Haluk Baylas

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.

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.

Expression Profile of Human Gingival Fibroblasts Induced by Interleukin-1β Reveals Central Role of Nuclear Factor-Kappa B in Stabilizing Human Gingival Fibroblasts During Inflammation

BACKGROUND Interleukin (IL)-1beta is a key cytokine in the pathogenesis of periodontitis, and it induces inflammatory mediators in periodontal diseases. We developed immortalized human gingival fibroblasts (HGFs), investigated the effects of IL-1beta on the gene expression using expression arrays containing approximately 40,000 genes, and tested the role of nuclear factor-kappa B (NF-kappaB) in maintaining an activated HGF population. METHODS Total RNA was isolated from IL-1beta-induced and mock-induced control cells. Gene expression analyses were performed using expression arrays and confirmed by quantitative real-time polymerase chain reaction. Western blot analysis to show inhibitor of kappa B-alpha (IkappaBalpha) phosphorylation and immunostaining of cells for NF-kappaB nuclear translocation were performed. Apoptosis was confirmed by assay of poly ADP-ribose polymerase (PARP) cleavage. RESULTS A total of 382 probe sets corresponding to 254 genes were differentially expressed in IL-1beta-induced cells (P <0.001). A total of 215 genes were upregulated, and 39 genes were downregulated. Most notable NF-kappaB pathway members (NFkappaB1, NFkappaB2, IkappaBalpha, IkappaBepsilon, IkappaBzeta, REL, RELB, and TA-NFKBH) were upregulated. IkappaBalpha was phosphorylated, and NF-kappaB accumulated in the nucleus. An IL-1beta-induced set of 27 genes was downregulated by an NF-kappaB inhibitor, leading to a decreased number of viable cells and suggesting an antiapoptotic role for NF-kappaB. CONCLUSIONS IL-1beta leads to a large number of significant expression changes consistent with a pathologic role in periodontitis, including enhancement of inflammatory cytokines, chemokines, transcription factors, matrix metalloproteinases, adhesion molecules, and especially NF-kappaB-dependent antiapoptotic genes. NF-kappaB activation blocks apoptosis, thereby stabilizing the HGF population in inflammation.

Dietary Supplementation of Omega-3 Fatty Acid and Circulating Levels of Interleukin-1β, Osteocalcin, and C-Reactive Protein in Rats

BACKGROUND In this study, we evaluated the effects of two different regimes of dietary supplementation of omega-3 fatty acid on serum levels of interleukin-1 beta (IL-1β), osteocalcin (OC), and C-reactive protein (CRP) in experimental periodontitis. METHODS Experimental periodontitis was induced by repeated injections of Escherichia coli lipopolysaccharide (LPS). Thirty-nine adult male Sprague-Dawley rats were divided into four study groups as follows: an LPS positive control group; a saline (negative) control group; and two different groups with omega-3 fatty acid dietary supplementation, one in which we gave the supplement subsequent to disease induction (TO3) and the other in which the agent was started prior to and continued subsequent to LPS injections (P + TO3). In the TO3 group, omega-3 fatty acid administration was performed for 14 days following induction of experimental periodontitis. In the P + TO3 group, omega-3 fatty acid was given for 14 days prior to the start of LPS injections and was continued for another 14 days subsequent to the induction of experimental periodontitis. On day 15 of the first LPS injection, serum samples were obtained and rats were sacrificed. Serum samples were analyzed for IL-1β, OC, and CRP concentrations by enzyme-linked immunosorbent assay. Defleshed jaws were analyzed morphometrically for alveolar bone loss. Data were evaluated statistically by non-parametric tests. RESULTS LPS injection resulted in statistically significantly more bone loss compared to the saline control group (P <0.05). None of the omega-3 fatty acid administration groups showed evidence that this fatty acid was effective in preventing LPS-induced alveolar bone loss. TO3 and P + TO3 groups revealed significantly higher IL-1β and OC levels than the LPS group (P <0.05). The study groups exhibited no significant differences in the serum CRP levels. CONCLUSIONS Omega-3 fatty acid administration does not seem to influence circulating levels of CRP. The significantly increased serum OC level observed in both omega-3 fatty acid regimes is curious and could have an effect on bone turnover, as could the further significant increase in serum IL-1β, which could counteract any osteoblastic induction by OC through promotion of osteoclast activity. The lack of a therapeutic benefit of omega-3 fatty acid in this study, despite the effects on OC and IL-1β, is difficult to explain, and further studies are required to more fully assess the potential role of this fatty acid in periodontal treatment.

Angiotensin-converting enzyme (ACE), angiotensinogen (AGT), and angiotensin II type 1 receptor (AT1R) gene polymorphisms in generalized aggressive periodontitis

AIM Host response to periodontopathic microorganisms can be modulated by genetic factors. Accumulated evidence highlighted the role of renin-angiotensin system (RAS) in inflammatory response thus potential implication of this molecular system in the pathogenesis of periodontitis can be suggested. The present study investigated common genetic variants of molecules within the RAS family namely angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II type 1 receptor (AT1R) in relation to generalized aggressive periodontitis (G-AgP). METHODS DNA was obtained from peripheral blood of 103 G-AgP patients and 100 periodontally healthy subjects. ACE I/D, AGT M235T and AT1R A1166C polymorphisms were genotyped by polymerase chain reaction and restriction fragment length polymorphism method. Chi-square, ANOVA and logistic regression were used in statistical analyses. RESULTS Both ACE I/D and AT1R polymorphisms were similar in G-AgP and healthy groups (p>0.05). G-AgP subjects exhibited decreased AGT TT genotype and T allele frequency as compared to healthy subjects (p<0.05). The same trend was also observed in the nonsmoker subgroup regarding investigated RAS polymorphisms. CONCLUSIONS Present findings suggest that AGT M235T TT genotype and T allele might be associated with decreased risk for G-AgP in Turkish population.

Gingival status, crevicular fluid tissue-type plasminogen activator, plasminogen activator inhibitor-2 levels in pregnancy versus post-partum.

BACKGROUND This study was conducted to evaluate a possible link between periodontal status of pregnant women and the plasminogen activator system in gingival crevicular fluid (GCF). METHODS GCF samples were obtained from four interproximal sites of anterior teeth in 43 women during the second trimester and also after delivery. Full mouth dental plaque, bleeding on probing (BOP) and probing depth (PD) values were recorded at six sites/tooth in each subject. GCF levels of tissue type plasminogen activator (t-PA) and its inhibitor, plasminogen activator-inhibitor-2 (PAI-2) were determined by ELISA. Data comparisons between pregnancy and post-partum were made by Wilcoxon signed rank test. RESULTS The number of pockets with a PD>4 mm and total volume of GCF sampled were reduced significantly after delivery (p=0.000 and p=0.013, respectively). No significant differences were detected in GCF concentrations of t-PA or PAI-2 between pregnancy and post-partum. CONCLUSIONS Our results suggest that GCF t-PA and PAI-2 concentrations are not affected by pregnancy. Reductions in PD values and GCF volume following delivery indicate a resolution of oedema in gingival tissues, possibly related to hormonal changes due to the ending of pregnancy.

The effect of adjunctive chlorhexidine mouthrinse on clinical parameters and gingival crevicular fluid cytokine levels in untreated plaque-associated gingivitis.

Abstract.Objective and design:To examine the effectiveness of chlorhexidine mouthrinse (CHX) in addition to daily plaque control on gingival inflammation.Methods:Fifty gingivitis patients were randomized to CHX or placebo groups. In addition to proper plaque control, CHX group rinsed with CHX, while placebo group rinsed with placebo mouthrinse for 4 weeks. Gingival crevicular fluid (GCF) samples were collected and clinical parameters including plaque index (PI), papillary bleeding index (PBI), calculus index and probing depth (PD) were recorded at baseline and repeated at 4 week. GCF IL-1α, IL-1β, IL-1Ra, and IL-8 levels were determined by ELISA.Results:Whole mouth clinical parameters were significantly improved in both groups at 4 weeks. CHX group showed greater reduction in the mean PI scores than placebo at 4 weeks (p < 0.05). GCF IL-8 levels of anterior sites significantly reduced in CHX and placebo group at 4 weeks (p < 0.05). GCF IL-1α, IL-1β, IL-1Ra levels remained unchanged at 4 weeks in both groups. GCF cytokine levels of CHX group were similar to those of placebo at 4 weeks.Conclusions:Within the limitations of this study, CHX mouthrinse as adjuncts to daily plaque control could be useful in management of plaque-associated gingivitis, although ineffective on GCF cytokine levels.

Renin-angiotensin gene polymorphisms in relation to severe chronic periodontitis.

AIM Evidence suggests that the ultimate product of the renin-angiotensin system (RAS), angiotensin II, exerts inflammatory actions. The present study aimed to evaluate the inter-relation between gene polymorphisms of the RAS components; angiotensin converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II type-I receptor (AT1R), and severe chronic periodontitis (CP). MATERIAL AND METHODS DNA was obtained from peripheral blood of 90 CP patients and 126 periodontally healthy subjects, and the clinical parameters were recorded. ACE I/D, AGT M235T and AT1R A1166C polymorphisms were genotyped by the PCR-RFLP method. Chi-square, anova and logistic regression methods were used in statistical analyses. RESULTS The frequency of the ACE D allele was significantly lower in the CP group than the healthy group (p(corr)=0.015). CP subjects exhibited increased C allele carriage and C allele frequency of the AT1R gene (p(corr)=0.03 and p(corr)=0.03, respectively). All clinical parameters of CP patients were found to be similar in variant allele-carrying and non-carrying subjects (p>0.05). CONCLUSIONS The present findings suggest that ACE I/D and AT1R polymorphisms might be associated with susceptibility to CP but not with disease severity. The D allele of ACE I/D might be associated with decreased, whereas the C variant of AT1R A1166C might be associated with an elevated risk for CP in Turkish population.

Effects of Selective Cyclooxygenase-2 Inhibitor and Omega-3 Fatty Acid on Serum Interleukin-1β, Osteocalcin, and C-Reactive Protein Levels in Rats

BACKGROUND The aim of this study was to evaluate the effects of selective cyclooxygenase-2 inhibitor, celecoxib, and omega-3 fatty acid on serum levels of interleukin 1-beta (IL-1β), osteocalcin (OC), and C-reactive protein (CRP) in experimental periodontitis. METHODS Experimental periodontitis in rats was induced by repeated injection of purified lipopolysaccharide (LPS) derived from Escherichia coli endotoxin. Forty-seven adult male Sprague-Dawley rats were divided into five study groups as follows: saline control, LPS, LPS + celecoxib, LPS + omega-3 fatty acid, and LPS + celecoxib + omega-3 fatty acid. Celecoxib and omega-3 fatty acid were given alone or in combination during 14 days of the experimental study period. At the end of the 2-week protocol, serum samples were obtained, and the rats were sacrificed. Serum samples were analyzed for IL-1β, OC, and CRP concentrations by enzyme-linked immunosorbent assay. Defleshed jaws were analyzed morphometrically for alveolar bone loss. Data were evaluated statistically by non-parametric tests. RESULTS According to the morphometric measurements, the LPS and drug treatment groups showed significantly higher bone loss than the saline control group (P <0.05). Omega-3 fatty acid, both alone and in combination with celecoxib, revealed significantly higher IL-1β levels than LPS and celecoxib groups (P <0.05). Individual and combined administration of celecoxib and omega-3 fatty acid significantly increased OC levels compared to the LPS group (P <0.05). There were no significant differences in serum CRP levels. CONCLUSIONS Celecoxib and/or omega-3 fatty acid administration does not significantly influence circulating levels of CRP. The significantly increased serum OC level observed after individual and combination administration suggests that celecoxib and omega-3 fatty acid may influence bone remodeling and thereby inhibit the progression of alveolar bone resorption. However, the failure to observe any significant inhibition of bone loss in celecoxib- and/or omega-3 fatty acid-treated rats compared to the LPS group suggests that their therapeutic effect may be reduced by other factors, such as increases in serum IL-1β promoting osteoclast activity.

Gingival crevicular fluid can degrade Emdogain and inhibit Emdogain-induced proliferation of periodontal ligament fibroblasts

BACKGROUND AND OBJECTIVE Emdogain (EMD), consisting mostly of amelogenin, is used in periodontal therapy to regenerate lost connective tissue. Emdogain is applied onto periodontally affected root surfaces, where it becomes exposed to proteolytic enzymes. In this study, we aimed to find out whether gingival crevicular fluid or matrix metalloproteinases (MMPs) could degrade EMD, and whether this degradation has consequences for in vitro cell proliferation. MATERIAL AND METHODS We studied the effects of 156 gingival crevicular fluid samples collected from subjects with different stages of periodontal disease and from healthy control subjects and the effects of MMP-1, -2, -8, -9, -13 and -14 on the degradation of EMD using EMD-embedded zymography. The effects of gingival crevicular fluid with or without EMD and the effects of amelogenin on the proliferation of cultured periodontal ligament fibroblasts were studied by cell proliferation enzyme-linked immunosorbent assay kit. RESULTS Degradation of Emdogain induced by gingival crevicular fluid was greater in samples from all stages of periodontal diseases compared with healthy control samples. Of the MMPs studied, only MMP-2 and MMP-8 showed limited EMD-degrading activities. One hundred micrograms per millilitre of EMD increased proliferation of periodontal ligament fibroblasts on average by 24% (confidence interval 0.60-0.64) and at 200 microg/mL by 30% (confidence interval 0.62-0.68) compared with control fibroblasts (confidence interval 0.48-0.52). However, gingival crevicular fluid (10 microg/mL) together with 100 microg/mL EMD induced the proliferation only by 6% (confidence interval 0.51-0.55) and with 200 microg/mL EMD by 12% (confidence interval 0.54-0.58). Amelogenin at 200 microg/mL decreased the proliferation of periodontal ligament fibroblasts by 54% (confidence interval 0.22-0.25). CONCLUSION We suggest that diseased gingival crevicular fluid containing various proteases leads to degradation of EMD and decreased proliferation of periodontal ligament fibroblasts.