Toll‐like receptors 2 and 5 in human gingival epithelial cells co‐operate with T‐cell cytokine interleukin‐17

Background/aim: Periodontitis begins as the result of perturbation of the gingival epithelial cells caused by subgingival bacteria interacting with the epithelial cells via pattern recognition receptors. Toll‐like receptors (TLRs) have been shown to play an important role in the recognition of periodontal pathogens so we have studied the interaction of TLR ligands with TLR2 and TLR5 for cytokine production in the cultures of gingival epithelial cells. Methods: Immunohistochemistry was used for the localization of TLR2 and TLR5 in tissue specimens. Enzyme‐linked immunosorbent assays were performed to detect the levels of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), released from gingival epithelial cell cultures following stimulation with TLR ligand alone or in combination with IL‐17. Results: Both TLR2 and TLR5 were increased in periodontitis (2128 ± 159 vs. 449 ± 59 and 2456 ± 297 vs. 679 ± 103, respectively, P < 0.001) including gingival epithelial cells that stained strongly. Cultured gingival epithelial cells stimulated with their respective ligands (HKLM, a TLR2 ligand that is also found in Porphyromonas gingivalis, and flagellin, a TLR5 ligand that is also found in Treponema denticola) produced both IL‐1β and TNF‐α. To mimic T‐cell help, IL‐17 was added. This further greatly enhanced TLR ligand‐induced IL‐1β (P < 0.001) and TNF‐α (P < 0.01) production. Conclusions: These findings show how pathogen‐associated molecular patterns, shared by many different periodontopathogenic bacteria, stimulate the resident gingival epithelial cells to inflammatory responses in a TLR‐dependent manner. This stimulation may be particularly strong in periodontitis and when T helper type 17 cells provide T‐cell help in intercellular cooperation.     

Comparison of CCL28, interleukin‐8, interleukin‐1β and tumor necrosis factor‐alpha in subjects with gingivitis,chronic periodontitis and generalized aggressive periodontitis

Background and Objective: Cytokines produced by various cells are strong local mediators of inflammation. Mucosa‐associated epithelial chemokine (CCL28), interleukin‐8 (IL‐8), interleukin‐1beta (IL‐1β) and tumor necrosis factor‐alpha (TNF‐α) are major cytokines that play important roles in the periodontal inflammatory process. In this study we aimed to compare the levels of CCL28, IL‐8, IL‐1β and TNF‐α in the gingival crevicular fluid of both periodontally healthy subjects and in subjects diagnosed with gingivitis, chronic periodontitis and generalized aggressive periodontitis. Material and Methods: A total of 84 subjects participated in the study: 21 subjects had gingivitis, 21 subjects had chronic periodontitis, 21 subjects had generalized aggressive periodontitis and 21 were periodontally healthy. The levels of CCL28, IL‐8, IL‐1β and TNF‐α were analyzed using enzyme‐linked immune sorbent assay (ELISA). Results: The total levels of CCL28 and IL‐8 in the gingival crevicular fluid of the generalized aggressive periodontitis group (324.74 ± 42.62 pg/30 s, 487.62 ± 49.21 pg/30 s) were significantly higher than those of the chronic periodontitis group (268.81 ± 28.64 pg/30 s, 423.65 ± 35.24 pg/30 s), the gingivitis group (146.35 ± 17.46 pg/30 s, 310.24 ± 48.20 pg/30 s) and the periodontally healthy group (92.46 ± 22.04 pg/30 s, 148.41 ± 24.64 pg/30 s). Similarly, the total levels of IL‐1β and TNF‐α in the generalized aggressive periodontitis group (110.23 ± 9.20 pg/30 s, 1284.46 ± 86.32 pg/30 s) were significantly higher than those in the chronic periodontitis group (423.65 ± 35.24 pg/30 s, 82.64 ± 9.12 pg/30 s), the gingivitis group (52.10 ± 7.15 pg/30 s, 824.24 ± 44.68 pg/30 s) and the periodontally healthy group (36.44 ± 8.86 pg/30 s, 628.26 ± 34.61 pg/30 s). Conclusion: CCL28, IL‐8, IL‐1β and TNF‐α may play key roles in the host response to inflammation in periodontal diseases. As the severity of periodontal diseases increases, destruction of periodontal tissues also increases. Inflammation is one among many factors that trigger periodontal tissue destruction. Identification of the mediators that influence the development and progression of inflammation in periodontal diseases may be very important in understanding the prognoses of periodontal diseases.     

Interleukin-4, a T-helper 2 cell cytokine, is associated with the remission of periodontal disease

Background and Objectives: Interleukin‐4 (IL‐4), secreted mainly by T‐helper 2 cells, is a key cytokine for the growth and proliferation of B lymphocytes. Previous studies have proved that IL‐4 has an anti‐inflammatory effect owing to its efficient inhibition of the production of proinflammatory cytokines such as tumour necrosis factor‐α (TNF‐α), IL‐1α, IL‐1β, IL‐6 and IL‐8 by monocytes/macrophages. The aim of the present study was to assess the relation between clinical parameters and concentrations of IL‐4 within gingival crevicular fluid from inflamed gingiva and periodontitis sites and, subsequently, after treatment of the periodontitis sites. Material and Methods: A total of 60 subjects were divided into three groups based on gingival index (GI), pocket probing depth and clinical attachment loss (CAL): healthy (group 1), gingivitis (group 2) and chronic periodontitis (group 3). A fourth group (group 4) consisted of 20 subjects from group 3, 6–8 weeks after treatment (i.e. scaling and root planing). Gingival crevicular fluid samples collected from each patient were quantified for IL‐4 using the enzymatic immunometric assay. Results: The highest mean concentration of IL‐4 was obtained for group 1 (99.39 ± 49.33 pg/mL) and the lowest mean concentration of IL‐4 was obtained for group 3 (15.78 ± 21.92 pg/mL). The mean IL‐4 concentrations for group 2 (64.34 ± 39.56 pg/mL) and group 4 (68.92 ± 42.85 pg/mL) were intermediate between the levels in healthy subjects and periodontitis subjects. Conclusion: The mean concentration of IL‐4 decreased from periodontal health to disease. Thus, we suggest that type 2 helper T cell cytokine, as represented by IL‐4, was associated with the remission or improvement of periodontal disease.     

Cytokine profile in the gingival crevicular fluid of rheumatoid arthritis patients with chronic periodontitis

The aim was to assess the cytokine profile in the gingival crevicular fluid (GCF) of rheumatoid arthritis (RA) patients with chronic periodontitis (CP). Databases were searched from 1991 to August 2013 using a combination of various keywords. Eight studies were included. The GCF concentrations of interleukin (IL)‐1β, IL‐4, IL‐10, matrix metalloproteinase (MMP)‐8, MMP‐13 and tumor necrosis factor‐alpha (TNF‐α) were reported to be higher in patients with RA than in healthy controls (HC) without CP. In one study, TNF‐α levels in GCF were significantly higher in HC than in RA patients receiving anti‐TNF‐α therapy. One study reported no significant difference in GCF TNF‐α levels among RA patients and HC regardless of anti‐TNF‐α therapy. One study reported no difference in IL‐1β and prostaglandin E₂ levels among RA patients and HC with CP. Raised levels of proinflammatory cytokines are exhibited in the GCF of RA patients with CP.     

Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils

Hiroshima Y, Bando M, Inagaki Y, Mihara C, Kataoka M, Murata H, Shinohara Y, Nagata T, Kido J. Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils. J Periodont Res 2012; 47: 554–562. © 2012 John Wiley & Sons A/S Background and Objective: Resistin is an adipocytokine that induces insulin resistance and is predominantly expressed in adipocytes and peripheral blood mononuclear cells. Resistin expression increases in inflammatory diseases as well as diabetes mellitus, and is upregulated by bacterial pathogens and proinflammatory cytokines. The aim of this study was to identify resistin in human gingival crevicular fluid, to compare the resistin levels in gingival crevicular fluid between subjects with and without periodontitis and diabetes mellitus and to investigate the regulation of resistin release from human neutrophils by Porphyromonas gingivalis lipopolysaccharide (P‐LPS). Material and Methods: Gingival crevicular fluid samples were collected from patients with chronic periodontitis (n = 24), patients with diabetes mellitus‐related periodontitis (n = 18) and healthy subjects (n = 21). Resistin in gingival crevicular fluid was determined using western blot analysis and an ELISA kit. The glycated hemoglobin (HbA_1c) value was obtained from patients with diabetes mellitus‐related periodontitis by a medical interview. Human neutrophils were cultured with P‐LPS (0–1000 ng/mL), or incubated with inhibitors of actin or microtubule polymerization in the absence or presence of P‐LPS. The medium and cellular fractions were used for determination of resistin by ELISA. Results: The resistin level in gingival crevicular fluid from patients with periodontitis or diabetes mellitus‐related periodontitis was significantly higher than that of healthy subjects. The resistin level in gingival crevicular fluid was correlated with gingival index score, but not blood HbA_1c value. The P‐LPS increased resistin release from human neutrophils, and its induction was decreased by actin polymerization inhibitors. Conclusion: We show, for the first time, the presence of resistin in gingival crevicular fluid. A high resistin level in gingival crevicular fluid samples from periodontitis patients may to some extent be related to P‐LPS‐induced resistin release from neutrophils.     

Does Obstructive Sleep Apnea Increase the Risk for Periodontal Disease? A Case‐Control Study

Background: A possible association between periodontitis and obstructive sleep apnea (OSA) has been suggested. The aim of this study is to compare periodontitis prevalence between controls and patients with OSA by assessing clinical periodontal parameters and gingival crevicular fluid (GCF) levels of interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, and high‐sensitive C‐reactive protein (hs‐CRP); serum hs‐CRP was also sampled. Methods: A case‐control study was performed that included 163 individuals: 83 individuals (18 females and 65 males) with OSA and 80 non‐OSA individuals (23 females and 57 males) as controls. The test group was classified according to OSA severity. Clinical periodontal measurements were recorded, and GCF samples were collected. GCF hs‐CRP, IL‐lβ, and TNF‐α levels were analyzed using an enzyme‐linked immunosorbent assay method. Serum hs‐CRP was measured by latex‐enhanced immunoturbidimetric assay. Results: Prevalence of periodontitis in the OSA group (96.4%) was significantly higher than in the control group (75% [P <0.001]). Severe periodontitis prevalence was higher in the OSA group than control group. All periodontal clinical parameters and GCF IL‐lβ concentrations were significantly higher in patients with OSA than in controls (P = 0.001). No significant differences were found between the mild OSA and moderate‐to‐severe OSA groups. Additionally, there was no significant difference in GCF TNF‐α and hs‐CRP levels between the groups (P >0.05). Serum hs‐CRP levels were significantly higher in patients with OSA. A significant correlation was found between GCF IL‐1β and all clinical parameters. Conclusions: Results demonstrated higher prevalence of periodontitis and higher levels of GCF IL‐1β and serum hs‐CRP in patients with OSA. However, there is still a need for randomized clinical trials testing oral care interventions.     

Is There an Interaction Between Polycystic Ovary Syndrome and Gingival Inflammation?

Background: The aim of this study is to evaluate the gingival crevicular fluid (GCF), saliva, and serum concentrations of tumor necrosis factor‐α (TNF‐α), TNF‐α receptor‐1 (TNF‐αR1), TNF‐αR2, and interleukin‐6 (IL‐6) in non‐obese females with polycystic ovary syndrome (PCOS) and either clinically healthy periodontium or gingivitis. Methods: Thirty‐one females with PCOS and healthy periodontium, 30 females with PCOS and gingivitis, and 12 systemically and periodontally healthy females were included in the study. GCF, saliva, and serum samples were collected, and clinical periodontal measurements, body mass index, and Ferriman‐Gallwey score (FGS) were recorded. Sex hormones, cortisol, and insulin levels were measured. TNF‐α, TNF‐αR1, TNF‐αR2, and IL‐6 were determined by enzyme‐linked immunosorbent assay. Kruskal‐Wallis followed by Bonferroni‐corrected post hoc Mann‐Whitney U tests were used to analyze the data. Results: The PCOS + gingivitis group revealed significantly higher GCF, saliva, and serum IL‐6 concentrations than the PCOS + healthy group (P <0.0001). The two PCOS groups exhibited significantly higher saliva TNF‐α concentrations than the control group (P = 0.024 and P = 0.013, respectively). The FGS index was significantly higher in the PCOS + gingivitis group than the PCOS + healthy group (P = 0.030). The PCOS + gingivitis group revealed significantly higher insulin concentration than the PCOS + healthy and control groups (P = 0.014 and P <0.0001, respectively). Serum TNF‐α, TNF‐αRs, and serum, GCF, and salivary IL‐6 levels correlated with the clinical periodontal measurements. Conclusions: PCOS and gingival inflammation appear to act synergistically on the proinflammatory cytokines IL‐6 and TNF‐α. Thus, PCOS may have an impact on gingival inflammation or vice versa. Additional studies are warranted to clarify the possible relationship between PCOS and periodontal disease.     

Alcohol Consumption and Periodontitis: Quantification of Periodontal Pathogens and Cytokines

Background: There are few studies on periodontal status related to microbiologic and immunologic profiles among individuals not or occasionally using alcohol and those with alcohol dependence. The aim of this study is to determine the effect of alcohol consumption on the levels of subgingival periodontal pathogens and proinflammatory cytokines (interleukin [IL]‐1β and tumor necrosis factor [TNF]‐α) in the gingival fluid among individuals with and without periodontitis. Methods: This observational analytic study includes 88 volunteers allocated in four groups (n = 22): individuals with alcohol dependence and periodontitis (ADP), individuals with alcohol dependence and without periodontitis (ADNP), individuals not or occasionally using alcohol with periodontitis (NAP), and individuals not or occasionally using alcohol without periodontitis (NANP). Levels of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Eikenella corrodens, and Fusobacterium nucleatum were determined by real‐time polymerase chain reaction on the basis of the subgingival biofilm, and IL‐1β and TNF‐α were quantified by enzyme‐linked immunosorbent assay in gingival fluid samples. Results: Individuals with alcohol dependence showed worse periodontal status and higher levels of P. intermedia, E. corrodens, F. nucleatum, and IL‐1β than non‐users. No significant correlations between TNF‐α and bacterial levels were observed. However, in the ADP group, higher levels of E. corrodens were correlated with higher levels of IL‐1β. Conclusion: A negative influence of alcohol consumption was observed on clinical and microbiologic periodontal parameters, as well as a slight influence on immunologic parameters, signaling the need for additional studies.     

MicroRNA‐126 Regulates Inflammatory Cytokine Secretion in Human Gingival Fibroblasts Under High Glucose via Targeting Tumor Necrosis Factor Receptor Associated Factor 6

Background: MicroRNAs (miRs) play a crucial role in inflammatory diseases, including periodontitis. Meanwhile, miRs act as biomarkers for predicting diabetes mellitus (DM). However, the regulatory mechanism of miR‐126 on development of periodontitis in patients with DM still remains unclear. Methods: Human gingival fibroblasts were cultured with low (5.5 mmol/L), medium (15 mmol/L), and high (25 mmol/L) glucose, respectively. Expressions of miR‐126, tumor necrosis factor (TNF) receptor associated factor (TRAF) 6, and related cytokines were analyzed by real‐time polymerase chain reaction (PCR). After transfection with miR‐126 mimic, PCR and western blot were performed to detect level of TRAF6, and luciferase reporter assay confirmed if TRAF6 is the direct target of miR‐126. Production of cytokines was measured using enzyme‐linked immunosorbent assay. Results: Increased glucose significantly suppressed miR‐126 expression in human gingival fibroblasts (P <0.05). Also, high glucose increased TRAF6, interleukin (IL)‐6, TNF‐α, and chemical chemokine ligand (CCL) 2 levels, whereas it decreased IL‐10 level. MiR‐126 mimic significantly decreased TRAF6 mRNA and protein levels under high glucose (P <0.05). Also, miR‐126 directly targeted TRAF6 through binding to its 3′ untranslated region in human gingival fibroblasts. Overexpression of miR‐126 significantly abrogated high glucose–induced secretion of proinflammatory cytokines such as IL‐6, TNF‐α, and CCL2 and promoted production of IL‐10. Conclusion: These data suggest that miR‐126 inhibits inflammation of human gingival fibroblasts under high glucose through targeting TRAF6, which may be a potential therapeutic target for periodontitis concomitant with DM.     

Cytokines and bone-related factors in systemically healthy patients with chronic periodontitis and patients with type 2 diabetes and chronic periodontitis

Background: This study compares the levels of cytokines and bone‐related factors in the gingival crevicular fluid (GCF) of systemically healthy patients with chronic periodontitis (CP); and better‐controlled, and poorly controlled patients with type 2 diabetes and CP. Methods: Thirty‐seven patients with type 2 diabetes and CP and 20 systemically healthy patients with CP were enrolled in this study. The patients with diabetes mellitus were categorized as better‐controlled (n = 17; HbA_1c levels ≤8%) or poorly controlled (n = 20; glycated hemoglobin values >8%). Levels of tumor necrosis factor‐α, interleukin (IL)‐4, interferon (IFN)‐γ, IL‐23, IL‐17, soluble receptor activator of nuclear factor‐kappa B ligand (sRANKL), and osteoprotegerin (OPG) in GCF of diseased sites were analyzed by enzyme‐linked immunosorbent assay. Results: Type 2 diabetes mellitus, as a whole, upregulates the levels of OPG, sRANKL, IFN‐γ, IL‐17, and IL‐23 and downregulates the production of IL‐4 in sites with CP (P <0.05). Better‐controlled individuals exhibited the highest levels of IFN‐γ, whereas poorly controlled patients presented the highest levels of IL‐17 (P <0.05). There were no differences in the levels of tumor necrosis factor‐α, OPG, and IL‐23 among systemically healthy, better‐controlled, and poorly controlled patients with diabetes (P >0.05). Conclusions: Increased levels of proinflammatory cytokines and RANKL were observed in the GCF of patients with type 2 diabetes with CP, compared to patients without diabetes. In addition, poor or good glycemic status seems to modulate osteo‐immunoinflammatory mediators in a different manner.     

Significance of elevated gingival crevicular fluid tumor necrosis factor‐α and interleukin‐8 levels in chronic hemodialysis patients with periodontal disease

Da? A, F?rat ET, Kadiro?lu AK, Kale E, Y?lmaz ME. Significance of elevated gingival crevicular fluid tumor necrosis factor‐α and interleukin‐8 levels in chronic hemodialysis patients with periodontal disease. J Periodont Res 2010; 45: 445–450. © 2010 John Wiley & Sons A/S Background and Objective: The prevalence of chronic renal disease in industrialized countries is increasing, and chronic renal disease and periodontitis can have significant, reciprocal effects. The aim of this study was to evaluate the associations between specific clinical parameters and the levels of tumor necrosis factor‐α (TNF‐α) and interleukin‐8 (IL‐8) in the gingival crevicular fluid of hemodialysis (HD) patients with periodontal disease. Material and Methods: Forty‐three HD patients and 43 systemically healthy subjects were enrolled in this study. Plaque index (PI), gingival index (GI) and pocket depth were used to determine periodontal status. Venous blood samples were obtained from each patient in the morning before the dialysis session and analyzed to determine the levels of inflammatory, biochemical and hematological parameters. Gingival crevicular fluid was collected from all subjects, and the levels of TNF‐α and IL‐8 were determined in the gingival crevicular fluid samples. Results: The following results were obtained from HD patients and controls: TNF‐α (pg/mL), 31.40 ± 1.46 and 3.06 ± 0.15 (p < 0.001); IL‐8 (pg/mL), 90.98 ± 94.03 and 35.05 ± 16.86 (p < 0.001); PI, 1.69 ± 1.02 and 0.04 ± 0.02 (p < 0.001); GI, 0.82 ± 0.06 and 0.04 ± 0.02 (p < 0.001); and pocket depth, 2.23 ± 0.63 and 1.51 ± 0.05 (p < 0.001), respectively. In addition, there were positive correlations between TNF‐α and PI (r = 0.642, p < 0.001), between TNF‐α and GI (r = 0.565, p < 0.001), between TNF‐α and pocket depth (r = 0.522, p < 0.001), between IL‐8 and PI (r = 0.402, p = 0.002), between IL‐8 and GI (r = 0.396, p = 0.002), and between IL‐8 and pocket depth (r = 0.326, p = 0.012). There were negative correlations between albumin and PI (r = ?0.491, p < 0.001), albumin and GI (r = ?0.406, p < 0.001), albumin and pocket depth (r = ?0.464, p < 0.001) and albumin and CRP (r = ?0.467, p = 0.002) and between the gingival crevicular fluid levels of TNF‐α and IL‐8, TNF‐α and hemoglobin (r = ?0.745, p < 0.001; r = ?0.285, p < 0.05) (respectively). Conclusion: The levels of TNF‐α and IL‐8 in gingival crevicular fluid were significantly higher in HD patients than in controls. There were strong, positive correlations between clinical periodontal parameters and the levels of inflammatory cytokines in gingival crevicular fluid from the HD patients.     

Crevicular fluid matrix metalloproteinase‐8, ‐13, and TIMP‐1 levels in type 2 diabetics

Oral Diseases (2010) 16 , 476–481 Objectives: To evaluate whether type 2 diabetes mellitus (DM) enlarged and if so the quantum of such increase in the gingival crevicular fluid (GCF) levels of matrix metalloproteinase‐8 (MMP‐8), MMP‐13 and tissue inhibitor of metalloproteinases‐1 (TIMP‐1). Methods: Subjects (n = 73) were divided into five groups as follows: 12 DM patients with gingivitis (DM‐G), 12 DM patients with periodontitis (DM‐P), 12 systemically healthy patients with gingivitis (H‐G), 13 systemically healthy patients with periodontitis (H‐P) and 24 periodontally, systemically healthy volunteer subjects (H‐C). Full‐mouth clinical periodontal measurements were performed at six sites per tooth. Gingival crevicular fluid samples were obtained from two sites representing the clinical periodontal diagnosis in single‐rooted teeth. Gingival crevicular fluid levels of MMP‐8, MMP‐13 and TIMP‐1 were analysed by immunofluorometric MMP assay (IFMA), enzyme‐linked immunosorbent assay (ELISA). Data were tested statistically by parametric tests. Results: All clinical periodontal measurements were similar in both diabetic and systemically healthy patients with periodontal disease (all P > 0.05). Total amounts of MMP‐8 in GCF samples were significantly lower in H‐C group than DM‐G, DM‐P, H‐P groups (all P < 0.05). Matrix metalloproteinase‐13, TIMP‐1 total amounts were similar in study groups (P > 0.05). Diabetes mellitus patients exhibited similar levels of MMP‐8, MMP‐13, TIMP‐1 with systemically healthy gingivitis/periodontitis patients (P > 0.05). Conclusions: Within the limits of this study, DM does not seem to significantly affect GCF levels of MMP‐8, MMP‐13, TIMP‐1 or clinical periodontal status.     

The Association Between Periodontal Inflammation and Labor Triggers (Elevated Cytokine Levels) in Preterm Birth: A Cross‐Sectional Study

Background: Periodontitis is considered to be a risk factor for preterm birth. Mechanisms have been proposed for this pathologic relation, but the exact pathologic pattern remains unclear. Therefore, the objective of the present study is to evaluate levels of four major labor triggers, prostaglandin E₂ (PGE₂), interleukin (IL)‐1β, IL‐6, and tumor necrosis factor (TNF)‐α, in gingival crevicular fluid (GCF) and serum samples between women with preterm birth (PTB) and full‐term birth (FTB) and correlate them with periodontal parameters. Methods: PGE₂, IL‐1β, IL‐6, and TNF‐α levels were estimated using enzyme‐linked immunosorbent assays in GCF and serum samples collected 24 to 48 hours after labor from 120 women (60 FTB, 60 PTB). Results: Women with PTB exhibited significantly more periodontitis, worse periodontal parameters, and increased GCF levels of IL‐6 and PGE₂ compared with the FTB group; there were no significant differences in serum levels of measured markers. GCF levels of IL‐1β, IL‐6, and PGE₂ and serum levels of TNF‐α and PGE₂ were significantly higher in women with periodontitis compared with periodontally healthy women. Serum levels of PGE₂ were positively correlated with probing depth (PD) and clinical attachment level (CAL) as well as with GCF levels of TNF‐α in women with PTB. Conclusions: Women with PTB demonstrated worse periodontal parameters and significantly increased GCF levels of IL‐6 and PGE₂ compared with those with FTB. Based on significant correlations among serum PGE₂ and PD, CAL, and GCF TNF‐α in PTB, periodontitis may cause an overall increase of labor triggers and hence contribute to preterm labor onset.     

Hyperglucose Contributes to Periodontitis: Involvement of the NLRP3 Pathway by Engaging the Innate Immunity of Oral Gingival Epithelium

Background: The NLRP3 inflammasome is essentially a family of intracellular innate immune sensors that can respond to bacterial challenge and initiate early host immunity responses. However, the involvement and possible molecular mechanism of the NLRP3 pathway in the context of chronic periodontitis (CP) and diabetes mellitus have yet to be fully elucidated. Methods: Gingival tissues were collected from patients with CP and/or type 2 diabetes mellitus (T2DM), and the expression of NLRP3 and interleukin (IL)‐1β was analyzed by immunohistochemistry. To explore the possible molecular mechanism, human gingival epithelial cells (HGECs) were established in vitro and challenged with lipopolysaccharide (LPS) and/or high glucose. High extracellular K⁺ was applied as an inhibitor of NLRP3. The NLRP3 pathway was analyzed by immunocytochemistry and quantitative polymerase chain reaction. Results: Compared with control individuals, NLRP3 and IL‐1β were significantly upregulated in oral gingival epithelium of patients with CP and/or T2DM (P <0.05). The expression of NLRP3 was significantly upregulated in HGECs when stimulated in vitro by LPS or high glucose (P = 0.00). The simultaneous stimulation of LPS and high glucose contributed to significant upregulation of NLRP3 expression versus LPS or high glucose alone (P = 0.00). Although expression of caspase 1 and IL‐1β protein were increased in HGECs when stimulated by LPS, they were partially inhibited after the NLRP3 was successfully blocked. Conclusion: For patients with T2DM and CP, hyperglycemic status may exacerbate the inflammation state of gingival tissue by activating the NLRP3 pathway, and this abnormal host inflammatory response may contribute to further tissue breakdown.     

Pro-inflammatory biomarkers during experimental gingivitis in patients with type 1 diabetes mellitus: a proof-of-concept study

Aim: To compare gingival crevicular fluid (GCF) biomarker levels and microbial distribution in plaque biofilm (SP) samples for subjects with type 1 diabetes (T1DM) versus healthy subjects without diabetes during experimental gingivitis (EG). Materials and Methods: A total of nine T1DM patients and nine healthy controls of age and gender similar to the T1DM patients were monitored for 35 days during EG. Hygiene practices were stopped for 3 weeks, and GCF, SP, plaque index (PI) and gingival index were determined. IL‐1β, IL‐8, MMP‐8 and MMP‐9 were quantified by enzyme‐linked immunosorbent assay, and SP samples were assessed by DNA–DNA hybridization for a panel of 40 subgingival microbial species. Results: IL‐1β levels in T1DM patients were elevated compared with healthy individuals, and showed differences between groups at 7–21 days while healthy patients showed IL‐1β increases from baseline to 14–21 days (p<0.05). Differences were observed in MMP‐9 levels between patients with and without T1DM at 7–14 days (p<0.05). Orange complex species and PI measurements displayed a superior correlation with biomarker levels when compared with other complexes or clinical measurements during EG. Conclusions: The mean GCF biomarker levels for IL‐1β and MMP‐8 were most significantly elevated in T1DM subjects compared with healthy individuals during EG, not resulting from differences in the mean PI or microbial composition.     

Synergistic effects of lipopolysaccharides from periodontopathic bacteria on pro‐inflammatory cytokine production in an ex vivo whole blood model

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia have been strongly associated with chronic periodontitis. This disease is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. The secretion of high levels of inflammatory cytokines by those cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of whole blood from periodontitis patients following challenges with whole cells of P. gingivalis, T. denticola, and T. forsythia or their lipopolysaccharides (LPS), individually and in combination. Whole blood collected from seven periodontitis patients was stimulated with whole cells or LPS and the production of interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor alpha (TNF‐α) were quantified by enzyme‐linked immunosorbent assays. The mono and mixed challenges with whole bacterial cells or LPS induced the secretion of high amounts of IL‐1β, IL‐6, IL‐8, and TNF‐α by the mixed leukocyte population from periodontitis patients. In addition, P. gingivalis LPS, T. denticola LPS, and T. forsythia LPS acted in synergy to induce high levels of IL‐1β and TNF‐α. This study suggests that P. gingivalis, T. denticola, and T. forsythia may contribute to the immunodestructive host response characteristic of periodontitis through synergistic effects of their LPS on the inflammatory response induced by a mixed population of leukocytes.     

Chemerin as a Novel Crevicular Fluid Marker of Patients With Periodontitis and Type 2 Diabetes Mellitus

Background: The objectives of the present study are to: 1) determine whether gingival crevicular fluid (GCF) chemerin is a novel predictive marker for patients with chronic periodontitis (CP) with and without type 2 diabetes mellitus (t2DM); 2) analyze the relationship between chemerin and interleukin (IL)‐6 in periodontally healthy individuals and in patients with CP and with and without t2DM; and 3) evaluate the effect of non‐surgical periodontal therapy on GCF chemerin levels. Methods: Eighty individuals were split into four groups: 20 who were systemically and periodontally healthy (CTRL), 20 with t2DM and periodontally healthy (DM‐CTRL), 20 systemically healthy with CP (CP), and 20 with CP and t2DM (DM‐CP). Individuals with periodontitis were treated with non‐surgical periodontal therapy. GCF sampling procedures and clinical periodontal measures were performed before and 6 weeks after treatment. Enzyme‐linked immunosorbent assay was used to measure chemerin and IL‐6 levels. Results: Greater values for GCF chemerin and IL‐6 levels were found in CP groups than in periodontally healthy groups, in DM‐CP than in CP, and in DM‐CTRL than in CTRL (P <0.008). GCF chemerin and IL‐6 levels decreased following therapy in CP groups (P <0.02). A comprehensive overview of all groups showed a statistically significant positive correlation of chemerin with IL‐6, glycated hemoglobin, sampled‐site clinical attachment level, and gingival index (P <0.05). Conclusions: In this study, periodontitis and t2DM induced aberrant secretion of chemerin, and non‐surgical periodontal therapy influenced the decrease of GCF chemerin levels in patients with CP with and without t2DM. Furthermore, it suggests GCF chemerin levels may be considered a potential proinflammatory marker for diabetes, periodontal disease, and treatment outcomes.