Expression of human caspase-4 in the gingival epithelium affected with periodontitis: Its involvement in Porphyromonas gingivalis-challenged gingival epithelial cells.

OBJECTIVE: Implication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs. DESIGN: Ex vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human ß-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR. RESULTS: Ex vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum. CONCLUSIONS: Caspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis.

The miR-223-3p Regulates Pyroptosis Through NLRP3-Caspase 1-GSDMD Signal Axis in Periodontitis.

Salivary exosomes contain various components and may play important roles in oral diseases. The purpose of this study was to verify the possible function of miR-223-3p from salivary exosomes in periodontitis. We isolated the salivary exosomes and found that the miR-223-3p content of salivary exosomes from periodontitis was less than the healthy control. Furthermore, we performed dual-luciferase reporter assay and real-time PCR to verify that (NOD)-like receptor (NLR) pyrin domain-containing 3 (NLRP3) was the target of miR-223-3p. When we knocked down the miR-223-3p expression in THP-1-derived macrophages, the expression of NLRP3 and the downstream inflammatory mediators interleukin-1ß (IL-1ß) and IL-6 were upregulated. By using integrated bioinformatics analysis, we found that pyroptosis and cytokine secretion participated in inflammatory gingival tissues. In addition, NLRP3, and the pyroptosis executioner, gasdermin D (GSDMD) was highly active in inflammatory gingival tissues compared with healthy controls by western blotting and immunohistochemistry. In summary, we speculated that miR-223-3p in salivary exosomes might regulate GSDMD-mediated pyroptosis by targeting NLRP3 in periodontitis. Detection of miR-223-3p expression in salivary exosomes could be used as an important non-invasive method to diagnose and evaluate the severity of periodontitis.

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.

Periodontitis and peri-implantitis tissue levels of Treponema denticola-CTLP and its MMP-8 activating ability.

BACKGROUND AND AIMS: Chymotrypsin-like-proteinase of Treponema denticola (Td-CTLP) can stimulate the protein expression and activation of matrix metalloproteinase (MMP)-8 (or collagenase-2), a potent tissue destructive enzyme from gingival cells in vitro. The aims of this study were 1) to demonstrate the proMMP-8 (or latent MMP-8) activation by Td-CTLP in vitro and 2) to detect Td-CTLP and MMP-8 protein levels in the tissue samples of peri-implantitis and periodontitis patients. MATERIALS AND METHODS: proMMP-8 activation by Td-CTLP was analyzed by immunoblots. Tissue specimens were collected from 38 systemically healthy and non-smoking patients; 14 of whom had moderate to severe periodontitis, 10 of whom were suffering from peri-implantitis, and finally 14 of whom showed no sign of periodontal inflammation nor radiological bone decay (control group). The immune-expression levels of MMP-8 and Td-CTLP in the epithelium and the connective tissue were analyzed immunohistochemically. A pixel color-intensity analyze was performed with ImageJ software (version 1.46c; Rasband WS, National Institutes of Health, Bethesda, MD, USA) to obtain a comparable numeral score for each patient's epithelium and connective tissue MMP-8 and Td-CTLP enzyme level. RESULTS: Td-CTLP activated proMMP-8 in vitro by converting the 70-75 kDa proMMP-8 to 65 kDa active MMP-8. Also, lower molecular size 25-50 kDa parts of MMP-8 were formed. There was no statistically significant difference between the study groups in terms of their MMP-8 and Td-CTLP levels in the epithelium or in the connective tissue. CONCLUSION: Regarding the limits of this study, it can thus be said that the Td-CTLP enzyme can activate the host proMMP-8 enzyme. Tissue protein levels of MMP-8 and Td-CTLP do not seem to be changed in peri-implantitis and in periodontitis.

Parathyroid hormone promotes the osteogenesis of lipopolysaccharide-induced human bone marrow mesenchymal stem cells through the JNK MAPK pathway.

Periodontitis is a series of inflammatory processes caused by bacterial infection. Parathyroid hormone (PTH) plays a critical role in bone remodeling. The present study aimed to investigate the influences of PTH on human bone marrow mesenchymal stem cells (HBMSCs) pretreated with lipopolysaccharide (LPS). The proliferative ability was measured using cell counting kit-8 (CCK-8) and flow cytometry. The optimal concentrations of PTH and LPS were determined using alkaline phosphatase (ALP) activity assay, ALP staining, and Alizarin Red staining. Osteogenic differentiation was further assessed by quantitative reverse-transcription polymerase chain reaction (RT-qPCR), Western blot analysis, and immunofluorescence staining. PTH had no effects on the proliferation of HBMSCs. Also, 100 ng/ml LPS significantly inhibited HBMSC osteogenesis, while 10-9 mol/l PTH was considered as the optimal concentration to reverse the adverse effects. Mechanistically, c-Jun N-terminal kinase (JNK) phosphorylation was activated by PTH in LPS-induced HBMSCs. SP600125, a selective inhibitor targeting JNK mitogen-activated protein kinase (MAPK) signaling, weakened the effects of PTH. Taken together, the findings revealed the role and mechanism of PTH and JNK pathway in promoting the osteogenic differentiation of LPS-induced HBMSCs, which offered an alternative for treating periodontal diseases.

The virulence factor GroEL promotes gelatinase secretion from cells in the osteoblast lineage: Implication for direct crosstalk between bacteria and adult cells.

OBJECTIVE: The aim of this study was to demonstrate the influence of the virulence factor GroEL on osteoblast behavior by characterizing the changes of secreted gelatinases. DESIGN: ELISA was performed to detect GroEL from samples from patients with or without apical periodontitis. An apical periodontitis model was established in rats and the expression of MMP-2, MMP-9 and NF-κB was evaluated by immunofluorescence staining. The primary osteoblasts and osteoblast-like MC3T3 cells were stimulated with recombinant GroEL, and gelatin zymography was used to determine the activity and expression of MMP-2 and MMP-9. Western blot was used to screen signaling pathways, and immunofluorescence staining was performed to confirm the activated signaling. RESULTS: First, we found expression of GroEL to be higher in oral saliva, gingival crevicular fluid and periradicular granulation tissue of patients with apical periodontitis than it was in healthy control patients. We next found that recombinant GroEL could increase the activity of the gelatinases, MMP-2 and MMP-9, which were secreted by both primary osteoblasts and MC3T3 cells. In a rat apical periodontitis model, strong expression of gelatinases was confirmed. Then, we found that GroEL-enhanced gelatinase activity was mediated through activation of NF-κB signaling. Acetylated NF-κB accumulated in the cell nucleus and bound to the promoter of MMP-2 and MMP-9 genes, thus initiating their high expression. CONCLUSION: This study reveals a direct interaction between oral bacteria and adult cells by demonstrating that gelatinase secretion is induced by GroEL, which partially explains bone resorption through gelatinase activation.

Suppressing ROS generation by apocynin inhibited cyclic stretch-induced inflammatory reaction in HPDLCs via a caspase-1 dependent pathway.

It has been reported that cyclic stretch could induce inflammatory reaction in human periodontal ligament cells (HPDLCs). Though reactive oxygen species (ROS) has been reported to be involved in pathogen-induced periodontal inflammatory reaction, its role in the force-related periodontal diseases has not been well clarified. This study inspected the role of ROS in the cyclic stretch-induced inflammatory reaction in HPDLCs and studied the inhibitory effect of antioxidant apocynin on this inflammatory reaction. Results confirmed that cyclic stretch induced inflammatory reaction and production of ROS in HPDLCs. This inflammatory reaction was inhibited by apocynin through blocking the production of ROS. The cyclic stretch also induced the expression of caspase-1 and NLRP3 inflammasome, which could also be inhibited by apocynin. Moreover, the cyclic stretch-induced inflammatory reaction was inhibited by caspase-1 inhibitor. Collectively, it is the first time that increased intracellular ROS was proved to play as an intermediate signal in the cyclic stretch-induced inflammatory reaction in HPDLCs, via a caspase-1-dependent pathway. The inhibitory effect of apocynin on the cyclic stretch-induced inflammatory reaction in HPDLCs shows the potential of antioxidants in the treatment of force-related periodontal inflammatory diseases.

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.

[Protein arginine deiminase of oral microbiome plays a causal role in the polyarthritis rheumatoid initiating]. / Peptidylarginine désiminases du microbiote buccal et polyarthrite rhumatoïde.

In the last decade, the association between the periodontitis and rheumatoid arthritis (RA) has been established, suggesting that oral microbiome plays a causal role by initiating this chronic autoimmune inflammatory disease of articulation. Both pathogenesis are similar in term of chronic inflammation, tissue breakdown and bone resorption. Molecular aspects have also revealed that citrullination, a post-translational modification catalyzed by peptidyl-arginine deiminases (PADs), is involved in both diseases. For RA, citrullinated proteins production leads to the synthesis the of anti-citrullinated protein antibodies triggering the loss of immune tolerance. In humans, five PADs have been identified. Recently, studies have found that only Porphyromonas species possess PAD. Thus, a major periodontal pathogen, Porphyromonas gingivalis, is able to generate citrullinated epitopes, and could consequently induce anti-citrullinated protein antibodies. In this review, citrullination process, periodontitis and RA are described to put them in relation with molecular, clinical and epidemiological studies establishing the association between periodontitis and RA.

TITLE: Peptidylarginine désiminases du microbiote buccal et polyarthrite rhumatoïde. ABSTRACT: Ces dernières années, des études se sont focalisées sur l'existence d'une association entre la parodontite et la polyarthrite rhumatoïde (PR), suggérant l'implication du microbiote buccal dans le déclenchement de cette maladie auto-immune des articulations. D'un point de vue clinique, les deux pathologies reposent sur un processus inflammatoire qui conduit à une érosion osseuse. Elles font également intervenir une modification post-traductionnelle appelée citrullination. Dans le cas de la PR, la citrullination de certains sites protéiques par les peptidylarginine désiminases (PAD) aboutit à la production d'auto-anticorps. C'est la découverte d'une PAD exprimée par la bactérie Porphyromonas gingivalis qui a orienté de nombreuses études vers l'analyse d'une association entre ces deux pathologies.

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.

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.

Levels of Myeloperoxidase and Metalloproteinase-9 in Gingival Crevicular Fluid from Diabetic Subjects with and without Stage 2, Grade B Periodontitis.

OBJECTIVE: The present study aimed to compare levels of matrix metalloproteinase-9 (MMP-9) and myeloperoxidase (MPO) in gingival crevicular fluid (GCF) from subjects with controlled and noncontrolled Type 2 Diabetes Mellitus (T2D), with and without stage 2 grade B periodontitis (POD2B) versus healthy (H) subjects. METHODS: The levels of both enzymes, from 80 GCF samples collected with PerioPaper strips, were analyzed by a Multiplex/Luminex assay. Five groups were formed, all current patients at the Institutional Dentistry Service, and distributed as follows: two groups of diabetics (one controlled and one poorly controlled); two groups with the previous conditions and diagnosed with POD2B; and one H group. RESULTS: The highest concentration of MMP-9 corresponded to the H group, while the lowest corresponded to the T2D controlled group. Regarding MPO levels, the highest levels were associated with the T2D controlled with POD2B group and the lowest with the T2D controlled group. CONCLUSIONS: No apparent relationship between the elevation of MMP-9 and MPO levels was observed among subjects with T2D, with and without POD2B, compared to H subjects.

Associations of chairside salivary aMMP-8 findings with periodontal parameters, potentially periodontal pathogenic bacteria and selected blood parameters in systemically healthy adults.

The aim of this cross-sectional study was to investigate associations between salivary active matrix-metalloproteinase 8 (aMMP-8) and periodontitis severity, potentially periodontal pathogenic bacteria as well as blood parameters in generally healthy participants. Therefore, 188 participants with a mean age of 48.9 ±â€¯8 years were examined. The periodontitis severity was assessed based on periodontal probing depth and clinical attachment loss. Both, aMMP-8 and microbiological analysis were performed using a validated, commercially available test system. Blood values were utilized from regular differential blood count. The aMMP-8 findings were associated with the periodontitis severity (P < 0.01), as well as with the prevalence of Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Parvimonas micra, Camphylobacter rectus and Eubacterium nodatum (Pi < 0.05). No associations between aMMP-8 and the examined blood parameters were found (Pi > 0.05). In conclusion, salivary aMMP-8 findings seem to reflect periodontal disease severity as a result of an immunoreaction, especially against bacteria with high periodontal pathogenic potential.

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.

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.

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.

Effects of diode laser application on inflammation and mpo in periodontal tissues in a rat model.

OBJECTIVE: In this study, we aimed to histologically and immunologically evaluate the effect of diode laser treatment when applied adjunctive to scaling and root planing (SRP) in an experimental periodontitis model. MATERIALS AND METHODS: We used Wistar-Albino rats (n=60) with average weight of 230 g. Experimental periodontitis was induced by ligature at the right and left first mandibular molar teeth in all rats. After 11 days, the ligature was removed and rats were divided into two groups. The control group (n=30) received only SRP treatment, while the laser group (n=30) received a diode laser (GaAlAs, 810 nm, 1 W, 10 J, 20 s) treatment adjunctive to SRP. Ten rats in each group were sacrificed after 7, 15, and 30 days. Histopathological examination was performed in the left mandible of rats. Myeloperoxidase (MPO) was evaluated by western blot in the gingival specimens from the right mandible. RESULTS: MPO levels in the laser group were statistically significantly lower compared with the control group (p≤0.05). There was no statistically significance at any time between MPO levels in the control group (p>0.05). MPO levels in the laser group at the 7th day were statistically significantly higher compared to the 15th (p≤0.05) and the 30th day (p≤0.05). Inflammatory cell infiltration decreased over time in both groups and was statistically significantly lower in the laser group than in the control group at all times (p≤0.01). CONCLUSIONS: Within the limits of this study, we suggest that diode laser application is an adjunctive treatment because it reduced inflammation and MPO when applied in addition to SRP. On the other hand, more studies are needed for the assessment of the effects of diode laser application to periodontal tissues.

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.

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.