The extent of periodontal disease and the IL-6 -174 genotype as determinants of serum IL-6 level

AIM: To study the extent of periodontal disease and the IL-6(-174) genotype as determinants of serum and mouthwash IL-6 concentration in subjects with moderate to severe periodontal disease. MATERIAL AND METHODS: Fifty-two generally healthy subjects volunteered to participate. Probing pocket depth (PD) and periodontal attachment level (AL) were clinically examined and alveolar bone level (BL) was measured on orthopantomographs. IL-6 concentrations in mouthwash, collected by rinsing with 3 ml saline for 30 s and in serum, obtained by venipuncture, were measured using ELISA. IL-6(-174) polymorphism was studied using a polymerase chain reaction. RESULTS: Eleven subjects carried the GG genotype, and 41 subjects, carried the CG/CC genotype. The mean (+/- SD) concentration of IL-6 in serum was 1.6 (+/- 1.5) pg/ml and, 2.8 (+/- 5.04) pg/ml in mouthwash. The serum concentration of IL-6 was higher in subjects with the GG genotype than with the CG/CC genotype. In regression analyses the percentages of sites with PD> or =6 mm, AL> or =6 mm and BL> or =8 mm, the IL-6(-174) genotype, body mass index and gender associated significantly with serum IL-6 concentration. CONCLUSIONS: The extent of moderate to severe periodontal disease and the IL-6(-174) genotype contribute significantly to serum IL-6 concentration.     

Interleukin 4 (IL-4) and IL-6-producing memory T-cells in peripheral blood and gingival tissues in periodontitis patients with high serum antibody titers to Porphyromonas gingivalis

We assessed cytokine production and proliferation of memory T-cells that were isolated from peripheral blood of adult periodontitis patients with high anti- Porphyromonas gingivalis titer. Memory T-cells were stimulated with P. gingivalis lipopolysaccharide, sonicates and formalin-killed whole cells. Interleukin 4 (IL-4)- and IL-6-producing cells were stained by immunocytochemically on peripheral blood smears and compared with cryostat sections of autologous gingival biopsies. Memory T-cells in the peripheral blood of patients rated significantly higher than in healthy subjects (32.3±7.1 vs 25,3±3.0%). Stimulation of patient-derived memory T-cells with P. gingivalis whole cells induced higher IL-4 production than in healthy subjects (4.4±4.1% vs 0.7±0.6%). Induction of IL-4-producing memory T-cells by P. gingivalis lipopolysaccharide and whole cells was respectively 1.37 and 1.56 times that induced by medium alone. IL-6 production did not differ between the groups. Proliferation of memory T-cells in healthy subjects tended to be more inhibited by P. gingivalis antigens than that in patients. In some patients, induction of IL-4- and IL-6-producing memory T-cells in peripheral blood and in autologous gingival biopsies tended to coincide. Memory T-cells with functional characteristics of Th2 could be a crucial cell population capable of reflecting individual susceptibility to periodontitis.     

IL—6对人牙髓细胞,牙周膜细胞生长影响的实验研究

目的：了解ＩＬ－６对构成牙髓、尖周组织主要细胞生长的影响，揭示ＩＬ－６在牙髓、尖周病变过程中的作用。方法：应用细胞培养技术，采用ＭＴＴ比色法进行细胞生长及存活状态测定。结果：ＩＬ－６抑制牙髓细胞、牙周膜细胞的生长，并呈时间和剂量依赖关系，两细胞间无显著差异。结论：ＩＬ－６可能对牙髓、尖周组织损伤的修复和代谢功能有一定影响     

Polymorphisms in the CD14 and IL-6 genes associated with periodontal disease

AIM: To compare the frequencies of cytokine and receptor molecule genotypes in patients with chronic periodontitis with the corresponding frequencies in a reference population and to study the relationship between periodontal disease severity and polymorphisms in the studied genes. SUBJECTS AND METHODS: CD14, IL-6, TNF-alpha, IL-10, IL-1alpha, IL-1beta, and TLR-4 polymorphisms of 51 periodontitis patients were studied using polymerase chain reaction. The genotype frequencies in the periodontitis patients and a reference population (n=178) were compared. Probing pocket depth (PD), periodontal attachment level (AL), and alveolar bone level (BL) were related to the genotypes. RESULTS: No statistically significant differences could be found between the frequencies of the cytokine genotypes in the periodontitis patients and in the reference group. The extent of periodontal disease was higher in subjects with the T-containing genotype of CD14(-260) and the GG genotype of IL-6(-174) when compared with the extent in the rest of the group. Subjects carrying the composite genotype of the above two were most severely affected by periodontal disease. CONCLUSION: According to the present results, an evident association exists between the carriage of the T-containing genotype of CD14(-260) and the GG genotype of IL-6(-174) and the extent periodontal disease.     

CD45RA and CD45RO positive CD4 cells in human peripheral blood and periodontal disease tissue before and after stimulation with periodontopathic bacteria

Flow cytometric analysis was used to examine naive and primed or memory CD4 cells extracted from periodontal lesions compared with cells from peripheral blood of healthy subjects before and after stimulation with the periodontopathic bacteria, Porphyromonas gingivalis and Fusobacterium nucleatum. In peripheral blood, approximately 60% and 40% of CD4 cells were CD45RO+ and CD45RA+ respectively at day 0. Phytohaemagglutinin (PHA) induced CD45RO expression on almost 100% of CD4 cells. However, P. gingivalis and F. nucleatum stimulation did not cause any significant change in percentage of CD45RO+ CD4 cells except for a loss of antigen at day 6 together with re-expression at day 7, which also occurred on cells cultured in medium only. CD45RA expression on PHA and bacterial-stimulated peripheral blood CD4 cells remained fairly stable for the 10-d culture period. Greater than 90% CD4 cells extracted from healthy or marginal gingivitis (H/MG) and adult periodontitis (AP) lesions were CD45RO+ and this was maintained on AP cells throughout the 6-d culture period, except for a small decrease in the percentage of positive cells induced by P. gingivalis at day 3. Approximately 9% CD4 cells from H/MG tissue were CD45RA+, but about 22% AP cells expressed this antigen, and this increased again in P. gingivalis- and F. nucleatum-stimulated cultures after 3 d. Therefore, in peripheral blood P. gingivalis and F. nucleatum do not act as nonspecific T-cell mitogens and, in AP cells, these bacteria induce changes in phenotype, supporting previous data that although they may be polyclonal B-cell activators, they activate antigen specific T-cells.     

Immunohistological analysis of memory T lymphocytes and activated B lymphocytes in tissues with periodontal disease

Memory T-cells and activated B-cells were identified in cryostat sections of adult periodontitis (AP) lesions and categorized in terms of frequency and distribution. Nineteen periodontitis biopsies were obtained at the time of periodontal surgery to remove residual periodontal pockets following the completion of initial preparation. Gingival tissues exhibited various degree of inflammation (Gl of 0-2) but probing depths of < 4 mm and < 5 mm loss of attachment. As a control, 5 gingivitis specimens (Gl of 1, probing depth and loss of attachment of ≤ 3 mm) were obtained from premolar and third molar sites requiring extraction for either orthodontic treatment or pericoronitis. Serial cryostat sections (6 μm in thickness) were prepared from each biopsy, on which a double staining avidin-biotin immunoperoxidase and avidin-biotin alkaline phosphatase technique was used to identify CD4+, CD45RO+ memory T-cells and activated CD19+ B-cells expressing CD23 or CD25. In periodontitis lesions the mean percentage of CD4+ cells expressing CD45RO was consistently high (65.9% in the crevicular (C) one-third (1/3), 61.2% in the middle (M) 1/3 and 62.5% in the oral (O) 1/3). This contrasts with the low mean percentage of CD4+, CD45RA+ naive T-cells (17.1% in the C 1/3, 14.8% in the M 1/3 and 12.4% in the O 1/3). In gingivitis specimens, the incidence of CD4+, CD45RO+ was 81.9% in the C 1/3, 81.1% in the M 1/3 and 89.0% in the O 1/3. This was higher than that of periodontitis biopsies. With CD4+, CD45RA+ the incidence was 10.0% in the C 1/3, 8.0% in the M 1/3, and 6.6% in the O 1/3 and the relationship to the periodontitis biopsies was reversed. However, the percentage of CD23+ and CD25+, CD19+ B-cells which were identified in 13 out of 19 samples from periodontitis varied significantly (0-100% for CD23, 0-36.2% for CD25) in spite of similar clinical status. The frequency of B-cells and activated B-cells in the gingivitis was much lower than that of periodontitis. These results indicate that both T-cells and B-cells were in active stage in periodontitis lesions. Differences of immunohistological features between gingivitis and periodontitis may be attributable to the heterogeneity of profiles of cytokine production by CD4+, CD45RO+“memory’ cells.     

牙龈卟啉菌和大肠杆菌内毒素对人牙周膜细胞分泌IL-6、TNF-α的影响

目的：研究龈卟啉菌、大肠杆菌内毒素对人牙周膜细胞（PDLC）分泌IL-6、TNF-α的影响。方法：采用细胞培养技术和ELISA方法，检测培养上清中IL-6、TNF-α水平。结果：在孵育6h后，即可在培养上清中检测到IL-6和TNF-α。IL-6在12h、TNF-α在24h内呈时间依赖性方式升高。结论：PDLC在内毒素作用下局部分泌IL-6、TNF-α，参与了牙周炎的发生、发展过程。     

力增强对大鼠牙周组织IL-6 mRNA表达影响的研究

目的 :检测不同牙合力状态下 ,大鼠牙周膜成纤维细胞和牙槽骨成骨细胞中IL - 6mRNA的动态表达 ,探讨IL - 6在牙周组织改建过程中的分子机制。方法 :选用Wistar大鼠建立正常牙合力、牙合力增强的动物模型 ,采用原位杂交的方法 ,观察成纤维细胞和成骨细胞中IL - 6mRNA表达的动态变化。结果 :牙合力增强诱导成纤维细胞和成骨细胞中IL - 6mRNA表达较正常牙合力时明显增强。结论 :牙合力增强 ,促使牙周组织中的成纤维细胞和成骨细胞产生IL - 6mRNA明显增多 ,且出现一定的规律性变化 ,提示IL - 6在牙合力影响牙周组织改建的过程中起着重要的调节作用。     

He力增强对大鼠牙周组织IL—6mRNA表达影响的研究

目的：检测不同He力状态下，大鼠牙周膜成纤细胞和牙槽骨成骨细胞中IL－6mRNA的动态表达，探讨IL－6在牙周组织改建过程中的分子机制。方法：选用Wistar大鼠建立正常He力、He力增强的动物模型，采用原位杂交的方法，观察成纤细胞和成骨细胞中I-6mRNA表达的动态变化。结果：He力增强诱导成纤维细胞和成骨细胞中IL-6mRNA表达较正常He力时明显增强。结论：He力增强，促使牙周组织中的成纤维细胞和成骨细胞产生IL－6mRNA明显增多，且出现一定的规律性变化，提示IL－6在He力影响牙周组织改建的过程中起着重要的调节作用。     

羧甲基壳聚糖对LPS干预人牙周膜成纤维细胞表达IL-6的影响

目的：脂多糖（LPS）干预下,不同浓度羧甲基壳聚糖（CMC）对人牙周膜成纤维细胞（HPDLFs）表达白细胞介素-6（IL-6）的影响。方法：取冻存的HPDLFs进行复苏培养,以浓度为10mg／L的LPS进行诱导,用不同浓度的羧甲基壳聚糖进行干预,采用免疫组织化学方法检测IL-6在HPDLFs中的表达变化。结果：不同浓度羧甲基壳聚糖能够下调同一浓度LPS诱导的HPDLFs表达的IL-6,随羧甲基壳聚糖浓度的增加,IL-6的表达量呈逐渐减弱趋势（P〈0．05）。结论：羧甲基壳聚糖可能对LPS诱导的HPDLFs炎症损伤过程中表达IL一6有重要的抑制作用,为牙周病治疗提供新的实验资料。     

In situ hybridization study on tissue inhibitors of metalloproteinases (TIMPs) mRNA-expressing cells in human inflamed gingival tissue

This study presents the exact cell types and localization of tissue inhibitors of metalloproteinases (TIMPs) production sites in periodontal diseased gingiva by means of in situ hybridization. Gingival tissue specimens were fixed, embedded and hybridized in situ with specific digoxigenin-labeled cRNA probes (386 and 496 bp). TIMP-1 and -2 mRNAs were expressed on macrophages, mononuclear cells, capillary endothelial cells and some fibroblasts throughout the gingival tissue. In periodontitis, TIMP-1 and -2 mRNA-expressing cells showed significantly different localization. TIMP-1 mRNA was broadly observed in the gingival connective tissue while TIMP-2 mRNA was predominantly expressed in the connective tissue adjacent to the pocket epithelium (p < 0.01). Fewer TIMPs mRNA were observed in minimal gingivitis than in periodontitis, especially in the middle zone of gingival tissue. Thus, TIMP-1 and TIMP-2 mRNA was detected differentially and site-specifically in periodontal diseased gingival tissue.     

IL-6−174 genotype associated with the extent of periodontal disease in type 1 diabetic subjects

Aim: The aim of this study was to investigate whether genetic polymorphism in certain cytokine and receptor molecule genes and diabetic status associate with the extent of periodontal disease in type 1 diabetes mellitus (DM).
Material and Methods: Eighty patients with type 1 DM participated. Visible plaque, bleeding on probing (BOP), probing pocket depth (PD) and attachment level (AL) were examined clinically and glycosylated haemoglobin (HbA1c) levels were used to assess the glycemic control of DM. CD-14, IL-6, TNF- α , IL-10, IL-1 α , IL-1 β and TLR-4 gene polymorphisms were studied using the polymerase chain reaction (PCR).
Results: The 3-year HbA1c was good (<7.5%) in 16%, acceptable (7.5–8.5%) in 36% and poor (>8.5%) in 48% of the subjects. IL-6⁻¹⁷⁴ genotype and 3-year GHbA1c associated significantly with BOP and PD4 mm, subjects with the GG genotype of the IL-6⁻¹⁷⁴ exhibiting more severe periodontal disease than those with the GC/CC genotype. After stratification by IL-6 genotype, associations between the extent of periodontal disease and 3-year HbA1c levels remained significant in subjects carrying the GC/CC but not the GG genotype.
Conclusions: In addition to the HbA1c level, the IL-6⁻¹⁷⁴ genotype is a significant susceptibility factor for periodontal disease among type 1 diabetics.     

Characterizing traditionally defined periodontal disease in HIV+ adults

Abstract – Background: Results have varied from previous studies examining the level and extent of periodontal disease (PD) in HIV‐1 infected (HIV+) adults. These studies used different methodologies to measure and define PD and examined cohorts with divergent characteristics. Inconsistent methodological approaches may have resulted in the underestimation of traditionally‐defined PD in HIV+ individuals. Objectives: To characterize the level, extent and predictors (i.e. immunologic, microbiologic, metabolic and behavioral) of PD in an HIV+ cohort during the era of highly active antiretroviral therapy (HAART). Study Design: Cross‐sectional study. Setting: HIV+ adults receiving outpatient care at three major medical clinics in Cleveland, OH. Subjects were seen from May, 2005 to January, 2008. Measurements: Full‐mouth periodontal examinations included periodontal probing depth (PPD), recession (REC) and clinical attachment level (CAL). Subgingival plaque was assessed for DNA levels of Porphyromonas gingivalis (Pg), Tannerella forsythia, and Treponema denticola by real‐time DNA PCR assays developed for each pathogen. Rather than using categories, we evaluated PD as three continuous variables based on the percent of teeth with ≥1 site per tooth with PPD ≥ 5mm, REC > 0 mm and CAL ≥ 4mm. Results: Participants included 112 HIV+ adults. Each subject had an average 38% (±24%) of their teeth with at least one site of PD ≥ 5 mm, 55% (±31%) of their teeth with at least one site of REC > 0 mm, and 50% (±32%) of their teeth with at least one site of CAL ≥ 4 mm. CD4+ T‐cell count <200 cells/mm³ was significantly associated with higher levels of REC and CAL, but not PPD. Greater levels of Pg DNA were associated with PPD, REC and CAL. By regression analysis, CD4+ T‐cell count <200 cells/mm³ had approximately twice the deleterious effect on CAL as did smoking (standardized β coefficient 0.306 versus 0.64). Annual dental visit compliance remained an independent predictor for lower levels of PD. Conclusions: The level and extent of PD were high in this cohort even though most patients were being treated with HAART. The definition of periodontal disease used and cohort characteristics examined can influence the level of periodontal disease reported in studies of persons with HIV. Traditional periodontal pathogens are associated with PD in this cohort. Those with CD4+ T‐cell counts <200 cells/mm³ are at greater risk for PD. Therefore, earlier HAART initiation may decrease exposure to immunosuppression and reduce PD morbidity. Continuity of dental care remains important for HIV+ patients even when they are being treated with HAART.     

Interleukin-6 production by human gingival fibroblasts

The ability of human gingival fibroblasts to synthesize interleukin-6 (IL-6) was studied using in vitro and immunohistochemical techniques. Culture supernatants of human gingival fibroblasts contained significant quantities of IL-6 activity which could be stimulated by fetal calf serum, recombinant interleukin-1 beta and lipopolysaccharide. The activity in the supernatants was specifically attributed to IL-6 since up to 97% of the activity could be inhibited by an anti-IL-6 antibody. Immunohistochemical studies on low-density human gingival fibroblast cultures indicated that the cells were associated with material reactive to the anti-IL-6 antibody. This localization was seen on the cell surface and in the cytoplasm of the cells. Immunoreactivity towards IL-6 was also noted in sections of human gingivae. Moderate staining was seen in the connective tissues and lower portions of the gingival epithelium, while intense staining was seen at foci of inflammation. The identification of IL-6 with human gingival tissues and cells implicates this lymphokine in the molecular events associated with the inflammatory periodontal diseases.     

Phenotypic analysis of B-cells extracted from human periodontal disease tissue

B-cells extracted from periodontal disease tissue were analyzed for the presence of activation markers using a range of monoclonal antibodies. In adult periodontitis (AP), 6% of B-cells expressed the IL-2 receptor (CD25) compared with 1-2% in peripheral blood and healthy or marginal gingivitis (H/MG) gingival B-cells. There was also an increase in the mean percentage of IgD-positive B-cells and a decrease in CD21 and CD22 expression. In both AP and H/MG lesions, 20-22% of the B-cells expressed CD23 compared with less than 5% in peripheral blood. As B-cells are activated by day 3 in culture and start differentiating into immunoglobulin-secreting cells by day 6, B-cell phenotypes were assayed at these times in this study. Following stimulation with the periodontopathic bacterium Porphyromonas gingivalis, the expression of CD23, CD21 and CD22 on B-cells extracted from AP lesions remained relatively constant over the 6-d culture period. However, with Fusobacterium nucleatum stimulation, there was a significant decrease in CD23, CD21 and CD22 expression after 3 d in culture, which corresponds to the activation time for B-cells. These results show that B-cells extracted from periodontal disease tissue display a range of activation markers and on stimulation, demonstrate differing responses to individual periodontopathic bacteria.     

T-cell lymphoma associated with periodontal disease and HIV infection

Abstract A case is described of gingival non-Hodgkins lymphoma presenting in a site previously diagnosed as HIV-periodontitis. The lymphoma presented along with other signs of HIV infection and AIDS, which taken together were compatible with increased immunosuppression and reactivation of Epstein-Barr virus. The implications of these findings are discussed, and it is suggested that areas of gingiva which show rapid localised recession, associated with HIV seropositivity, should be monitored closely and considered for biopsy if abnormal signs are detected.     

Antigen-presenting cells in human periodontal disease tissues

T cells are present in the inflammatory infiltrates of periodontal disease lesions and require antigen presentation by antigen-presenting cells (APCs). While it is still not known whether Th1 or Th2 cells predominate in these lesions, it has been reported that different APCs may induce activation of different T-cell subsets. An immunoperoxidase technique was used to investigate the presence of CD1a+, CMRF-44+, CMRF-58+ and CD83+ dendritic cells, CD14+ macrophages or dendritic cell precursors and CD19+ B cells in gingival biopsies from 21 healthy or gingivitis and 25 periodontitis subjects. The samples were divided into three groups according to the size of infiltrate (group 1, small infiltrates; group 2, medium infiltrates; group 3, extensive infiltrates). The presence of numerous CD1a+ Langerhans cells was noted in the epithelium with no differences between the healthy/gingivitis and periodontitis groups. The percentage of CD83+ dendritic cells in the infiltrates was higher than the percentage of CD1a+, CMRF-44+ or CMRF-58+ dendritic cells. Endothelial cells positive for CD83 were found predominantly in areas adjacent to infiltrating cells, CD83+ dendritic cells being noted in the region of CD83+ endothelium. The percentage of CD14+ cells in the inflammatory infiltrates was similar to that of CD83+ dendritic cells. B cells were the predominant APC in group 2 and 3 tissues. The percentage of B cells in group 3 periodontitis lesions was increased in comparison with group 1 periodontitis tissues and also in comparison with group 3 healthy/gingivitis sections. Functional studies are required to determine the roles of different APC subpopulations in periodontal disease.     

槲皮素对LPS刺激的人牙周膜细胞表达IL-6的影响

目的研究槲皮素对接受大肠杆菌脂多糖(lipopolysaccharide,LPS)刺激的人牙周膜细胞(human periodontal ligament cells,h PLDCs)表达IL-6(interleukin-6)的影响,探讨槲皮素(quercetin,Que)的抗炎作用。方法使用酶消化组织块法培养人牙周膜细胞并鉴定,根据ELISA(enzyme-linked immunosorbent assay,酶联免疫吸附实验)法测定不同时间点以及不同浓度下槲皮素对人牙周膜细胞抑制LPS诱导细胞表达IL-6的作用。结果 LPS刺激24 h左右时人牙周膜细胞分泌IL-6达到峰值,而槲皮素处理后的细胞再加入LPS刺激所产生的IL-6与单纯的LPS组相对比明显降低,有统计学意义。经不同浓度槲皮素处理细胞后加入LPS刺激所产生的IL-6无统计学差异。结论槲皮素可以抑制LPS诱导的人牙周膜细胞表达IL-6,提示槲皮素可能通过某些途径调节影响IL-6的合成和分泌,因而在炎症的早期发挥其减轻炎症反应的作用。     

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目的通过观察分析中重度牙周炎患者血清中对氧磷酶-1（PON1）活性及白细胞介素-6（IL-6）在牙周基础治疗前后的变化,探讨PON1和IL-6与牙周病之间的相关性以及两者在牙周病病情变化中的作用。方法2012年5月至2013年2月在辽宁省人民医院体检中心体检的牙周健康人群30名（牙周健康组）及口腔科就诊的中重度牙周炎患者39例（牙周炎组）,对所有受试者进行基础治疗,检查治疗前后PON1、IL-6以及临床牙周检查指标。结果牙周健康组与牙周炎组在基础治疗前比较：牙周炎症指标[出血指数（BI）、牙周探诊深度（PD）]及血清IL-6差异均有统计学意义（P〈0．05）,血清中PON1活性差异无统计学意义（P〈0．05）。牙周健康组在牙周基础治疗前后各项指标差异均无统计学意义（P〉0．05）。牙周炎组在牙周基础治疗前后比较：牙周炎症指标BI、PD、血清PON1及IL-6与治疗前比较差异均有统计学意义（均P〈0．05）。结论牙周健康人群和中重度牙周炎患者血清PON1活性无差异;牙周基础治疗可改善中重度牙周病患者牙周健康状况,提高血清中PON1活性,降低IL-6水平;中重度牙周炎患者在牙周基础治疗前后血清中PON1活性和IL-6水平的变化存在负相关性。