to the cystatin family. It is consistently and dramatically upregulated in a variety of fibrotic diseases. However, little is known about the correlation between cystatin C and cyclosporine A-induced gingival overgrowth. The aim of this study was to compare cystatin C expression in normal, healthy gingival tissues and cyclosporine A-induced gingival overgrowth specimens and further explore the potential mechanism that may result in cystatin C expression. Material and Methods: Fifteen cyclosporine A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Three human gingival fibroblast (HGF) strains were established from crown-lengthening surgery. The reverse transcriptase-polymerase chain reaction was used to investigate the effects on HGFs exposed to cyclosporine A. In addition, predominant periodontal pathogens (Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis) and proinflammatory cytokines (interleukin-1α and tumor necrosis factor-α) were added to seek the possible regulatory mechanisms of cystatin C expression. Results: The cystatin C staining in gingival tissue was stronger in the cyclosporine A-induced gingival overgrowth group than in the normal gingival group (p

Aim To investigate the receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. Methodology The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1α and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student’s t-test. Results IL-1α was found to upregulate RANKL production in U2OS cells (P

Background and Objective: Cyclosporin A is used as an immunosuppressive agent and its prominent side-effect is the induction of fibrous gingival overgrowth. The progression of fibrous gingival overgrowth results from the accumulation of extracellular matrix. Type I plasminogen activator inhibitor (PAI-1) acts as the inhibitor of extracellular matrix degradation and is involved in some fibrotic diseases. However, little is known about the correlation between PAI-1 and cyclosporin A-induced gingival overgrowth. The aim of this study was to investigate the effects of cyclosporin A on the expression of PAI-1 mRNA and protein in human gingival fibroblasts human gingival fibroblasts in vitro and to compare PAI-1 expression in normal healthy gingival tissues and cyclosporin A-induced gingival overgrowth specimens in vivo. Material and Methods: Quantitative reverse transcription–polymerase chain reaction and western blot assay were used to investigate the effects on human gingival fibroblasts exposed to cyclosporin A. In addition, 10 cyclosporin A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Results: Investigations of the time dependence of PAI-1 mRNA expression in human gingival fibroblasts treated with 200 ng/ml of cyclosporin A revealed a rapid accumulation of the transcript: a significant signal was first detectable after 1 h of exposure and the signal remained elevated throughout the 24-h incubation period (p

To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002.The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity.The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P0.05).Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA.

Aim To compare tissue-type plasminogen activator (t-PA) expression in normal human pulp and inflamed human pulp tissue specimens. Methodology Thirty pulpal tissue specimens (13 normal and 17 inflamed pulps) were obtained from extracted third molars. The levels of t-PA between normal pulp and inflamed pulp tissues were compared using the quantitative reverse-transcriptase polymerase chain reaction analysis. In addition, immunohistochemistry was used to identify the in situ localization of t-PA expression in pulp specimens. Wilcoxon–Mann–Whitney rank sum test was applied for the statistical analysis of the results. Results t-PA mRNA gene was found more in inflamed pulps when compared with normal pulp tissue (P

The objective of this study was to determine the cytocompatibility of three different extracts of gingival retraction cords and to compare the cytotoxic effect of these materials on human gingival fibroblasts. Gingival retraction cords impregnated with aluminium sulphate (Gingi-Aid), dl-adrenaline HCl (Gingi-Pak) and non-drug-impregnated cord (Gingi-Plain) were eluted with culture medium for 10 min and 24 h. Cytotoxicity was judged using a tetrazolium bromide reduction assay. Our data demonstrated that gingival retraction cords applied alone almost completely inhibited cell viability (P0.05). In addition, the results also showed that the eluates from aluminium sulphate-impregnated cord, dl-adrenaline HCl-impregnated cord and non-drug-impregnated cord were cytotoxic to primary human gingival fibroblast cultures (P0.05). The cell viability of incubation of gingival fibroblasts containing 10-min eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 61, 21 and 70%, respectively. The cell viability of incubation of gingival fibroblasts containing 24 h eluates of aluminium sulphate, dl-adrenaline HCl and non-drug-impregnated cord was 68, 58 and 72%, respectively. It was found that dl-adrenaline HCl-impregnated gingival retraction cord was the most toxic gingival retraction cord among the materials tested in all cultures (P0.05). The cytotoxicity decreased in an order of dl-adrenaline HCl-impregnated cordaluminium sulphate-impregnated cordnon-drug-impregnated cord. The extent or degree of the cytotoxicity depended on the materials tested. Gingival retraction cords have significant potential for gingival toxicity. Careful management of gingiva retraction cords would lower the risk of potential gingival tissue damage during clinical application procedure and thus increase the success of prosthodontic procedures.

Aim To investigate the effect of black-pigmented Bacteroides on the expression of interleukin (IL)-8 gene in human pulp fibroblasts and osteoblasts. Methodology The supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL-8 gene expression in human pulp fibroblasts and osteoblasts. The levels of mRNAs were measured by the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Results Investigations of the time-dependence of IL-8 mRNA expression in black-pigmented Bacteroides-treated pulp fibroblasts and osteoblasts revealed a rapid accumulation of the transcript after 2 h of exposure, and remained elevated throughout the 24-h incubation period. In addition, IL-8 mRNA gene expression was also found in human osteoblasts stimulated with black-pigmented Bacteroides. However, black-pigmented Bacteroides was found to be more effective in the induction of IL-8 mRNA gene expression in osteoblasts than in pulp fibroblasts (P

Aim To investigate the effect of pro-inflammatory cytokines and black-pigmented Bacteroides on the expression of IL-6 gene in human pulp fibroblasts. Methodology IL-1α, tumour necrosis factor-α (TNF-α) and the supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL-6 gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse-transcriptase polymerase chain reaction analysis. Results Investigations of the time dependence of IL-6 mRNA expression in pro-inflammatory cytokines-treated cells revealed a rapid accumulation of the transcript after 2 h of exposure and remained elevated throughout the 24-h incubation period. In addition, black-pigmented Bacteroides also induced IL-6 gene expression in human pulp fibroblasts. Conclusions Pro-inflammatory cytokines and black-pigmented Bacteroides may be involved in developing pulpal inflammation through the stimulation of IL-6 production.

To investigate the cytotoxicity of five different dentine-bonding agents on human pulp cells in vitro.Set specimens from Clearfil SE Bond (CB), Heliobond (HB), PrimeBond NT (PB), Single Bond (SB), and Syntac Single Component (SC) were eluted with culture medium for 2 and 5 days. Cytotoxicity was judged using tetrazolium bromide reduction assay on human primary pulp cells.Elutes from five dentine-bonding agents were cytotoxic to primary human pulp cells (P0.05). CB was the least toxic sealer amongst the chemicals tested. The cytotoxic response decreased in an order of SBPBSCHBCB.The influence of the cytotoxicity depended on the materials tested. Dentine-bonding agents have significant potential for pulpal toxicity.

Aim The purpose of this study was to determine the cytotoxicity of three different types of root canal sealer on human periodontal ligament (PDL) cells and a permanent hamster cell line (V79 cells). Methodology Set specimens from two resin based sealers (AH26 and AHPlus), three zinc oxide–eugenol-based sealers (Canals, Endomethansone and N2) and one calcium hydroxide-based sealer (Sealapex) were eluted with culture medium for 1, 2, 3 and 7 days. Cytotoxicity was judged using tetrazolium bromide reduction assay on human primary PDL cells and V79 cells derived from a Chinese hamster. Results The results showed that elutes from resin-based, zinc oxide–eugenol-based, and calcium hydroxide-based sealers were cytotoxic to primary human PDL cultures and V79 cells. Calcium hydroxide-based sealer was the least toxic sealer amongst the chemicals tested in both cultures. The cytotoxic response decreased in an order of N2 > Endomethansone > AH26 > AHplus > Canals > Sealapex. Conclusions The sensitivity of toxicity depended on the materials tested and the cell culture system used. Thus, the use of both permanent and primary cells is recommended for screening of the cytotoxic effects of root canal sealers. In addition, the results confirmed that root canal sealers constantly dissolve when exposed to an aqueous environment for extended periods, possibly causing moderate or severe cytotoxic reactions. Use of calcium hydroxide-based material as a root canal sealer initially may result in a more favourable response to periradicular tissues.

Background In childhood acute lymphoblastic leukemia (ALL), t(1;19)(q23;p13.3) with TCF3-PBX1 fusion is one of the most frequent translocations. Historically, it has been associated with poor prognosis. Intensive treatment, however, has improved its outcome. We determined the outcome of children with this genotype treated with contemporary intensive chemotherapy in Taiwan. Procedure In Taiwan Pediatric Oncology Group 2002 ALL studies, genotypes were determined by cytogenetic analysis and/or reverse transcriptase polymerase chain reaction assay. Based on presenting features, immunophenotype and genotype, patients were assigned to one of the three risk groups: standard risk (SR), high risk (HR), or very high risk (VHR). The patients with t(1;19)/TCF3-PBX1 were treated in the HR arm receiving more intensive chemotherapy. The outcomes of patients with t(1;19)/TCF3-PBX1 were compared to that of patients with other subtypes of B-precursor ALL (B-ALL). Results Of the 1,129 patients with B-ALL, 64 (5.7%) had t(1;19)/TCF3-PBX1; 51 of whom were treated in the HR arm, but 11 were treated in the VHR and 2 in the SR arm because of physician's preference. As a group, 64 patients with t(1;19)/TCF3-PBX1 had similar 5-year event-free survival (83.3 ± 4.8%) as those with TEL-AML1 (85.2 ± 3.4%, P = 0.984) or those with hyperdiploidy >50 (84.0 ± 3.1%, P = 0.748). The cumulative risk of any (isolated plus combined) central nervous system relapse among patients with t(1;19)/TCF3-PBX1 (8.7 ± 3.8%) tended to be higher than that of patients with TEL-AML1 (5.8 ± 2.3%, P = 0.749) or those with hyperdiploidy (4.1 ± 1.8%, P = 0.135), albeit the differences did not reach statistical significance. Conclusions With contemporary intensive chemotherapy, children with t(1;19)/TCF3-PBX1 fared as well as those with favorable genotypes (TEL-AML1 or hyperdiploidy).