Gül Atilla

Expression and regulation of the NALP3 inflammasome complex in periodontal diseases

Periodontitis is an infectious process characterized by inflammation affecting the supporting structures of the teeth. Porphyromonas gingivalis is a major oral bacterial species implicated in the pathogenesis of periodontitis. Processing of interleukin (IL)‐1 family cytokines is regulated by an intracellular innate immune response system, known as the NALP3 [nacht domain‐, leucine‐rich repeat‐, and pyrin domain (PYD)‐containing protein 3] inflammasome complex. The aim of the present study was to investigate by quantitative real‐time polymerase chain reaction (PCR) the mRNA expression of NALP3, its effector molecule apoptosis associated speck‐like protein (ASC), its putative antagonist NLRP2 (NLR family, PYD‐containing protein 2), IL‐1β and IL‐18 (i) in gingival tissues from patients with gingivitis (n = 10), chronic periodontitis (n = 18), generalized aggressive periodontitis (n = 20), as well as in healthy subjects (n = 20), (ii) in vitro in a human monocytic cell line (Mono‐Mac‐6), in response to P. gingivalis challenge for 6 h. The clinical data indicate that NALP3 and NLRP2, but not ASC, are expressed at significantly higher levels in the three forms of inflammatory periodontal disease compared to health. Furthermore, a positive correlation was revealed between NALP3 and IL‐1β or IL‐18 expression levels in these tissues. The in vitro data demonstrate that P. gingivalis deregulates the NALP3 inflammasome complex in Mono‐Mac‐6 cells by enhancing NALP3 and down‐regulating NLRP2 and ASC expression. In conclusion, this study reveals a role for the NALP3 inflammasome complex in inflammatory periodontal disease, and provides a mechanistic insight to the host immune responses involved in the pathogenesis of the disease by demonstrating the modulation of this cytokine‐signalling pathway by bacterial challenge.

Gingival Crevicular Fluid Levels of Cathelicidin LL-37 and Interleukin-18 in Patients With Chronic Periodontitis

BACKGROUND Cathelicidin LL-37, an antimicrobial peptide, is part of the host innate immune response in the oral cavity. Interleukin (IL)-18, a proinflammatory cytokine, could play a role in the progression of the inflammatory response. The aim of the present study was to determine the levels of cathelicidin LL-37 and IL-18 in the gingival crevicular fluid (GCF) of patients with gingivitis and chronic periodontitis. METHODS Fifty-nine subjects were included in the present study. Probing depth (PD), clinical attachment level (CAL), plaque index (PI), bleeding on probing, and papilla bleeding index (PBI) were assessed in patients with chronic periodontitis or gingivitis and in healthy controls. GCF levels of cathelicidin LL-37 and IL-18 were analyzed by enzyme-linked immunosorbent assay. RESULTS GCF levels of cathelicidin LL-37 were significantly elevated in patients with chronic periodontitis compared to the other groups (P <0.05). No significant difference was found in the total amount of GCF IL-18 among the study groups (P >0.05). Spearman correlation analysis revealed a positive relationship between levels of GCF cathelicidin LL-37 and PD, CAL, PI, and PBI at the sampled sites (P <0.01), whereas no correlation was found between the total amount of GCF IL-18 and clinical periodontal parameters at the sampled sites (P >0.05). CONCLUSION Elevated levels of GCF cathelicidin LL-37 in chronic periodontitis suggest that it may play a role in the host innate immune response during periodontal inflammation.

Effect of MMP‐1 promoter polymorphisms on GCF MMP‐1 levels and outcome of periodontal therapy in patients with severe chronic periodontitis

AIMS The aims of this study were to investigate (1) the matrix metalloproteinase-1 (MMP-1) promoter polymorphisms in severe chronic periodontitis (CP), (2) the relationship of periodontal therapy outcome with these genotypes, and (3) the gingival crevicular fluid (GCF) MMP-1 levels-MMP-1 genotype correlation. MATERIAL AND METHODS Genomic DNA was obtained from the peripheral blood of 102 patients with severe CP and 98 periodontally healthy subjects. MMP-1 -519A/G and -1607 1G/2G polymorphisms were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Fifty-eight CP patients received non-surgical periodontal therapy and were followed for 6 months. Clinical periodontal parameters and GCF samples were collected at baseline and at 6 months. GCF MMP-1 levels were analysed by enzyme-linked immunosorbent assay (ELISA). RESULTS The distribution of MMP-1 genotypes did not significantly differ between the study groups. On the other hand, the -1607 2G allele frequency of severe CP patients was higher than that of healthy subjects. MMP-1 -519G allele carriers had higher GCF MMP-1 levels and percentage of sites with 4-6 mm clinical attachment level (CAL) compared with AA genotypes after non-surgical periodontal therapy (p<0.05). CONCLUSIONS These data suggest that the -1607 2G polymorphic allele of the MMP-1 gene could be associated with susceptibility to severe CP in the Turkish population. It seems that -519AG and GG genotypes could play a role in the outcome of periodontal therapy.

Matrix Metalloproteinases, Tissue Inhibitor of Matrix Metalloproteinase-1, and Laminin-5 γ2 Chain Immunolocalization in Gingival Tissue of Endotoxin-Induced Periodontitis in Rats: Effects of Low-Dose Doxycycline and Alendronate

BACKGROUND Matrix metalloproteinases (MMPs) play important roles in tissue destruction mechanisms of periodontitis. MMP-8 and -13 are the major collagenases that act in extracellular matrix degradation in periodontal tissues. MMP-14 is a membrane-type MMP, and laminin (Ln)-- is a basal membrane component. The aim of the present study was to evaluate the effects of doxycycline and alendronate on gingival tissue expression of MMP-8, -13, and -14; tissue inhibitors of MMP (TIMP)-1; and Ln-5 γ2 chain in experimental periodontitis induced by Escherichia coli endotoxin (LPS) in rats. METHODS Experimental periodontitis was induced by repeated injection of LPS. Forty-four adult male Sprague-Dawley rats were divided into five study groups: saline control, LPS, LPS + doxycycline, LPS + alendronate, and LPS + doxycycline + alendronate. Doxycycline and alendronate were given as a single agent or as combination therapy during the 7 days of the experimental study period. On day 7, the rats were sacrificed, and the gingival tissues were analyzed immunohistochemically for expression of MMP-8, -13, and -14, Ln-- γ2 chain, and TIMP-1. Alveolar bone loss was evaluated morphometrically under a stereomicroscope. Data were tested statistically by Kruskal-Wallis and Mann-Whitney tests and Spearman correlation analysis. RESULTS Alveolar bone loss was significantly higher in the LPS, doxycycline, alendronate, and combination groups than in the saline control group (all P <0.01). MMP-8 expression was significantly higher in the LPS group than in the saline control group (P = 0.001). Individual administration of doxycycline or alendronate significantly decreased the expression of MMP-8 compared to LPS (P = 0.01). Combined drug administration reduced MMP-14 significantly compared to doxycycline (P = 0.004). No significant differences in Ln-5 γ2 chain expression were found between the study groups (P >0.05). MMP-14 significantly correlated with the Ln-5 γ2 chain in the LPS + alendronate group (P = 0.04) and with the amount of alveolar bone loss in the LPS + doxycycline + alendronate group (P = 0.03). CONCLUSIONS Our findings suggest that alendronate and/or doxycycline may inhibit MMP-8 expression significantly; particularly, their combined administration may provide beneficial effects in periodontal treatment. Moreover, individual administration of alendronate and doxycycline results in significant increases in TIMP-1 expression in gingiva. However, these effects of combined low-dose doxycycline and alendronate on MMPs and TIMP should be verified by clinical human trials before these agents are used in dental practice.

Subantimicrobial-Dose Doxycycline and Cytokine-Chemokine Levels in Gingival Crevicular Fluid

BACKGROUND The present randomized, double-masked, placebo-controlled, parallel-arm study examines the impact of adjunctive subantimicrobial-dose doxycycline (SDD) on the local inflammatory response through cytokine and chemokine levels in gingival crevicular fluid (GCF) samples from patients with chronic periodontitis. METHODS Forty-six patients with chronic periodontitis received scaling and root planing with or without adjunctive SDD. GCF samples were collected and clinical parameters including probing depth, clinical attachment level, gingival index, and plaque index were recorded every 3 months for 12 months. GCF tumor necrosis factor-α, interleukin (IL)-6, IL-4, IL-10, IL-13, IL-17, macrophage inhibitory protein 1α, macrophage inhibitory protein 1β, monocyte chemoattractant protein 1, and regulated on activated normal T-cell expressed and secreted protein levels were determined by xMAP multiplex immunoassay. RESULTS Significant improvements were observed in all clinical parameters in both groups over 12 months (P <0.0125), whereas the SDD group showed significantly better reduction in gingival index, probing depth, and gain in clinical attachment compared to the placebo group (P <0.05). Decrease in IL-6 in the SDD group was significantly higher compared to the placebo group at 6 and 9 months in deep pockets (P <0.05), whereas tumor necrosis factor-α was significantly reduced in moderately deep pockets (P <0.05). SDD resulted in a stable IL-4 and IL-10 response while reducing the monocyte chemoattractant protein 1 levels at 3 months (P <0.05). CONCLUSIONS These results show that SDD, as an adjunct to non-surgical periodontal therapy, stabilizes the inflammatory response by promoting the suppression of proinflammatory cytokines and increasing the anti-inflammatory cytokines. The chemokine activity would account for the regulation of the inflammatory response to SDD therapy.

Effects of Menstrual Cycle on Periodontal Health and Gingival Crevicular Fluid Markers

BACKGROUND Fluctuations in sex steroid hormones, which are also noticeable through the menstrual cycle of women, may impact periodontal health. The aim of this study is to evaluate the effect of hormonal changes occurring in the menstrual cycle on gingival inflammation and the gingival crevicular fluid (GCF) levels of interleukin 6 (IL-6), prostaglandin E(2) (PGE(2)), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-2 (PAI-2). METHODS Twenty-five gingivitis patients and 25 periodontally healthy subjects having regular menstrual cycles were seen at menstruation (ME) (1 to 2 days of menstruation), ovulation (OV) (12 to 14 days), and premenstrual phases (PM) (22 to 24 days). GCF and saliva samples were collected and clinical parameters including plaque index and bleeding on probing were recorded at each menstrual phase. Salivary estrogen and progesterone levels were analyzed to determine exact menstrual cycle days. GCF levels of IL-6, PGE(2), t-PA, and PAI-2 were measured by enzyme-linked immunosorbent assay. RESULTS The percentages of sites with bleeding on probing were significantly higher in ME (60.85 +/- 18.36) and OV (58.92 +/- 25.04) than in the PM (40.12 +/- 20.10) phase in the gingivitis group (P <0.001; repeated measures analysis of variance), whereas it was similar for all phases in the healthy group (P >0.05; repeated measures analysis of variance). GCF levels of IL-6 were significantly elevated in gingivitis patients compared to healthy subjects in all phases (P = 0.004, P = 0.041, and P = 0.046 for ME, OV, and PM, respectively; Mann-Whitney U test). GCF levels of IL-6, PGE(2), t-PA, and PAI-2 were unchanged in different menstrual phases in both groups (P >0.05; Friedman test). CONCLUSION The present study suggests that changes in the sex steroid hormones during menstrual cycles might have a limited effect on the inflammatory status of gingiva, but GCF cytokine levels were not affected.

Tumor Necrosis Factor-α-converting Enzyme (TACE) Levels in Periodontal Diseases:

Tumor necrosis factor-α-converting enzyme (TACE) is a metalloprotease which can shed several cytokines from the cell membrane, including receptor activator of NF-κB ligand (RANKL). This study aimed to investigate the hypothesis that TACE would be elevated in the gingival crevicular fluid (GCF) of persons with periodontitis. Total TACE amounts in GCF were higher in persons with chronic and aggressive periodontitis than in those with gingivitis or in healthy persons. TACE concentrations in GCF were higher in persons with chronic and aggressive periodontitis than in those with gingivitis, although not significantly higher than in healthy persons. Persons with chronic periodontitis receiving immunosuppressive treatment exhibited over 10-fold lower TACE levels than the other periodontitis groups. TACE was positively correlated with probing pocket depth, clinical attachment levels, and RANKL concentrations in GCF. In conclusion, the increased GCF TACE levels in persons with periodontitis and their positive correlation with RANKL may indicate an association of this enzyme with alveolar bone loss, and may warrant special attention in future therapeutic approaches.

Effect of azithromycin, as an adjunct to nonsurgical periodontal treatment, on microbiological parameters and gingival crevicular fluid biomarkers in generalized aggressive periodontitis

UNLABELLED Emingil G, Han B, Özdemir G, Tervahartiala T, Vural C, Atilla G, Baylas H, Sorsa T. The effect of azithromycin, as an adjunct to nonsurgical periodontal treatment, on microbiological parameters and gingival crevicular fluid biomarkers in generalized aggressive periodontitis. J Periodont Res 2012; 47: 729-739.

Effect of periodontal treatment on receptor activator of NF-κB ligand and osteoprotegerin levels and relative ratio in gingival crevicular fluid.

AIM Receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) have an established role in the pathogenesis of periodontitis, which is characterized by an increased RANKL/OPG ratio. The present study aims to investigate changes of RANKL, OPG and their relative ratio in gingival crevicular fluid (GCF) of periodontitis patients after non-surgical periodontal treatment. MATERIALS AND METHODS GCF was obtained from chronic periodontitis (n=14), generalized aggressive periodontitis (G-AgP; n=13) patients at baseline. The patients received scaling and root planing and were recalled after 2, 3 and 4 months for follow-up clinical examination and sampling. The total amounts and concentrations of RANKL and OPG in GCF were measured by enzyme-linked immunosorbent assay, and their relative ratio was calculated. RESULTS The RANKL/OPG ratio remained unchanged and did not correlate with clinical parameters throughout the monitoring period, despite the improved clinical outcome. This trend was similar in both chronic and G-AgP. CONCLUSIONS Although the RANKL/OPG ratio has a potential diagnostic value for untreated periodontitis, it may not be a suitable predictor of clinically successful treatment outcome. As conventional therapy does not negatively modulate this ratio, the host could still be susceptible to further periodontal tissue destruction, warranting the consideration of adjunctive treatments.

Azithromycin as an Adjunctive Treatment of Generalized Severe Chronic Periodontitis: Clinical, Microbiologic, and Biochemical Parameters

BACKGROUND This study examines the efficacy of azithromycin in combination with non-surgical periodontal therapy on clinical and microbiologic parameters and gingival crevicular fluid (GCF) matrix metalloproteinases-8 (MMP-8) levels over 6 months in patients with severe generalized chronic periodontitis (CP). METHODS Twenty-eight of 36 patients with severe generalized CP were included in this randomized, double-masked, placebo-controlled, parallel-arm study. They were randomly assigned to azithromycin or placebo groups (500 mg, once daily for 3 days). Probing depth (PD), clinical attachment level, dichotomous presence or absence of supragingival plaque accumulation, and bleeding on probing were recorded. GCF samples were obtained from one single-rooted tooth with PD ≥ 6 mm, whereas microbiologic samples were collected from two single-rooted teeth with PD ≥ 6 mm. Microbiologic parameters were analyzed by quantitative real-time polymerase chain reaction for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Prevotella intermedia, and total bacteria. GCF MMP-8 levels were determined by immunofluorescence assay. RESULTS Azithromycin and placebo groups demonstrated similar but significant improvements in all clinical parameters (P <0.05). A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. intermedia, and total bacteria significantly decreased over the 6-month period in both groups, whereas F. nucleatum was significantly reduced in all visits in the azithromycin group, with the levels also being lower compared with those of the placebo group (P <0.05). The azithromycin and placebo groups exhibited significant reduction in GCF MMP-8 levels at the post-treatment visit and at 2 weeks (P <0.05). CONCLUSION On the basis of the present findings, it can be concluded that adjunctive azithromycin provides no additional benefit over non-surgical periodontal treatment on parameters investigated in patients with severe generalized CP.