Marinella Holzhausen

Protease-activated Receptor-2 (PAR2) in Human Periodontitis

No evidence for the role of protease-activated receptor-2 (PAR2) in human periodontal disease has been demonstrated so far. Thus, we sought to investigate the expression of PAR2 mRNA in chronic periodontitis, and to examine whether its expression is related to the presence of PAR2 potential activators. Microbiological and gingival crevicular fluid samples were collected from individuals with chronic periodontitis and control individuals, and the presence of neutrophil serine proteinase 3 (P3) and Porphyromonas gingivalis was evaluated. PAR2 mRNA expression was higher (p < 0.001) in those with chronic periodontitis compared with control individuals, and it was statistically decreased (p = 0.0006) after periodontal treatment. Furthermore, those with chronic periodontitis presented higher (p < 0.05) levels of IL-1α, IL-6, IL-8, and TNF-α, total proteolytic activity, P. gingivalis prevalence, and P3mRNA expression compared with control individuals. We conclude that PAR2 mRNA expression and its potential activators are elevated in human chronic periodontitis, therefore suggesting that PAR2 may play a role in periodontal inflammation.

Essential oils in one-stage full-mouth disinfection: double-blind, randomized clinical trial of long-term clinical, microbial and salivary effects.

AIM This randomized clinical trial evaluated the effects of an essential oils-containing mouthrinse for full-mouth disinfection. MATERIAL AND METHODS Fifty patients were assigned to receive full-mouth disinfection with either essential oils or placebo. At baseline, 2 and 6 months of treatment the primary outcomes probing depth (PD), plaque index (PlI) and modified gingival index (MGI) were monitored. Additional monitoring included bacterial presence (by polymerase chain reaction) in subgingival, saliva and tongue samples; flows, pH, total protein and alkaline phosphatase salivary levels. The following statistics were used: ANOVA, Students t-test, chi(2) and Kruskal-Wallis (p<0.05). RESULTS Mean PD>or=3.5 mm was reduced over time in both the placebo and the test groups, but there was no difference in PD reduction between groups at 2 and 6 months. At 2 and 6 months, PlI and MGI showed greater reductions in the test group than in the placebo group. Porphyromona gingivalis was not reduced in any site. At 6 months, Campylobacter rectus increased in both groups, while Tannerella forsythensis decreased subgingivally in the test group. S. sanguinis increased, except subgingivally, in the placebo group. Salivary pH and flows were not altered. Total protein reduced only in the test group. Alkaline phosphatase did not change in either group. CONCLUSIONS Essential oils for full-mouth disinfection showed clinical benefits, namely reducing plaque and gingival inflammation without altering basic salivary parameters.

Porphyromonas Gingivalis is Associated With Protease-Activated Receptor-2 Upregulation in Chronic Periodontitis

BACKGROUND We previously reported a higher expression of protease-activated receptor-2 (PAR(2)) together with higher interleukin (IL)-1α, IL-6, IL-8, and tumor necrosis factor-α levels, total proteolytic activity, Porphyromonas gingivalis (Pg) prevalence, and neutrophil-protease 3 messenger RNA (mRNA) expression in patients with chronic periodontitis compared to healthy control patients. The aim of the present study is to expand this observation by considering the site level according to the presence of Pg. METHODS Microbiologic and gingival crevicular fluid samples were collected from patients with chronic periodontitis. Pg presence was evaluated by polymerase chain reaction and PAR(2) mRNA expression was evaluated by reverse-transcription polymerase chain reaction. Total proteolytic activity in the crevicular fluid was analyzed by using a specific substrate benzoylarginine nitroanilide, and the proinflammatory mediators IL-1α, IL-6, IL-8, and tumor necrosis factor-α were analyzed by enzyme-linked immunosorbent assay. RESULTS In Pg-positive periodontal sites, the mean probing depth and clinical attachment level, the prevalence of bleeding on probing sites, and crevicular fluid volume were higher (P <0.05) compared to Pg-negative sites. In addition, with the exception of IL-8, all other inflammatory mediators were positively (P <0.05) associated with Pg presence. Pg presence was also positively associated with a higher proteolytic activity (P = 0.0037) and higher PAR(2) mRNA expression (P = 0.0271). CONCLUSIONS We conclude that in chronic periodontitis, periodontal pockets presenting Pg show an upregulation of PAR(2) gene expression, and higher proinflammatory profile associated with advanced clinical destruction, therefore suggesting that Pg plays a pivotal role on PAR(2)-mediated periodontal inflammation in humans.

Prevalence and distribution of serotype-specific genotypes of Aggregatibacter actinomycetemcomitans in chronic periodontitis Brazilian subjects

OBJECTIVE Previous studies have suggested that Aggregatibacter actinomycetemcomitans is involved in the aetiology of aggressive periodontitis as well as chronic periodontitis. In addition, some authors have also reported that serotype-specific antigens of A. actinomycetemcomitans determine the severity of disease. This study aimed to elucidate the prevalence of A. actinomycetemcomitans and the distribution of A. actinomycetemcomitans serotypes in Brazilian subjects with chronic periodontitis. DESIGN A total of 486 individuals were enrolled in this survey. All patients received clinical examinations that included periodontal pocket depth, clinical attachment loss, plaque, and gingival indexes. Subgingival samples were taken for microbial analysis. The genomic DNA of A. actinomycetemcomitans was provided by PCR. RESULTS Out of 486 subjects examined, A. actinomycetemcomitans was isolated in 85 (17.5%) individuals. Out of 85 positive samples, 68 were infected by at least 1 serotype, 7 by mixed infection, and 10 were non-serotyped. Serotypes d and f were not detected. Serotype c showed the highest prevalence (52.9%), followed by serotype a (31.8%). CONCLUSIONS Intragroup analysis revealed that, in slight/moderate periodontitis, serotypes c and a were significantly more prevalent than serotypes b and d-f; the prevalence of serotype c in severe periodontitis was significantly greater than that of serotypes a and b. Our data were similar in Asian and Eurasian populations.

Periodontal Treatment Downregulates Protease-Activated Receptor 2 in Human Gingival Crevicular Fluid Cells

ABSTRACT Protease-activated receptor 2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases, including periodontitis; it can be activated by gingipain and produced by Porphyromonas gingivalis and by neutrophil protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. The effects of periodontal treatment on PAR2 expression and its association with levels of proinflammatory mediators and activating proteases were investigated in chronic periodontitis patients. Positive staining for PAR2 was observed in gingival crevicular fluid cells and was reflective of tissue destruction. Overexpression of PAR2 was positively associated with inflammatory clinical parameters and with the levels of interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, matrix metalloprotease 2 (MMP-2), MMP-8, hepatocyte growth factor, and vascular endothelial growth factor. Elevated levels of gingipain and P3 and decreased levels of dentilisin and the protease inhibitors secretory leukocyte protease inhibitor and elafin were also associated with PAR2 overexpression. Healthy periodontal sites from individuals with chronic periodontitis showed diminished expression of PAR2 mRNA and the PAR2 protein (P < 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression and correlated with decreased expression of inflammatory mediators and activating proteases. We concluded that periodontal treatment resulted in decreased levels of proteases and that proinflammatory mediators are associated with decreased PAR2 expression, suggesting that PAR2 expression is influenced by the presence of periodontal infection and is not a constitutive characteristic favoring periodontal inflammation.

Local and cardiorenal effects of periodontitis in nitric oxide-deficient hypertensive rats

OBJECTIVE in this study we have assessed the renal and cardiac consequences of ligature-induced periodontitis in both normotensive and nitric oxide (NO)-deficient (L-NAME-treated) hypertensive rats. MATERIALS AND METHODS oral L-NAME (or water) treatment was started two weeks prior to induction of periodontitis. Rats were sacrificed 3, 7 or 14 days after ligature placement, and alveolar bone loss was evaluated radiographically. Thiobarbituric reactive species (TBARS; a lipid peroxidation index), protein nitrotyrosine (NT; a marker of protein nitration) and myeloperoxidase activity (MPO; a neutrophil marker) were determined in the heart and kidney. RESULTS in NO-deficient hypertensive rats, periodontitis-induced alveolar bone loss was significantly diminished. In addition, periodontitis-induced cardiac NT elevation was completely prevented by L-NAME treatment. On the other hand L-NAME treatment enhanced MPO production in both heart and kidneys of rats with periodontitis. No changes due to periodontitis were observed in cardiac or renal TBARS content. CONCLUSIONS in addition to mediating alveolar bone loss, NO contributes to systemic effects of periodontitis in the heart and kidney.

Human β-defensin 2 and protease activated receptor-2 expression in patients with chronic periodontitis

OBJECTIVE Some previous studies have shown that gingipains, trypsin-like proteases produced by Porphyromonas gingivalis, up-regulate human β defensin-2 (HBD-2) mRNA expression through protease-activated receptor-2 (PAR(2)) in gingival epithelial cells. This study aimed at investigating salivary HBD-2 levels and crevicular PAR(2) mRNA expression in human chronic periodontitis and evaluating whether periodontal treatment affected this process. METHODS Salivary and gingival crevicular fluid (GCF) samples were collected from periodontally healthy (control) and chronic periodontitis patients at baseline and 50 days after non-surgical periodontal treatment. Salivary HBD-2, and GCF TNF-α levels were analysed by ELISA, and PAR(2) mRNA at the GCF was evaluated by RT-PCR. RESULTS P. gingivalis was significantly (p<0.05) more prevalent in patients with chronic periodontitis when compared to controls. This prevalence decreased after periodontal therapy (p<0.0001). The control group showed statistically significant lower levels of HBD-2, TNF-α, and PAR(2) expression when compared to the chronic periodontitis group. In addition, periodontal treatment significantly reduced PAR(2) expression and HBD-2 levels in chronic periodontitis patients (p<0.001). CONCLUSIONS Our results suggest that salivary HBD-2 levels and PAR(2) mRNA expression from GCF are higher in subjects with chronic periodontitis than in healthy subjects, and that periodontal treatment decreases both HBD-2 levels and PAR(2) expression.

Clinical status and detection of periodontopathogens and Streptococcus mutans in children with high levels of supragingival biofilm

Knowledge about the presence of some important oral pathogens is an important step in better identifying children at risk for periodontal and/or caries diseases in later life. The purpose of this study was to detect the presence of Streptococcus mutans (Sm), Aggregatibacter actinomycetemcomitans (Aa), Campylobacter rectus (Cr), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), and Tannerella forsythia (Tf) in gingival biofilm samples from 196 children, and to assess whether any of these pathogens are more associated with gingival inflammation extension and the Decayed/Missing/Filled teeth (DMFT/dmft) index. The subjects presented plaque index greater than 80% and were divided in 3 groups according to the bleeding index (BI): I) Low bleeding (< 30%), II) Medium bleeding (31 - 59%) and III) High bleeding (> 60%). The presence of each pathogen was determined by PCR. The prevalence of Sm was 71.9% and the mean dmft/DMFT was 6.68. The prevalence in low, medium and high bleeding groups was 43.5%, 34.5% and 46.7% for Aa; 43.5%, 37.9%, and 36.7% for Cr; 99.1%, 100%, and 96.7% for Pg; 56.5%, 56.9%, and 66.7% for Pi; and 58.3%, 60.3%, and 56.7% for Tf, respectively. Pg (99.0%) was the most prevalent periodontal pathogen detected followed by Tf (58.7%), Pi (58.2%), Aa (41.3%) and Cr (40.8%). Our study indicated that in this high plaque index population studied, a high prevalence of Sm and high mean DMFT were observed. In addition, the presence of Pi was associated with the presence of inflammation (P < 0.05) whereas Cr was associated with periodontal health (P < 0.05).

Expression of Protease Activated Receptor-1 in Chronic Periodontitis

BACKGROUND Protease activated receptor-1 (PAR1) activation by thrombin may play a role in repair and homeostasis of periodontal tissues. The main objective of this study is to investigate PAR1 expression in patients with periodontitis, before and after non-surgical periodontal treatment, and to associate its expression with the presence of inflammatory biomarkers and PAR2 expression. METHODS Gingival crevicular fluid (GCF) samples and clinical parameters, including probing depth, clinical attachment level, bleeding on probing, and gingival and plaque indices, were collected from periodontally healthy individuals and patients with moderate chronic periodontitis (CP) before and 6 weeks after periodontal non-surgical treatment. PAR1 and PAR2 messenger RNA (mRNA) at the GCF were evaluated by quantitative polymerase chain reaction (qPCR). Flow cytometry analysis identified the GCF PAR1-expressing cells. GCF inflammatory biomarkers were also determined. RESULTS Clinical parameters were significantly improved after therapy (P <0.01). The qPCR analysis showed that, before therapy, PAR1 mRNA levels in CP were similar to controls. Periodontal treatment led to increased PAR1 expression in CP (P <0.05). PAR1 expression was inversely correlated to PAR2 expression and with interleukins 6 and 8, tumor necrosis factor-α, interferon-γ, and matrix metalloproteinase-2 levels. CONCLUSIONS Periodontal treatment results in PAR1 overexpression in the GCF, and PAR1 expression is associated with decreased expression of inflammatory biomarkers and inversely correlated to PAR2 expression in the GCF. Therefore, the data suggest the importance of PAR1 mediating the known anabolic actions of thrombin in the periodontium.

Influence of Parstatin on Experimental Periodontal Disease and Repair in Rats

BACKGROUND Parstatin is a 41-amino acid peptide, formed by proteolytic cleavage on activation of the protease activated receptor-1, with antiangiogenic properties. The purpose of this study is to evaluate the influence of synthetic parstatin on experimental periodontal disease and repair in rats. METHODS Ligature-induced periodontitis was established in rats and the influence of parstatin administration was assessed after 8 and 15 days for periodontal disease and 24 hours and 8 days after repair after ligature removal. RESULTS Parstatin administration significantly inhibited gingival myeloperoxidase activity, interleukin (IL)-1β, tumor necrosis factor-α, and IL-6 levels and led to suppression of macrophages and collagen degradation. At periodontal tissues under repair, parstatin increased the gingival levels of endostatin and decreased vascular endothelial growth factor expression and blood vessel number but did not influence histologic healing. In addition, the tomographic linear bone loss was significantly reduced at 15 days of periodontitis when the rats were treated with parstatin compared to their respective phosphate-buffered saline-treated controls. CONCLUSIONS Parstatin suppresses the periodontal tissue breakdown followed by experimental periodontitis in rats and did not impair periodontal tissue repair, despite its antiangiogenic effect. Parstatin may represent a novel approach to modulate host response in chronic periodontal disease.