Effect of non-surgical periodontal therapy on salivary histone deacetylases expression: A prospective clinical study.

AIM: To evaluate the effect of non-surgical periodontal therapy (NSPT) on salivary histone deacetylases (HDACs) gene expression in patients with Stage III-IV periodontitis at baseline and at 3 and 6 months post NSPT treatment. MATERIALS AND METHODS: Twenty patients completed the study. Periodontitis (as well as the corresponding staging and grading) was diagnosed according to the 2017 World Workshop Classification. Clinical measures were recorded and whole unstimulated saliva was collected at baseline and at 3 and 6 months after NSPT. The expression of 11 HDACs was determined using reverse-transcription PCR, and the respective changes over time were evaluated. RESULTS: Six months after NSPT, significant improvements in all clinical periodontal parameters were observed, concomitant with significant up-regulation of HDAC2, 4, 6, 8, 9 and 11 expressions. Subgroup analyses of non-responders and responders revealed no significant differences in HDACs mRNA expression between groups at any time point. CONCLUSIONS: This prospective clinical study identified longitudinal changes in salivary HDACs expression in response to NSPT, which provides new insights into the epigenetic mechanisms underlying the pathobiology of periodontitis and creates avenues for the discovery of novel biomarkers.

Active matrix metalloproteinase-8 (aMMP-8) point-of-care test (POCT) in the COVID-19 pandemic.

INTRODUCTION: Active matrix metalloproteinase (aMMP)-8 utilized in point-of-care testing (POCT) is regarded as a potential biomarker for periodontal and peri-implant diseases. Various host and microbial factors eventually influence the expression, degranulation, levels and activation of aMMP-8. The type of oral fluids (saliva, mouthrinse, gingival crevicular, and peri-implant sulcular fluids [GCF/PISF], respectively) affect the analysis. AREAS COVERED: With this background, we aimed to review here the recent studies on practical, inexpensive, noninvasive and quantitative mouthrinse and GCF/PISF chair-side POCT lateral flow aMMP-8 immunoassays (PerioSafe and ImplantSafe/ORALyzer) and how they help to detect, predict, monitor the course, treatment and prevention of periodontitis and peri-implantitis. The correlations of aMMP-8 POCT to other independent and catalytic activity assays of MMP-8 are also addressed. EXPERT OPINION: The mouthrinse aMMP-8 POCT can also detect prediabetes/diabetes and tissue destructive oral side-effects due to the head and neck cancers' radiotherapy. Chlorhexidine and doxycycline can inhibit collagenolytic human neutrophil and GCF aMMP-8. Furthermore, by a set of case-series we demonstrate the potential of mouthrinse aMMP-8 POCT to real-time/online detect periodontitis as a potential risk disease for coronavirus disease 2019 (COVID-19). The clinical interdisciplinary utilization of aMMP-8 POCT requires additional oral, medical, and interdisciplinary studies.

Inhibition of transglutaminase activity in periodontitis rescues periodontal ligament collagen content and architecture.

BACKGROUND AND OBJECTIVE: Periodontal disease (PD) afflicts approximately 50% of the population in the United States and is characterized by chronic inflammation of the periodontium that can lead to loss of the periodontal ligament through collagen degradation, loss of alveolar bone, and to eventual tooth loss. Previous studies have implicated transglutaminase (TG) activity in promoting thin collagen I fiber morphology and decreased mechanical strength in homeostatic PDL. The aim of this study was to determine whether TG activity influenced collagen assembly in PDL in the setting of periodontal disease. MATERIAL AND METHODS: A ligature model was used to induce clinically relevant PD in mice. Mice with ligature were assessed at 5 and 14 days to determine PDL collagen morphology, transglutaminase (TG) activity, and bone loss. The effects of inhibition of TG on PDL were assessed by immunohistochemistry and second-harmonic generation (SHG) to visualize collagen fibers in native tissue. RESULTS: Ligature placement around the 2nd molar resulted in significant bone loss and a decrease in total collagen content after 5 days of ligature placement. A significant increase in thin over thick fibers was also demonstrated in mice with ligature at 5 days associated with apparent increases in immunoreactivity for TG2 and for TG-mediated N-ε-γ-glutamyl cross-links in PDL. Inhibition of TG activity increased total collagen and thick collagen fiber content over vehicle control in mice with ligature for 5 days. SHG of PDL was used to visualize and quantify the effects of TG inhibition on enhanced collagen fiber organization in unfixed control and diseased PDL. CONCLUSION: These studies support a role of TG in regulating collagen fiber assembly and suggest that strategies to inhibit TG activity in disease might contribute to restoration of PDL tissue integrity.

Methodological evaluation of gingival crevicular fluid volume and neutrophil elastase levels: sequential sampling, length of sampling time and two different sampling methods.

Objectives: The mechanisms underlying the formation and composition of gingival crevicular fluid (GCF) and its flow into and from periodontal pockets are not understood very well. The aim of this study was to evaluate the length of sampling time and sequential sampling of GCF neutrophil elastase (NE) enzyme levels by using intracrevicular and orifice methods.Material and methods: Twenty adults (mean age of 41.8 years, ranged 31-60 years, 18 males and 2 females) with chronic periodontitis were enrolled and all completed the 3-d study. GCF was collected by both intracrevicular and intrasulcular methods, 720 samples of GCF were collected. In first, second and third day, the length of sampling time in seconds (s) and order were '5- 10-30-s'; '10- 30- 5-s' and '30- 5- 10-s,' respectively. GCF elastase levels were determined by hydrolysis of neutrophil specific substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide.Results: NE activity (µU) and NE activity/volume (µU/µl) were significantly different for order of sampling (p < .05), but not for the length of sampling time (p>.05).Conclusions: Within the limits of this study, the choice of sampling technique in GCF-profile studies seems to be a critical decision as it has the potential to affect the GCF volume and NE activity.

Salivary Biomarkers and Their Application in the Diagnosis and Monitoring of the Most Common Oral Pathologies.

Saliva is a highly versatile biological fluid that is easy to gather in a non-invasive manner-and the results of its analysis complement clinical and histopathological findings in the diagnosis of multiple diseases. The objective of this review was to offer an update on the contribution of salivary biomarkers to the diagnosis and prognosis of diseases of the oral cavity, including oral lichen planus, periodontitis, Sjögren's syndrome, oral leukoplakia, peri-implantitis, and medication-related osteonecrosis of the jaw. Salivary biomarkers such as interleukins, growth factors, enzymes, and other biomolecules have proven useful in the diagnosis and follow-up of these diseases, facilitating the early evaluation of malignization risk and the monitoring of disease progression and response to treatment. However, further studies are required to identify new biomarkers and verify their reported role in the diagnosis and/or prognosis of oral diseases.

Tyrosine-protein phosphatase non-receptor type 2 inhibits alveolar bone resorption in diabetic periodontitis via dephosphorylating CSF1 receptor.

Tyrosine-protein phosphatase non-receptor type 2 (PTPN2) is an important protection factor for diabetes and periodontitis, but the underlying mechanism remains elusive. This study aimed to identify the substrate of PTPN2 in mediating beneficial effects of 25-Hydroxyvitamin D3 (25(OH)2D3 ) on diabetic periodontitis. 25(OH)2D3 photo-affinity probe was synthesized with the minimalist linker and its efficacy to inhibit alveolar bone loss, and inflammation was evaluated in diabetic periodontitis mice. The probe was used to pull down the lysates of primary gingival fibroblasts. We identified PTPN2 as a direct target of 25(OH)2D3 , which effectively inhibited inflammation and bone resorption in diabetic periodontitis mice. In addition, we found that colony-stimulating factor 1 receptor (CSF1R) rather than JAK/STAT was the substrate of PTPN2 to regulate bone resorption. PTPN2 direct interacted with CSF1R and dephosphorylated Tyr807 residue. In conclusion, PTPN2 dephosphorylates CSF1R at Y807 site and inhibits alveolar bone resorption in diabetic periodontitis mice. PTPN2 and CSF1R are potential targets for the therapy of diabetic periodontitis or other bone loss-related diseases.

Regulation of tyrosine hydroxylase in periodontal fibroblasts and tissues by obesity-associated stimuli.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the synthesis of catecholamines and has been connected to aggravated progression of periodontal disease under chronic stress. Obesity is known to increase the risk of periodontitis and adipokines have been suggested to be a pathomechanistic link. This study examines if obesity-associated stimuli have regulatory effects on TH levels in periodontal cells and tissues. Human periodontal ligament fibroblasts were cultured in the presence of leptin or visfatin for up to 2 days. Untreated cells served as control. TH regulation was analyzed by real-time PCR, immunocytochemistry and ELISA. TH gene expression in periodontal tissues of normal-weight and obese rodents was determined. Examination of gingival biopsies from rats and patients with and without periodontal disease was performed by real-time PCR or immunohistochemistry. For statistics, ANOVA and post hoc tests were applied (p < 0.05). In vitro, TH gene expression and protein levels were increased by leptin and visfatin. In vivo, TH gene expression was upregulated in periodontal tissues of obese rodents as compared to normal-weight animals. Additionally, increased TH gene expression was found in rat gingival biopsies with experimental periodontitis. Human gingival biopsies from sites of periodontitis confirmed the animal data by demonstrating elevated TH levels at periodontally diseased sites. This study provides original evidence that obesity-associated stimuli induce a TH upregulation in periodontal cells and tissues. Since TH levels were also increased at periodontitis sites, our in vitro and animal findings suggest that this enzyme could represent a pathomechanism whereby obesity contributes to periodontitis.

Up-regulated ferritin in periodontitis promotes inflammatory cytokine expression in human periodontal ligament cells through transferrin receptor via ERK/P38 MAPK pathways.

OBJECTIVE: Ferritin, an iron-binding protein, is ubiquitous and highly conserved; it plays a crucial role in inflammation, which is the main symptom of periodontitis. Full-length cDNA library analyses have demonstrated abundant expression of ferritin in human periodontal ligament. The aims of the present study were to explore how ferritin is regulated by local inflammation, and to investigate its functions and mechanisms of action in the process of periodontitis. METHODS: Human gingival tissues were collected from periodontitis patients and healthy individuals. Experimental periodontitis was induced by ligature of second molars in mice. The expression of ferritin light polypeptide (FTL) and ferritin heavy polypeptide (FTH) were assessed by immunohistochemistry. Meanwhile, after stimulating human periodontal ligament cells (HPDLCs) with P. gingivalis-lipopolysaccharide (LPS), interleukin (IL)-6, and tumor necrosis factor-α (TNF-α), the expression of FTH and FTL were measured. Then, IL-6 and IL-8 were measured after incubation with different concentrations of apoferritin (iron-free ferritin) and several intracellular signaling pathway inhibitors, or after knockdown of the transferrin receptor. RESULTS: Both FTH and FTL were substantially higher in inflamed periodontal tissues than in healthy tissues. The location of the elevated expression correlated well with the extent of inflammatory infiltration. Moreover, expression of FTH and FTL were enhanced after stimulation with P. gingivalis-LPS, IL-6, TNF-α. Apoferritin induced the production of IL-6 and IL-8 in a dose-dependent manner partly through binding to the transferrin receptor and activating ERK/P38 signaling pathways in HPDLCs. CONCLUSIONS: Ferritin is up-regulated by inflammation and exhibits cytokine-like activity in HPDLCs inducing a signaling cascade that promotes expression of pro-inflammatory cytokines associated with periodontitis.

Gingival Tissue Inflammation Promotes Increased Matrix Metalloproteinase-12 Production by CD200Rlow Monocyte-Derived Cells in Periodontitis.

Irreversible tissue recession in chronic inflammatory diseases is associated with dysregulated immune activation and production of tissue degradative enzymes. In this study, we identified elevated levels of matrix metalloproteinase (MMP)-12 in gingival tissue of patients with the chronic inflammatory disease periodontitis (PD). The source of MMP12 was cells of monocyte origin as determined by the expression of CD14, CD68, and CD64. These MMP12-producing cells showed reduced surface levels of the coinhibitory molecule CD200R. Similarly, establishing a multicellular three-dimensional model of human oral mucosa with induced inflammation promoted MMP12 production and reduced CD200R surface expression by monocyte-derived cells. MMP12 production by monocyte-derived cells was induced by CSF2 rather than the cyclooxygenase-2 pathway, and treatment of monocyte-derived cells with a CD200R ligand reduced CSF2-induced MMP12 production. Further, MMP12-mediated degradation of the extracellular matrix proteins tropoelastin and fibronectin in the tissue model coincided with a loss of Ki-67, a protein strictly associated with cell proliferation. Reduced amounts of tropoelastin were confirmed in gingival tissue from PD patients. Thus, this novel association of the CD200/CD200R pathway with MMP12 production by monocyte-derived cells may play a key role in PD progression and will be important to take into consideration in the development of future strategies to diagnose, treat, and prevent PD.

Endogenous acid ceramidase protects epithelial cells from Porphyromonas gingivalis-induced inflammation in vitro.

Ceramidases are a group of enzymes that degrade pro-inflammatory ceramide by cleaving a fatty acid to form anti-inflammatory sphingosine lipid. Thus far, acid, neutral and alkaline ceramidase isozymes have been described. However, the expression patterns of ceramidase isoforms as well as their role in periodontal disease pathogenesis remain unknown. In this study, expression patterns of ceramidase isoforms were quantified by real-time PCR and immunohistochemistry in gingival samples of patients with periodontitis and healthy subjects, as well as in EpiGingivalTM-3D culture and OBA-9 gingival epithelial cells both of which were stimulated with or without the presence of live Porphyromonas gingivalis (ATCC 33277 strain). A significantly lower level of acid ceramidase expression was detected in gingival tissues from periodontal patients compared to those from healthy subjects. In addition, acid-ceramidase expression in EpiGingival™ 3D culture and OBA-9 cells was suppressed by stimulation with P. gingivalis in vitro. No significant fluctuation was detected for neutral or alkaline ceramidases in either gingival samples or cell cultures. Next, to elucidate the role of acid ceramidase in P. gingivalis-induced inflammation in vitro, OBA-9 cells were transduced with adenoviral vector expressing the human acid ceramidase (Ad-ASAH1) gene or control adenoviral vector (Ad-control). In response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9 cells showed significantly lower mRNA expressions of caspase-3 as well as the percentage of Annexin V-positive cells, when compared with OBA-9 cells transduced with Ad-control vector. Furthermore, in response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9 cells produced less TNF-α, IL-6, and IL1ß pro-inflammatory cytokines than observed in OBA-9 cells transduced with Ad-control vector. Collectively, our data show the novel discovery of anti-inflammatory and anti-apoptotic effects of acid ceramidase in host cells exposed to periodontal bacteria, and the attenuation of the expression of host-protective acid ceramidase in periodontal lesions.

Increased citrullination and expression of peptidylarginine deiminases independently of P. gingivalis and A. actinomycetemcomitans in gingival tissue of patients with periodontitis.

BACKGROUND: A relationship between rheumatoid arthritis (RA) and periodontitis has been suggested from findings that individuals with RA are prone to have advanced periodontitis and vice versa. In search of possible common pathogenetic features of these two diseases, we investigated the presence of citrullinated proteins and expression of endogenous peptidylarginine deiminases (PAD2 and PAD4), in periodontal tissue of individuals with periodontitis and healthy controls, in relation to the periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), producing leukotoxin as virulence factor. These two oral bacteria have been suggested to be linked to anti-citrullinated protein antibodies in patients with RA. METHODS: Gingival tissue biopsies were obtained from 15 patients with periodontitis and 15 individuals without periodontal disease. Presence of CD3-positive lymphocytes, citrullinated proteins, PAD2, PAD4, P. gingivalis as well as A. actinomycetemcomitans and Mannheimia haemolytica produced leukotoxins were analysed by immunohistochemistry, followed by triple-blind semi-quantitative analysis. Mann-Whitney and Fisher's exact tests were used to analyse differences between groups. PADI2 and PADI4 mRNA levels were assessed by RT-qPCR and analysed using Wilcoxon signed rank test. RESULTS: Increased staining of citrullinated proteins was observed in gingival connective tissue from subjects with periodontitis (80%, 12/15) compared to healthy gingival tissue (27%, 4/15), whereas no differences were observed in gingival epithelium. There was also an increased staining of the citrullinating enzymes PAD2 and PAD4 in gingival connective tissue of patients with periodontitis whereas similar levels of PAD2 and PAD4 were observed in the gingival epithelium of the two groups. Similarly, the mRNA levels of PADI2 and PADI4 were also increased in the gingival tissue of patients with periodontitis compared to healthy controls. Furthermore, presence of P. gingivalis and leukotoxins was comparable in both epithelium and connective tissue, from the different investigated individuals with and without periodontitis, and there were no correlations between the presence of periodontal pathogens and the expression of citrullinated proteins or PAD enzymes. CONCLUSION: Chronic gingival inflammation is associated with increased local citrullination and PAD2 and PAD4 expression in periodontitis. The increased citrullination and PAD2 and PAD4 expression in periodontitis were, however, independent of the presence of periodontal pathogen P. gingivalis and A. actinomycetemcomitans leukotoxin.

Soluble urokinase-type plasminogen activator receptor is associated with signs of periodontitis in adolescents.

Owing to its molecular stability in body fluids, soluble urokinase-type plasminogen activator receptor (suPAR) is used as a biomarker for the level of systemic inflammation. This study compares the suPAR levels in serum with those in the saliva of adolescents and evaluates their association with the periodontal conditions. Adolescents identified as screen positive (n = 87) or screen negative (n = 73) for periodontitis had saliva and serum samples taken, along with subgingival plaque samples. The concentrations of suPAR were determined in saliva and serum, and 18 microbial species and the immunoglobulin response to them was evaluated. Factor analyses were used to reduce the number of variables within each of the domains of clinical, microbiological, and immunological findings. The median salivary suPAR concentration was 13.18 [(interquartile range (IQR): 6.20-23.36] µg l-1 and was not associated with the serum suPAR levels (median 2.05; IQR: 1.62-2.46 µg l-1 ). Linear regression analysis showed that the log10 (salivary suPAR concentration) was statistically significantly positively associated with the clinical phenotype 'Periodontitis Extent' (ß = 0.28; 95% CI: 0.16-0.39) along with 'Putative periodontopathogens' (ß = 0.65; 95% CI: 0.51-0.79). The study represents the first determination of salivary suPAR concentration in a larger well-defined adolescent population. Our results suggest the potential for clinical use of suPAR in saliva as an inflammatory risk indicator/biomarker of periodontitis.

Effects of a cyclic NSAID regimen on levels of gingival crevicular fluid prostaglandin E2and Interleukin-1ß: A 6-month randomized controlled clinical trial.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for inflammation control and pain relief. However, while the adjunct use of NSAIDs is avoided for periodontal therapy because of related side effects, cyclic administration of NSAIDs may reduce or eliminate these effects. We evaluated the effect of a cyclic diclofenac potassium (DP) regimen on clinical parameters and levels of prostaglandin E2 (PGE2) and interleukin-1ß (IL-1ß) in the gingival crevicular fluid (GCF) of individuals with periodontitis. MATERIALS AND METHODS: The study protocol was approved by the Ethics Committee (2000/071). Forty-one individuals with chronic periodontitis (33 men, 8 women) were divided into two groups (test and control) after initial periodontal therapy. During this 6-month, randomized, double-blind, placebo-controlled study, test (n = 28) and control (n = 13) groups were administered a cyclic regimen of DP (50 mg, twice daily) or placebo. Clinical measurements and GCF sample collections were made at baseline, 2, 4, and 6 months. GCF levels of PGE2and IL-1ß were determined using enzyme immunoassay and enzyme-linked immunoassay kits, respectively. RESULTS: At baseline, no significant differences existed between groups for plaque indices, gingival indices, bleeding on probing, probing depth (PD), or attachment levels (P > 0.05). Compared with the control group, cyclic regimen in the test group suppressed increased levels of PGE2found in GCF at the end of the study (P < 0.05). Significant differences for PD and relative attachment gain were also noted in favor of the test group (P < 0.05). CONCLUSIONS: These results suggest that a cyclic regimen of DP may be efficacious in the management of chronic periodontitis in adults.

Evaluation of a novel point-of-care test for active matrix metalloproteinase-8: agreement between qualitative and quantitative measurements and relation to periodontal inflammation.

BACKGROUND AND OBJECTIVES: Severe periodontitis affects about 10% of the world population. In addition, associations between periodontitis and systemic diseases exist. Therefore, the diagnosis should be made quickly and at an early stage. Matrix metalloproteinase-8 (MMP-8) is the most prominent collagenase found in inflamed periodontal tissues. Its active form (aMMP-8) is increasingly used as a diagnostic biomarker. Aim of the present study is to evaluate the diagnostic accuracy of a novel aMMP-8 point-of-care (POC) test in comparison to the standard laboratory test to diagnose the disease rapidly and reliably. MATERIAL AND METHODS: In a prospective, mono-center, double-blinded, case-control study, participants with healthy gums (n = 35), gingivitis (n = 60) and periodontitis (n = 35) were investigated before and after therapy. Beside clinical variables for plaque and inflammation, aMMP-8 concentrations were determined in oral rinsing specimens by the enzyme-linked immunosorbent assay (ELISA) and by POC. Positive and negative percent agreements with their exact one-sided lower 95% confidence limits were calculated. RESULTS: Of 130 participants, 111 finished the study. Overall, positive percent agreements were 75.8% (57.7-88.9) before treatment and 73.7% (56.9-86.6) after treatment. Negative percent agreements were 92.8% (85.7-97.0) before and 93.3% (85.1-97.8) after treatment. Positive test results (POC and ELISA) ranged from 5.7% to 8.6% in healthy patients, 25.0-29.8% in patients with gingivitis and 40.0-48.1% in patients with periodontitis. Patients who had positive aMMP-8 test results (POC) showed higher scores for plaque and inflammation. CONCLUSIONS: The novel POC test to detect aMMP-8 has proved to agree strongly with the standard method, ELISA. The test can be recommended to screen patients at risk for periodontitis in dental offices, at the general practitioner and at specialists for associated diseases.

Active matrix metalloproteinase-8 and periodontal bacteria depending on periodontal status in patients with rheumatoid arthritis.

BACKGROUND AND OBJECTIVES: The aim of this clinical cross-sectional study was to determine the level of active matrix metalloproteinase-8 (aMMP-8) and periodontal pathogenic bacteria in gingival crevicular fluid in patients with rheumatoid arthritis (RA) with varying periodontal conditions. MATERIAL AND METHODS: In total, 103 patients with RA and 104 healthy controls (HC) were included. The assessment of periodontal status included periodontal probing depth, bleeding on probing and clinical attachment loss. Periodontal disease was classified as healthy/mild, moderate or severe. For the determination of aMMP-8 levels using enzyme-linked immunosorbent assay and periodontal pathogenic bacteria using polymerase chain reaction, samples of gingival crevicular fluid were taken from the deepest gingival pockets. The statistical analyses used included a Mann-Whitney U-test, a chi-squared test or a Fisher's exact test, and the significance level was set at α = 5%. RESULTS: We found that 65% of patients with RA and 79% of HC had moderate to severe periodontal disease (p = 0.02). The prevalence of periodontal pathogens was almost equal (p > 0.05). Furthermore, depending on periodontal disease severity only minor differences in bacterial prevalence were detected. With increasing severity of periodontal disease, higher aMMP-8 levels were observed. Accordingly, a significant difference in patients with moderate periodontal disease (RA: 15.3 ± 13.8; HC: 9.1 ± 9.1; p ≤ 0.01) and severe periodontal disease (RA: 21.7 ± 13.3; HC: 13.1 ± 8.6; p = 0.07) was detected, with a greater tendency in the latter group. CONCLUSION: The increased aMMP-8 levels in the RA group indicate that the presence of RA appears to have an influence on the host response at a comparable level of bacterial load and periodontal disease severity.

Association of salivary peroxidase activity and concentration with periodontal health: A validity study.

AIM: Whereas the relationship between myeloperoxidase and periodontitis has been widely examined that between salivary peroxidase and periodontitis has received little attention. We examined how periodontitis depends on both salivary peroxidase activity and concentration. MATERIALS AND METHODS: A full mouth, clinical assessment of probing depth was performed in a sample of 46 participants aged 25-54 years. To minimise bias, these data were corrected by data from the general population (Study of Health in Pomerania). Using five repeated measurements of activity and concentration over 1 day, we assessed daily biological variability and increased the reliability of salivary peroxidase measurements. RESULTS: Salivary peroxidase activity was associated with probing depth (interquartile range effect = -0.48; robust estimates of 95% confidence interval: -0.90 to -0.31; p = .0052), and its effect was not confounded by salivary peroxidase concentration. In turn, the effect of salivary peroxidase concentration was confounded by salivary peroxidase activity, and it was smaller than that of activity. CONCLUSIONS: We found an inverse association between salivary peroxidase activity and probing depth. Thus, our results imply that salivary peroxidase activity could be a protective factor against periodontitis. However, large, well-designed studies are needed to explore the causal mechanisms of this association.

Role of poly(ADP-ribose) polymerase activation in the pathogenesis of periodontitis in diabetes.

AIM: The aetiology of progressive periodontitis in diabetes has not yet been elucidated. We previously demonstrated that nitrosative stress is increased in diabetic rats with periodontitis. Nitrosative stress induces poly(ADP-ribose) polymerase (PARP) activation. Here, we demonstrated the involvement of PARP activation in diabetic periodontitis and detailed the therapeutic effects of PARP inhibitor. MATERIALS AND METHODS: Experimental periodontitis was induced by placing a nylon thread ligature. Half of the normal and diabetic rats received the PARP inhibitor, 1,5-isoquinolinediol, for 2 weeks. Gingival PARP activation was detected by immunostaining for poly(ADP-ribose). Periodontitis was evaluated by gingival inflammatory cell infiltration, inflammatory gene expressions and micro-CT analyses. RESULTS: Although both periodontitis and the presence of diabetes increased PARP activation in the gingiva, diabetic rats with periodontitis had the highest activation of PARP. Diabetic rats with periodontitis also showed significant increases in monocyte/macrophage invasion into the gingiva, inflammatory gene expressions, nitrotyrosine-positive cells in the gingiva and alveolar bone loss, all of which were suppressed by treatment with the PARP inhibitor. CONCLUSIONS: These results indicate the involvement of PARP activation in the pathogenesis and aggravation of periodontal disease in diabetes and suggest the therapeutic potential of PARP inhibition for treating periodontal disease, especially in patients with diabetes.

Role of Cathepsin S in Periodontal Inflammation and Infection.

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1ß and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1ß and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

Evaluation of Metalloproteinase-8 Levels in Crevicular Fluid of Patients with Healthy Implants or Periodontitis.

Evaluation of periodontal and peri-implant tissue condition is mainly based on clinical examination and imaging diagnostics. Some data imply that Metalloproteinase-8 (MMP-8) level examination in peri-implant sulcular fluid (PISF) might be useful for evaluating the condition of peri-implant tissues and monitoring a development of peri-implant inflammation, including both mucositis and peri-implantitis. Hence, in this study, we decided to evaluate the level of MMP-8 in PISF obtained from patients without clinical symptoms of mucositis or peri-implantitis and compare it with MMP-8 level in gingival crevicular fluid (GCF) obtained from patients with healthy periodontium and those with varying severity of periodontitis. A total of 189 subjects were included in the study, and GCF/PISF samples were analysed for MMP-8 level by ELISA test. We documented that MMP-8 level in PISF obtained from patients without symptoms of mucositis or peri-implantitis was significantly higher not only than in GCF of periodontally healthy patients but also, which seems to be very interesting, than in GCF of patients with varying degrees of periodontal inflammation, consistent with earlier studies. Our observation might imply that monitoring of MMP-8 level in PISF could help to diagnose mucositis/peri-implantitis in an early stage, prior to clinical manifestations, which may allow for quick start of appropriate therapy.

A novel mechanism of latency in matrix metalloproteinases.

The matrix metalloproteinases (MMPs) are a family of secreted soluble or membrane-anchored multimodular peptidases regularly found in several paralogous copies in animals and plants, where they have multiple functions. The minimal consensus domain architecture comprises a signal peptide, a 60-90-residue globular prodomain with a conserved sequence motif including a cysteine engaged in "cysteine-switch" or "Velcro" mediated latency, and a catalytic domain. Karilysin, from the human periodontopathogen Tannerella forsythia, is the only bacterial MMP to have been characterized biochemically to date. It shares with eukaryotic forms the catalytic domain but none of the flanking domains. Instead of the consensus MMP prodomain, it features a 14-residue propeptide, the shortest reported for a metallopeptidase, which lacks cysteines. Here we determined the structure of a prokarilysin fragment encompassing the propeptide and the catalytic domain, and found that the former runs across the cleft in the opposite direction to a bound substrate and inhibits the latter through an "aspartate-switch" mechanism. This finding is reminiscent of latency maintenance in the otherwise unrelated astacin and fragilysin metallopeptidase families. In addition, in vivo and biochemical assays showed that the propeptide contributes to protein folding and stability. Our analysis of prokarilysin reveals a novel mechanism of latency and activation in MMPs. Finally, our findings support the view that the karilysin catalytic domain was co-opted by competent bacteria through horizontal gene transfer from a eukaryotic source, and later evolved in a specific bacterial environment.