IL-17A stimulates the expression of inflammatory cytokines via celecoxib-blocked prostaglandin in MC3T3-E1 cells

Objective

The prostaglandins (PGs) released from osteoblasts can alter the process of bone remodelling. Recently, we showed that compressive force induced the expression of pro-inflammatory cytokine interleukin (IL)-17s and their receptors in osteoblastic MC3T3-E1 cells and that IL-17A was expressed most highly. Consequently, in the current study we examined the effect of IL-17A and/or celecoxib on PGE₂ production and the expression of cyclooxygenases (COXs) and inflammatory cytokines in MC3T3-E1 cells. We also examined the effects of PGE₂ and cyclohexamide on the expression of inflammatory cytokines.

Methods

Cells were cultured with or without IL-17A (0.1, 1.0, or 10 ng/ml) in the presence or absence of 10 μM celecoxib, a specific inhibitor of COX-2, for up to 72 h. Cells were pretreated with or without 10 μg/ml cycloheximide, protein synthesis inhibitor, for 30 min, and then cultured with 10 ng/ml IL-17A for 24 h. Cells were also cultured with or without 1.5 ng/ml PGE₂ for 24 h. PGE₂ production was determined by ELISA. The expression of COX-1, COX-2, IL-1α, IL-6, IL-8, IL-11, and TNF-α mRNAs and proteins was determined by real-time PCR and ELISA, respectively.

Results

The expression of COX-2, IL-1α, IL-6, IL-8, IL-11, and TNF-α, as well as PGE₂ production increased in the presence of IL-17A, whereas COX-1 expression did not change. Celecoxib blocked the stimulatory effect of IL-17A on the expression of COX-2, IL-1α, IL-6, IL-8, and IL-11 as well as PGE₂ production, whereas it did not block TNF-α expression. Cycloheximide pretreatment suppressed the expression of IL-17-induced inflammatory cytokines. The expression of IL-1α, IL-6, IL-8, and IL-11 increased by the addition of PGE₂, whereas TNF-α expression was not affected.

Conclusion

These results suggest that IL-17A stimulates the expression of bone resorption-related inflammatory cytokines through an autocrine mechanism involving celecoxib-blocked PGs, mainly PGE₂, in osteoblasts.     

Reducing bacterial counts in dental unit waterlines: distilled water vs. antimicrobial agents

BACKGROUND: This study evaluated five chemical disinfectants to compare their abilities to improve dental unit waterline quality and assess their effects, if any, on the biofilm layer. METHODS: Sixty new dental units, with a closed-circuit water system, were used to compare microbial levels in DUWLs treated with five antimicrobials: Listerine, Bio 2000, Rembrandt, Dentosept, and sodium fluoride to a control group of sterile distilled water alone over a six-week period. For all units, the waterlines were filled with solution, left overnight, and then flushed for 30 seconds with sterile distilled water the following morning prior to patient treatment. Waterlines were examined for biofilm buildup using scanning electron microscopy and colony-forming-unit counts. RESULTS: The sodium fluoride and the four chemical antimicrobials reduced the microbial count to 200 cfu/ml or less. Only samples taken from dental units receiving the control treatment (distilled water with no added antimicrobial) failed to meet ADA's stated goal. Examination of the SEMs revealed an apparent decrease in the biofilm mass but not elimination, despite repeated treatment with the four antimicrobial materials. CONCLUSIONS: Even in a closed-circuit water system, distilled water alone cannot reduce microbial contamination of dental treatment water from dental unit waterlines to the 200 cfu/ml ADA stated goal. However, water treated with Listerine mouthrinse, Rembrandt mouthrinse, Bio 2000, 0.5 percent sodium fluoride and Dentosept, did meet the microbial reduction goal. The biofilm apparently was reduced in volume, but not entirely eliminated. CLINICAL SIGNIFICANCE: The ADA goal of a maximum of 200 cfu/ml was achieved using any of five chemical antimicrobials and distilled water in a closed-water system. Despite the successful reduction in microbial contamination of the dental treatment water, the biofilm was not completely eliminated. Biofilm elimination and prevention would be needed through some other means.     

Effect of curing modes of dual-curing luting systems and root regions on retention of translucent fiber posts in root canals

PURPOSE: To evaluate the effect of different curing modes of dual-curing luting systems and root regions on the push-out strength of fiber posts to intraradicular dentin. MATERIALS AND METHODS: Forty-two extracted premolars with a single root canal were sectioned at the cementoenamel junction and the roots were endodontically treated. The roots were divided into two groups according to two dual-curing luting systems: (1) XP BOND-Dual Cure/Calibra resin cement; (2) XP BOND-Dual Cure/FluoroCore 2. For each luting system, three different curing modes were applied to the dentin adhesive and resin cement: "Self-cure and Self-cure (SC&SC)", "Self-cure and Dual-cure (SC&DC)", and "Dual-cure and Dual-cure (DC&DC)". Translucent Easy fiber posts (Dentsply Maillefer) were luted in the roots. A thin-slice push-out test was performed, and the data of push-out strength were analyzed using three-way ANOVA with luting system, curing mode, and root region (apical, middle and coronal) as factors. Tukey's test was used for post-hoc comparisons. RESULTS: The push-out strength of XP BOND-Dual Cure/Calibra was significantly lower when Calibra resin cement was self-cured than when it was dual-cured (SC&SC: 6.04 +/- 2.65 MPa; SC&DC: 10.69 +/- 3.01 MPa; DC&DC: 10.72 +/- 3.63 MPa; p < 0.05). The curing modes did not affect the push-out strength of XP BOND-Dual Cure/FluoroCore 2 (SC&SC: 7.90 +/- 3.94 MPa; SC&DC: 8.32 +/- 2.73 MPa; DC&DC: 9.27 +/- 4.12 MPa; p > 0.05). The coronal push-out strength was significantly higher than the apical push-out strength (p < 0.05). CONCLUSION: Retention of fiber posts in root canals was affected by the curing modes of dual-curing luting system and root regions.     

牙龈卟啉单胞菌Hgp44基因的克隆表达与纯化

目的 克隆表达牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)牙龈素中黏附素区域的Hgp44基因,并纯化目的蛋白.方法 通过聚合酶链反应(PCR)和基因重组技术,克隆得到PgATCC33277的Hgp4基因,插入到克隆载体pMD18-T中并测序鉴定.经过酶切后将目的基因片段与表达载体pET22b相连,构建出表达质粒pET22b-Hgp44.将重组质粒转化到感受态细胞BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)诱导表达融合蛋白,通过SDS聚丙烯酰胺凝胶电泳及蛋白质印迹法进行分析.用固定化金属亲和层析法对重组蛋白进行分离纯化.结果 目的基因片段约为1100 bp,与预期大小相符,测序结果与GenBank中ATCC33277国际标准菌株的RgpA的等位基因序列U15282一致.IPTG诱导后的菌体经SDS聚丙烯酰胺凝胶电泳后形成一个以包涵体形式存在的44 000的融合蛋白.蛋白质印迹法检测证实其具有免疫原性.用镍离子金属螯合层析柱纯化出了目的蛋白,最后得到约3.5 mg/L的目的蛋白.结论 成功克隆表达了PgHgp44基因,并纯化出了目的蛋白.     

Ti—75铸造后机械性能测试的研究

目的：研究及比较Ｔｉ－７５金属在牙科铸造后的机械性能。方法：利用Ｉｎｓｔｒｏｎ拉伸试验机，对Ｔｉ－７５金属牙科铸造后部分力学性能进行了测试研究，并与Ｔｉ－６Ａｌ－４Ｖ及临床常用钴铬合金进行了比较。结果：Ｔｉ－７５铸造后抗拉强度为８５６．７ＭＰａ，屈服强度为５４０ＭＰａ延伸率为７．３％。Ｔｉ－６Ａｌ－４Ｖ铸造后抗拉强度为９５６．７ＭＰａ，屈服强度为５９０ＭＰａ，延伸率为５．２％。Ｃｏ－Ｃｒ合金铸造后抗拉强度为６５１．２ＭＰａ，屈服强度为４７８．８ＭＰａ，延伸率为６．１％。结论：Ｔｉ－７５金属铸造后抗拉强度，屈服强度及延伸率等性能指标均优于钴铬合金，延伸率也高于Ｔｉ－６Ａｌ－４Ｖ合金。     

减阻牵张正畸牙移动过程中牙周组织超微结构变化的研究

目的：研究减阻牵张正畸牙移动过程中牙周组织超微结构的变化，为此方法矫治正畸牙提供理论依据。方法：将6只杂种犬随机分为2周、3周、6周二组，拔除下颌两侧第二前磨牙，随机选择一侧为实验侧，实施减阻牵张措施加快牙齿移动速度，另一侧为空白对照侧，实验侧加力2周、固定保持1周、4周时，制备移动牙标本，进行透射电镜观察。结果：对照侧无明显成骨及破骨迹象；加力2周组张力侧成骨细胞、成纤维细胞的胞浆内高尔基体、内质网等细胞器非常丰富，分泌功能旺盛；压力侧破骨细胞增多，高尔基体发达，线粒体、溶酶体增多，成纤维细胞细胞器发达，间充质细胞、活跃的巨噬细胞增多；随着固定保持时间的延长，实验侧与对照侧超微结构的差异变小。结论：正畸牙压力侧减阻后，在强、高频率加力方式作片用下，牙周组织细胞功能旺盛、改建活跃，无不可逆的细胞变性坏死变化。     

A comparison of enamelin and amelogenin expression in developing mouse molars

Amelogenin and enamelin are structural proteins in the enamel matrix of developing teeth. The temporal and spatial patterns of enamelin expression in developing mouse molars have not been characterized, while controversy remains with respect to amelogenin expression by odontoblasts and cementoblasts. Here we report the results of in situ hybridization analyses of amelogenin and enamelin expression in mouse molars from postnatal days 1, 2, 3, 7, 9, 14, and 21. Amelogenin and enamelin mRNA in maxillary first molars was first observed in pre-ameloblasts on the cusp slopes at day 2. The onsets of amelogenin and enamelin expression were approximately synchronous with the initial accumulation of predentin matrix. Both proteins were expressed by ameloblasts throughout the secretory, transition, and early maturation stages. Enamelin expression terminated in maturation stage ameloblasts on day 9, while amelogenin expression is still detected in maturation stage ameloblasts on day 14. No amelogenin expression was observed in day 21 mouse molars. Amelogenin and enamelin RNA messages were restricted to ameloblasts. No expression was observed in pulp, bone, or along the developing root. We conclude that amelogenin and enamelin are enamel-specific and do not directly participate in the formation of dentin or cementum in developing mouse molars.     

Establishment of cell free conversion system with biotin-labelled recombinant PrPsen expressed in E. coli

Objective To report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope. Methods A hamster PrP protein （HaPrP） was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrP^sen preparation from scrapie strain 263K. Results Protease-resistant bands were detected after four-day incubation. Conclusion The new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.     

Immune responses to trichloroethylene and skin gene expression profiles in Sprague Dawley rats

Objective To characterize the immune reaction in SD rats exposed to trichloroethylene （TCE） and to identify the gene expression profiles involved in skin after TCE exposure. Methods Fifteen percent of TCE was injected intradermally into the rat back （100 μL/120 g） at intervals of 7 days. Whole blood was collected 24 h after the fifth or seventh intradermic administration of TCE. The percentages of CD4＋ and CD8＋ of T lymphocytes were measured by a flow cytometer. The concentrations of IFN-gamma and 1L-4 in the serum were semi-quantified by ELISA. Total RNAs of skin samples at 3 h or 24 h after the seventh dose of TCE in SD rats were extracted, and gene expression proftles of these tissues were analyszed by rat toxicology U34 array of Affymetrix. Results Obvious decline of CD4＋ in T lymphocytes was observed in the TCE-administer group. No significant concentration differences in IFN-gamma and IL-4 were found between TCE-treated and control rats. Gadd45a and Mel were significantly up regulated in skin tissue 24 h after TCE exposure. The expression regulation of immune response factors was as active as proteins associated with lipid metabolism and synthesis process in these skin samples of SD rats exposed to TCE. Conclusion T-helper type 1 cells mediate immune response can not be elicited in TCE-treated SD rats, but certain immune disorder can be induced.