Porphyromonas gingivalis lipopolysaccharide promotes T-hel per17 cell differentiation by upregulating Delta-like ligand 4 expression on CD14+ monocytes.

BACKGROUD: To investigate the effect and mechanism of Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) on Th17 cell differentiation mediated by CD14+ monocytes. METHODS: P. gingivalis LPS-activated CD14+ monocytes were co-cultured with CD4+T cells in different cell ratios. An indirect co-culture system was also established using transwell chambers. Furthermore, anti- Delta-like ligand 4 (Dll-4) antibody was used to investigate the role of Dll-4 in Th17 cell response. The mRNA expression was analyzed using quantitative reverse transcription-polymerase chain reaction, and secreted cytokines in culture supernatant were detected using enzyme-linked immunosorbent assay. Flow cytometry was used to determine the frequencies of Th17 cells. IL-17 protein expression levels were determined using western blotting assay. RESULTS: P. gingivalis LPS increased the expressions of interleukin (IL)-1ß, IL-6, IL-23 and transforming growth factor (TGF)-ß in CD14+ monocytes. Th17 cell frequency upregulated, which is not solely cytokine-dependent but rather requires cell-cell contact with activated monocytes, particularly in the 1:10 cell ratio. Furthermore, P. gingivalis LPS increased t he expression of Dll-4 on CD14+ monocytes, whereas the anti- Dll-4 a ntibody decreased the response of Th17 cells. The results suggest that P. gingivalis LPS enhances Th17 cell response via Dll-4 upregulation on CD14+ monocytes.

TLR-4 targeting contributes to the recovery of osteoimmunology in periodontitis.

OBJECTIVE: The aim of this study was to determine the potential role of TLR-4 in the osteoimmunological imbalance of periodontitis. BACKGROUND: Although current evidence supports that TLR-4 plays an important role in the inflammatory response of periodontal tissues triggered by microorganisms, little information is available regarding the function of TLR-4 in the osteoimmune regulation of homeostasis in periodontitis. METHODS: Human gingival epithelial cells (HGEC) were isolated from the gingival tissues of 3 healthy volunteers and the expression of osteoclastogenic cytokines was evaluated by ELISA and real time RT-PCR. In addition, 30 C57BL/6 mice were used and randomly divided into three groups: control group, periodontitis group (CP) and periodontitis+TAK-242 (a specific inhibitor of TLR-4) group (TAK-242) and the expression of osteoclastogenic cytokines and the osteoclast density in the periodontal tissue were evaluated by immunohistochemical staining and tartrate resistant acid phosphatase staining. Moreover, micro-computed tomography (Micro-CT) was used to assess bone resorption. RESULTS: The in vitro results showed that TAK-242 blocked the overproduction of IL-1, IL-6, TNF-α and RANKL in HGEC treated with LPS. The in vivo results revealed that TAK-242 also effectively decreased these osteoclastogenic cytokines in periodontal tissue of mice with periodontitis. More importantly, Micro-CT analysis showed a significant reduction of the alveolar bone loss in the TAK-242 group compared with the CP group. Furthermore, the TRAP staining showed a significant lower density of osteoclasts in the alveolar bone area of the TAK-242 group. CONCLUSION: TLR-4 inhibition decreased the differentiation of osteoclast through the inhibition of the overproduction of osteoclastogenic cytokines and the prevention of the alveolar bone absorption in mouse periodontitis models. Therefore, the use of TAK-242 might contribute to the recovery of the osteoimmunological homeostasis and might provide a potential strategy to treat periodontal diseases.

The role of PHF8 and TLR4 in osteogenic differentiation of periodontal ligament cells in inflammatory environment.

BACKGROUND: Histone methylation is considered to play an important role in the occurrence and development of periodontitis. Plant homeodomain finger protein 8 (PHF8), a histone demethylase, has been shown to regulate inflammation and osteogenic differentiation of bone marrow stromal cells (BMSCs). This study aimed to detect the functions of PHF8 and TLR4 in osteogenic differentiation in an inflammatory environment induced by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) METHODS: A periodontitis mouse model was established, and the mice were treated with TAK-242. Immunohistochemical staining was used to detect the expression of PHF8 in periodontal tissue. Periodontal ligament cells (PDLCs) were treated with mineralization induction medium supplemented with Pg-LPS and/or TAK-242, and a Cell Counting Kit-8 (CCK-8) assay was used to detect the proliferation of PDLCs. Real-time PCR and western blotting were used to detect the mRNA and protein expression levels, respectively, of PHF8, toll-like receptor 4 (TLR4) and the other osteogenic markers alkaline phosphatase (ALP), osteocalcin (OCN), Special AT-rich sequence-binding protein 2 (Satb2) and Runt-related transcription factor 2 (Runx2) RESULTS: Periodontitis reduced PHF8 expression in periodontal tissue, and TAK-242 partially reversed this downregulation. An in vitro experiment revealed that the mRNA and protein expression levels of PHF8 were significantly upregulated during the osteogenic differentiation of PDLCs. Alizarin red staining showed that the mineralized nodules of PDLCs in osteogenic induction group were more than those in control group. Real-time PCR and western blot results indicated that Pg-LPS inhibited PHF8 expression and upregulated TLR4 expression in PDLCs. TAK-242 inhibited TLR4 and partially reversed the inhibition of PHF8 expression and osteogenic differentiation induced by Pg-LPS in PDLCs CONCLUSION: PHF8 and TLR4 play important roles in periodontitis. Pg-LPS inhibits the expression of PHF8 via upregulation of TLR4 and might further inhibit the osteogenic differentiation of PDLCs. However, the specific mechanisms involved remain to be explored.

Effect of interleukin-22 on osteogenic differentiation and the osteoclastogenic response of human periodontal ligament fibroblasts in vitro.

BACKGROUND: Interleukin-22 (IL-22) exerts extensive biological effects, playing both protective and pathological roles in autoimmune and infectious diseases. However, the specific role and mechanism of IL-22 in the pathogenesis of periodontitis have not been clarified. The aim of this study was to analyze the possible roles of IL-22 in the osteoclastogenesis and osteogenesis of periodontitis. METHODS: Human periodontal ligament fibroblasts (hPDLFs) were treated with IL-22 and/or lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS), and the mRNA and protein expression of RANKL and OPG were measured by qRT-PCR and Western blotting, respectively. Western blotting was also used to examine the phosphorylated and total protein expression of MAPK signaling molecules. The role of the MAPK pathway in osteoclastogenesis marker expression was further confirmed by inhibition assays. For osteogenic assays, the mRNA expression of osteoblastic markers was quantified by qRT-PCR, the alkaline phosphatase (ALP) activity of hPDLFs was measured by an ALP assay, and the mineralized nodules formed by hPDLFs were determined by Alizarin Red S staining. RESULTS: IL-22 promoted the expression of RANKL in hPDLFs via the MAPK signaling pathway and further upregulated RANKL expression together with Pg-LPS via the p38 MAPK pathway. IL-22 could enhance the ALP activity and mineralized nodule formation of hPDLFs in the early period of osteogenic induction, while exhibiting no profound effect on the expression of osteoblastic markers. CONCLUSION: IL-22 plays regulatory roles in bone homeostasis, and it is likely to contribute to osteoclastogenesis as a proinflammatory cytokine in the pathogenesis of periodontitis.

Porphyromonas gingivalis lipopolysaccharide promotes T- helper 17 cell differentiation from human CD4+ naïve T cells via toll-like receptor-2 in vitro.

OBJECTIVES: The persistence of T-helper 17 (Th17) cells has been shown to support chronic inflammation and mediate tissue destruction in periodontitis. However, little is known regarding the underlying mechanisms that regulate Th17 cell differentiation in the periodontal inflammatory context. The objective of this study was to explore the possible effect and mechanism of lipopolysaccharide (LPS) from Porphyromonas gingivalis on Th17 cell differentiation. METHODS: Activated human CD4+CD45RA+ naïve T cells were stimulated with different doses of LPS from virulent and avirulent P. gingivalis strains combined with Th17 driven cytokines in vitro. Flow cytometry was used to analyze the differentiation ratio of Th17 cells. IL-17A protein expression and IL-17, retinoid-related orphan receptor C (RORC) and toll-like receptor 2 (TLR2) mRNA transcription were analysed by ELISA and real-time qPCR, respectively. The role of TLR2 in Th17 cell differentiation was further confirmed by TLR2 blocking assay. RESULTS: LPS from P. gingivalis (Pg-LPS) up-regulated Th17 cell differentiation ratios, expression of IL-17 and RORC mRNA, and IL-17 concentration in culture supernatant, with 0.1 µg/mL LPS from the virulent P. gingivalis strain being the most effectively. Furthermore, Pg-LPS also up-regulated expression of TLR2 on T cells during Th17 differentiation, and the differentiation was attenuated by treatment with TLR2 antibody. CONCLUSIONS: These results suggest that Pg-LPS promotes Th17 cell differentiation in vitro, and TLR2 signalling may be involved in this process. LPS from the virulent P. gingivalis strain up-regulated Th17 cell differentiation more effectively, which may be associated with the pathogenicity of different P. gingivalis strains.

A simple way to intrude overerupted upper second molars with miniscrews.

Various methods of using skeletal anchorage for the intrusion of overerupted maxillary molars have been reported; however, it is difficult to intrude the overerupted upper second molars because of the low bone density in the region of the tuberosity. This article illustrates a new treatment method using partial fixed edgewise appliances and miniscrews to intrude the overerupted upper second molars. The miniscrews were applied to reinforce the anchorage of the upper first molar. The intrusive force was generated by the Ni-Ti wire. The clinical results showed a significant intrusion effect without root resorption or periodontal problems. This report demonstrates that the combination of partial conventional fixed appliances with miniscrews is a simple and effective treatment option to intrude overerupted upper second molars, especially in situations where miniscrews cannot be inserted directly next to the second molar.

[Detection and functional analysis of CD-4+ CD-25+ regulatory T cells in the peripheral blood of patients with generalized aggressive periodontitis].

OBJECTIVE: To investigate the changes of proportion and suppression function of CD-4+ CD-25+ regulatory T cells in the peripheral blood of patients with aggressive periodontitis. METHODS: Flow cytometric analysis was used to detect the frequency of CD-4+ CD-25+ regulatory T cells in the peripheral blood of 16 patients with generalized aggressive periodontitis and 17 patients with chronic periodontitis, as well as 17 periodontal healthy controls. Furthermore, CD-4+ CD-25+ regulatory T cells and CD-4+ CD-25- T cells were separated from peripheral blood of each enrolling subject using magnetic cell sorting technology. The direct suppression effect of CD-4+ CD-25+ regulatory T cells on CD-4+ CD-25- T lymphocytes proliferation was performed by the mixed lymphocytes reaction and measured by 3H-thymidine radioactive assay. RESULTS: The patients with generalized aggressive periodontitis had a lower frequency of CD4+ CD-25+ regulatory T cells (9.71 +/- 4.01)% in the peripheral blood than periodontal healthy controls [(14.72 +/- 3.51)%] and chronic periodontitis patients [(17.01 +/- 5.16 )%], P < 0.05. A significant decrease was found in the suppression function of CD-4+ CD-25+ regulatory T cells from peripheral blood of patients with generalized aggressive periodontitis when co-cultured with CD-4+ CD-25- T lymphocytes in the proportion of 2 : 1, 1 : 1 and 1 : 2 as compared with chronic periodontitis patients and periodontal healthy controls (P < 0.05). CONCLUSIONS: Diminished numbers and decreased suppression function of CD-4+ CD-25+ regulatory T cells were found in patients with generalized aggressive periodontitis.

[Association of TNFA-308 gene polymorphisms with susceptibility to chronic periodontitis in Chinese patients].

PURPOSE: The purpose of this study was to investigate the relationship between TNFalpha-308 gene polymorphisms and susceptibility to chronic periodontitis in Chinese patients. METHODS: DNA samples with buccal swabs were collected from 63 severe CP patients, 103 initial to moderate CP patients, and 80 healthy controls in Chinese Han nationality. The TNFA-308 gene polymorphisms were analyzed with PCR-RFLP. The data were analyzed by X(2) test using SPSS10.0. RESULTS: There were no significant genotype distribution differences of TNFA-308 between either two of the three subject groups. CONCLUSION: Further study was needed since we did not find the relationship between TNFA-308 gene polymorphism and periodontitis in this present study.

[Bioassay of the soluble human tumor necrosis factor receptor I recombinant plasmid expression in vitro].

OBJECTIVE: To detect the expression of recombinant plasmid PcDNA3.1-sTNFRI in vitro and evaluate the bioactivity of expressed sTNFRI. METHODS: CHO cells were transfected with recombinant plasmid PcDNA3.1-sTNFRI by liposome. sTNFRI in cell culture supernatant was detected by ELISA and sTNFRI blockage of TNF-alpha cytotoxicity in L929 cells evaluated by MTT assay. RESULTS: The expression of sTNFRI in transfected cell culture supernatant was higher than control groups (P < 0.001). The expressed sTNFRI could significantly neutralize TNF-alpha cytotoxicity in L929 cells. CONCLUSIONS: These results showed that the recombinant plasmid PcDNA3.1-sTNFRI can be expressed in mammalian cells and the recombinant sTNFRI has biological function.

[Extraction and purification of porcine amelogenin and preparation for the polyclonal amelogenin antibody].

OBJECTIVE: To prepare the polyclonal antibody to amelogenin. METHODS: The fetal porcine dental enamel was collected. Enamel matrix protein was extracted in 4M guanidine HCl (pH 7.4) with protease inhibitors present. Polyacrylamide gel filtration was included to isolate amelogenin from the initial dissociated extraction. The purified amelogenin conjugated with or without complete/incomplete Freund's adjuvant was then used to immunize the rabbits subcutaneously or intravenously. The specific IgG antibody was further purified by DE-52 cellulose. The working concentration of IgG antibody was determined through ELISA test. RESULTS: The Gel filtration showed that amelogenin components is at molecular weights of 15 kD and 13 kD apparently, which was consistent with those described before. The ELISA results showed that the working concentration for IgG was 1:1000. CONCLUSION: The antibody prepared in this study can be used for the detection of amelogenin.

[Construction of the eukaryotic expression vector PsecTaq2A-AMG for human amelogenin].

OBJECTIVE: The purpose of this study was to construct a eukaryotic expression vector for human amelogenin (AMG). METHODS: PCR was performed to amplify the AMG encoding region. Amplified fragments for human AMG were recovered and inserted into eukaryotic expression vectors PsecTaq2A. The recombinant plasmid PsecTaq2A-AMG was constructed and their positive clones were identified. RESULTS: 1. Amplified products were checked by electrophoresis and the results were satisfactory. 2. The recombinant plasmid PsecTaq2A-AMG was analyzed by restriction endonuclease mapping and DNA sequencing. The results of sequencing were consistent with those from GenBank. CONCLUSION: The recombinant plasmid PsecTaq2A-AMG was successfully constructed with properly inserted DNA sequence encoding mature amelogenin.