Hypercitrullination and anti-citrullinated protein antibodies in chronic apical periodontitis, a laboratory investigation. Does autoimmunity contribute to the pathogenesis?

AIM: The presence of Gram-negative anaerobic bacteria, in particular, Porphyromonas gingivalis (P. gingivalis) in periapical granulomas predicts the generation of citrullinated proteins in the lesion. Citrullination of proteins may lead to the formation of anti-citrullinated autoantibodies (ACPA-s) initiating the formation of an autoimmune loop which may contribute to the perpetuation of inflammatory reactions and tissue damage in chronic apical periodontitis. The objective of this study was to demonstrate the formation of citrullinated proteins in chronic apical periodontitis and whether they can act as autoantigens. METHODOLOGY: Twenty-five periapical granulomas (n = 25) were investigated in the study. Healthy periodontal tissue samples served as normal control tissue (n = 6). The peptidyl-citrulline level was determined with the dot blot method. ACPA levels were analysed using anti-citrullinated cyclic peptide (anti-CCP) EDIA kit. Differences between periapical granuloma and control samples were assessed using Mann-Whitney U tests. p Values <.05 were considered as statistically significant. RESULTS: Protein concentrations, peptidyl-citrulline levels and anti-CCP ratios were compared between periapical granuloma and healthy control groups. Multiple linear regression analysis revealed significant (p = .042) hypercitrullination in periapical granuloma samples. Moreover, there was a significant difference in the ACPA ratios between periapical granuloma (2.03 ± 0.30) and healthy control (0.63 ± 0.17) groups (p = .01). Seventeen of 25 periapical granuloma samples (17/25; 68%), whereas one out of six control samples (1/6; 17%) were shown to be positive for the presence of ACPA. CONCLUSIONS: This is the first study detecting the presence of citrullinated peptides and APCA in periapical granuloma, suggesting the contribution of autoimmune reactions in the pathogenesis and perpetuation of chronic apical periodontitis.

Exosomal miR-205-5p derived from periodontal ligament stem cells attenuates the inflammation of chronic periodontitis via targeting XBP1.

INTRODUCTION: Chronic periodontitis (CP) is an inflammatory periodontal disease with high incidence and complex pathology. This research is aimed to investigate the function of exosomal miR-205-5p (Exo-miR-205-5p) in CP and the underlying molecular mechanisms. METHOD: Exo-miR-205-5p was isolated from miR-205-5p mimics-transfected periodontal ligament stem cells (PDLSCs), and subsequently cocultured with lipopolysaccharide (LPS)-induced cells or injected into LPS-treated rats. The mRNA expression of inflammatory factors and Th17/Treg-related factors were measured by quantitative real-time PCR. The contents of inflammatory factors and the percentages of Th17/Treg cells were measured by enzyme-linked immunosorbent assay and flow cytometry, respectively. Besides, the target relation between miR-205-5p and X-box binding protein 1 (XBP1) was explored. RESULTS: MiR-205-5p was downregulated in LPS-induced PDLSCs and corresponding exosomes. Exo-miR-205-5p inhibited inflammatory cell infiltration, decreased the production of TNF-α, IL-1ß, and IL-6, and decreased the percentage of Th17 cells in LPS-treated rats. In addition, XBP1 was a target of miR-205-5p. Overexpression of XBP1 weakened the effects of Exo-miR-205-5p on inhibiting inflammation and regulating Treg/Th17 balance in LPS-induced cells. CONCLUSIONS: Exo-miR-205-5p derived from PDLSCs relieves the inflammation and balances the Th17/Treg cells in CP through targeting XBP1.

Effectiveness of Platelet-Rich Fibrin with Decalcified Freeze-Dried Bone Allograft Compared to Decalcified Freeze-Dried Bone Allograft Alone in Mandibular Grade-II Furcation Defects: A Quasi-Experimental Study

ABSTRACT Objective: To assess the effectiveness of platelet-rich fibrin (PRF) with decalcified freeze-dried bone allograft (DFDBA) compared to DFDBA alone in mandibular grade-II furcation defects. Material and Methods: A quasi-experimental study was conducted on nine patients with chronic periodontitis, each having two almost identical mandibular grade II furcation defects. Test sites (left mandibular first molars) were treated with open flap debridement (OFD), DFDBA, and PRF, whereas control sites (right mandibular first molars) received OFD and DFDBA alone. Clinical parameters (plaque index (PI), gingival index (GI), vertical clinical attachment level (VCAL) and horizontal clinical attachment level (HCAL) into the furcation defect) and radiographic measurements (mean alveolar bone defect) were done at baseline and after six months postoperatively. Results: The gain in relative horizontal clinical attachment level (RHCAL) in the test sites was 2.94±0.52 mm compared to 1.33±0.35 mm in control sites (p=0.01). Improvement in mean alveolar bone defect (MABD) (was 1.21±0.5 mm2 at test sites compared to 1.15±0.7 mm2 at control sites) probing pocket depth (PPD), recession, relative vertical attachment level (RVCAL), and percentage of bone fill was found in the test sites compared to control, which statistically insignificant. Conclusion: The test sites had better outcomes than control sites, which was significant for the parameter RHCAL. Therefore, combining the biological benefits of autologous PRF with DFDBA is an efficient and economical treatment modality for the management of mandibular grade II furcation defects.

Comparison of Oncostatin M in Patients with Chronic Periodontitis with and without Diabetes

Abstract Objective: To compare the Oncostatin M (OSM) concentrations in tissues of patients with chronic periodontitis with and without diabetes. Material and Methods: Sixty-four subjects visiting the dental outpatient department were categorized as "healthy" (Group 1), "periodontitis" (Group 2), and "diabetes with periodontitis" (Group 3) groups. The clinical oral examination included assessment of plaque, gingivitis, probing depth, clinical attachment level. Blood glucose was assessed for group 3 patients. OSM concentration in the tissues was assessed using ELISA in all groups. Results: The mean OSM was 0.02 ± 0.04 pg/mg in the healthy group, 0.12 ± 0.09 pg/mg in the chronic periodontitis group and 0.13 ± 0.10 pg/mg in the diabetes-periodontitis group. A significantly higher mean OSM was seen in Group 2 and Group 3 than Group 1. The amount of OSM positively correlated with probing depth and clinical attachment level. Conclusion: Periodontal disease causes a rise in Oncostatin M, independent of the diabetic status. Expression of OSM in the gingival tissues can serve as an inflammatory marker.

lncRNA FGD5-AS1 and miR-130a Can Be Used for Prognosis Analysis of Patients with Chronic Periodontitis.

BACKGROUND: lncRNA and microRNA affect the occurrence and development of many diseases, so they are expected to become diagnostic or predictive indicators. But the relationship between lncRNA FGD5-AS1 and miR-130a and the prognosis of chronic periodontitis is still unclear. The purpose of this study is to explore the prognostic value of the two in chronic periodontitis. OBJECTIVE: This study set out to investigate the prognostic value of lncRNA FGD5-AS1 and miR-130a in chronic periodontitis. METHODS: Eighty-seven patients with chronic periodontitis who visited our hospital from March 2016 to August 2017 were collected as an observation group (OG), and 72 subjects with periodontal health who underwent physical examination at the same time were collected as a control group (CG). The FGD5-AS1 and miR-130a expression levels of subjects in the two groups were compared, and prognosis of 87 patients who were reviewed one year later was counted. The expression levels of patients with different prognoses were compared when they were admitted to our hospital. We drew the ROC curve and explored the prognostic value of FGD5-AS1 and miR-130a. The risk factors for adverse prognosis were analyzed through logistic regression. RESULTS: FGD5-AS1 was lowly expressed in patients, while miR-130a was highly expressed. FGD5-AS1 and miR-130a had certain diagnostic and predictive value in chronic periodontitis and patient prognosis. The higher the periodontal pocket, the higher the attachment loss. Lower FGD5-AS1 and higher miR-130a levels were independent prognostic risk factors. CONCLUSION: lncRNA FGD5-AS1 is lowly expressed in patients with chronic periodontitis, while miR-130a is highly expressed. Both of them have certain diagnostic and prognostic value in chronic periodontitis and may be potential diagnostic and prognostic indicators.

Elevated salivary activity of mast cell chymase of periodontitis patients, and a new bradykinin generation cascade, mediating the cross-talks between mast cell and gingival fibroblast.

Activated-mast cells (MCs) within gingival-tissue of chronic-periodontitis (CP) patients, release various inflammatory-factors. Bradykinin is a nine-amino-acid peptide and pro-inflammatory mediator, produced through factor-XII-cascade or tryptase-cascade. The ability of MC-chymase in bradykinin generation has not been discussed yet. This study investigated the salivary levels of MC-chymase, high molecular weight kininogen (HMWK) and bradykinin of CP patients; examined the potential of MC-proteases in bradykinin production using biochemistry-models; and explored the effects of bradykinin on gingival fibroblasts (GFs). Saliva-samples were collected; MC-protease activities were detected; HMWK cleavage was assessed by western-blot and SDS-PAGE; bradykinin levels were measured using immunoassay. Primary GFs were extracted and cultured with or without bradykinin; cell-viability, gelatine-zymography and flow-cytometry were applied. Immunocytochemistry and western-blot were used to detect intracellular protein expressions of bradykinin-stimulated GFs. The data showed that the salivary-levels of MC-proteases, bradykinin, HMWK, and lactoferrin of CP-patients were increased. HMWK was cleaved by MC-chymase in-vitro, resulting in bradykinin generation. Bradykinin promoted cell proliferation, cell cycle and matrix-metalloproteinase-2(MMP-2) activity, and increased intracellular expressions of nuclear-factor-kappa-B(NF-κB), focal-adhesion-kinase(FAK), transforming-growth-factor-ß(TGF-ß), P38, P53 of GFs. MC-chymase promotes bradykinin production to stimulate GFs and to continue inflammation during CP development. A new BK-generation cascade found in this study provides a new basis for the pathogenesis of CP and the mechanism of continuous inflammation. The activation of MC-chymase/bradykinin-generation cascade depends on HMWK level and MC-chymase activity under inflammatory condition. MC-chymase contributes to bradykinin production, mediating the cross-talks between MCs and GFs. MC-chymase can be used as a therapeutic target and a salivary biomarker in this case.

Leptin regulates OPG and RANKL expression in Gingival Fibroblasts and Tissues of Chronic Periodontitis Patients.

Objective: Chronic periodontitis is a bone-destructive disease affecting periodontal support structures. Although leptin has a protective effect against periodontitis, the underlying mechanism remains unclear. Therefore, this study aimed to investigate the possible role of leptin by examining its relationship with OPG and RANKL in human gingival tissues obtained from patients with chronic periodontitis. Method: Twenty-two patients with chronic periodontitis were enrolled (10 with moderate periodontitis and 12 with severe periodontitis) in the experimental group, and 12 healthy individuals were enrolled in the control group. Gingival tissue samples were collected, and the protein levels and localization of leptin, OPG, and RANKL were studied using immunohistochemistry (IHC). The staining intensities of leptin, OPG, and RANKL were correlated with the periodontal clinical index. Moreover, real-time quantitative PCR (RT-qPCR) was used to determine OPG and RANKL mRNA levels in gingival fibroblasts stimulated with gradient concentrations of leptin protein in vitro. Result: Leptin, OPG, and RANKL were located in the cytoplasm of gingival epithelial cells and the connective tissue. Leptin was widely and significantly expressed in the control group, whereas it was lightly stained in the severe group. RANKL was lightly stained in the control group, whereas it was widely and significantly expressed in the severe group. The control and the moderate groups had similar OPG levels, which were significantly higher than that in the severe group. Leptin was positively correlated with OPG(r = 0.905, p < 0.01) and negatively correlated with RANKL (r = -0.635, p < 0.01). In vitro low concentrations of leptin led to an increased OPG/RANKL mRNA ratio, whereas the opposite effect was observed at high concentrations. Conclusion: Leptin can regulate OPG and RANKL expression in gingival fibroblasts and may thus play a role in the development of chronic periodontitis by modulating the OPG/RANKL ratio.

Effect of Psoralen on the Intestinal Barrier and Alveolar Bone Loss in Rats With Chronic Periodontitis.

To study the effects of psoralen on the intestinal barrier and alveolar bone loss (ABL) in rats with chronic periodontitis. Fifty-two 8-week-old specific pathogen-free (SPF) male Sprague-Dawley (SD) rats were randomly divided into the following four groups: Control group (Control), psoralen group of healthy rats (Pso), periodontitis model group (Model), and psoralen group of periodontitis rats (Peri+Pso). The alveolar bone resorption of maxillary molars was observed via haematoxylin-eosin staining and micro-computed tomography. The expression level of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in periodontal tissues was evaluated by immunofluorescence staining. The changes in serum tumour necrosis factor (TNF)-α, interleukin (IL)-10, IL-6, intestinal mucosal occludin, and claudin-5 were detected using enzyme-linked immunosorbent assay (ELISA). The level of intestinal mucosal NOD2 was detected using immunohistochemical methods. DNA was extracted from the intestinal contents and the 16s rRNA gene was sequenced using an Illumina MiSeq platform. The expression of NOD2 protein in the intestinal tract of periodontitis rats decreased after intragastric psoralen administration. Psoralen increased the intestinal microbiota diversity of rats. The level of serum pro-inflammatory factor TNF-α decreased and the level of anti-inflammatory factor IL-10 increased. ABL was observed to be significantly decreased in rats treated with psoralen. Psoralen decreased the RANKL/OPG ratio of periodontitis rats. Psoralen may affect the intestinal immune barrier and ecological barrier, mediate immune response, promote the secretion of anti-inflammatory factor IL-10, and reduce the secretion of the pro-inflammatory factor TNF-α, thus reducing ABL in experimental periodontitis in rats.

Subgingival microbiome dynamic alteration associated with necrotizing periodontal disease: A case report.

RATIONALE: Necrotizing periodontal diseases (NPDs) are a group of infectious diseases varying in severity, and microorganisms are responsible for these diseases. Currently, the oral microbiota in early disease has been poorly investigated; thus, the causative pathogen and dynamic alteration of the microbiome in NPDs remain unclear. PATIENT CONCERNS: We report a case of a 33-year-old female patient with severe gingival pain and localized necrotizing ulcerative gingival lesions. Conventional therapy was performed, but the necrotizing lesion continued to develop. DIAGNOSES: X-ray examination showed marginal alveolar bone loss in the involved teeth. Histological examination of a biopsy from the gingival lesion showed chronic inflammatory cell infiltration in the tissue, and no cancer cells were observed. Subgingival swabs were taken from the ulcerative gingiva and the gingiva that was not yet affected, and the composition of the microbiota was analyzed by targeted pyrosequencing of the V3-V4 hypervariable regions of the small subunit ribosomal RNA. We found that Neisseria spp., Corynebacterium spp., and Prevotella spp. were clearly enriched in the lesion site. However, Fusobacteria was more abundant in the not-yet-affected gingiva, and Leptotrichia spp. were the most abundant phylotype. INTERVENTIONS: After clinical assessment, a tooth with poor prognosis was extracted, and minocycline hydrochloride was locally administered in the involved tooth pocket every day. Additionally, the patient received 100 mg of hydrochloric acid doxycycline twice per day. OUTCOMES: Remarkable improvement was obtained after 3 days, and the lesion completely healed after 1 week. The follow-up examination 1 year later showed a complete recovery with no recurrent episodes of pain. LESSONS: Changes in the subgingival microbiome can occurr before clinical symptoms appears, and Fusobacteria may be involved in the imbalance of the subgingival flora in the early stage of NPDs. Moreover, Neisseria is a potential bacterial candidate that deserves further study.

Knockdown of MALAT1 Inhibits the Progression of Chronic Periodontitis via Targeting miR-769-5p/HIF3A Axis.

PURPOSE: Chronic periodontitis (CP) is a long-lasting inflammatory disease that seriously affects oral health. This study is aimed at investigating the regulatory mechanism of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in CP. METHODS: Primary human periodontal ligament cells (PDLCs) were treated with P. gingivalis lipopolysaccharide (LPS) to establish a CP model. Quantitative real-time PCR (qRT-PCR) was used to measure the expression of MALAT1 and miR-769-5p in gingival tissues of patients with CP and LPS-treated PDLCs. Cell viability was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory cytokines. The protein levels of caspase-3, Bax, Bcl-2, and hypoxia-inducible factor (HIF) 3A were determined by western blot assay. Dual-luciferase reporter (DLR) assay was applied to validate the target relationships between miR-769-5p and MALAT1/HIF3A. RESULTS: The expression of MALAT1 and HIF3A was enhanced, and the expression of miR-769-5p was reduced in gingival tissues of patients with CP and LPS-treated PDLCs. MALAT1 knockdown promoted cell viability and inhibited inflammation and cell apoptosis in LPS-treated PDLCs. MALAT1 targeted miR-769-5p and negatively regulated miR-769-5p expression. miR-769-5p overexpression promoted cell viability and inhibited inflammation and cell apoptosis in LPS-treated PDLCs. Besides, miR-769-5p targeted HIF3A and negatively modulated HIF3A expression. Both miR-769-5p inhibition and HIF3A overexpression reversed the inhibitory effects of MALAT1 silencing on LPS-induced PDLC injury in vitro. CONCLUSION: MALAT1 knockdown attenuated LPS-induced PDLC injury via regulating the miR-769-5p/HIF3A axis, which may supply a new target for CP treatment.

MMP-8, TRAP-5, and OPG Levels in GCF Diagnostic Potential to Discriminate between Healthy Patients', Mild and Severe Periodontitis Sites.

Biomarkers represent promising aids in periodontitis, host-mediate diseases of the tooth-supporting tissues. We assessed the diagnostic potential of matrix metalloproteinase-8 (MMP-8), tartrate-resistant acid phosphatase-5 (TRAP-5), and osteoprotegerin (OPG) to discriminate between healthy patients', mild and severe periodontitis sites. Thirty-one otherwise healthy volunteers with and without periodontal disease were enrolled at the Faculty of Dentistry, University of Chile. Periodontal parameters were examined and gingival crevicular fluid was sampled from mild periodontitis sites (M; n = 42), severe periodontitis sites (S; n = 59), and healthy volunteer sites (H; n = 30). TRAP-5 and OPG were determined by commercial multiplex assay and MMP-8 by the immunofluorometric (IFMA) method. STATA software was used. All biomarkers showed a good discrimination performance. MMP-8 had the overall best performance in regression models and Receiver Operating Characteristic (ROC) curves, with high discrimination of healthy from periodontitis sites (area under the curve (AUC) = 0.901). OPG showed a very high diagnostic precision (AUC ≥ 0.95) to identify severe periodontitis sites (S versus H + M), while TRAP-5 identified both healthy and severe sites. As conclusions, MMP-8, TRAP-5, and OPG present a high precision potential in the identification of periodontal disease destruction, with MMP-8 as the most accurate diagnostic biomarker.

Hematogenous apoptotic mechanism in salivary glands in chronic periodontitis.

OBJECTIVE: The aim of the study is to investigate the apoptotic mechanism in salivary glands in the rat experimental periodontitis model. DESIGN: A rat periodontitis model was prepared by using a ligature around the second upper molar. In the salivary (parotid and submandibular) glands and blood samples, putative apoptotic factors and pathway molecules were investigated in vivo and in vitro. RESULTS: Four weeks of ligation (chronic periodontitis) demonstrated significant apoptotic atrophy of the salivary gland, but one week of ligation (initial periodontitis) did not. In the blood plasma, tumor necrosis factor-α (TNF-α) was increased in the periodontitis model, but interleukin-1ß and -6 were not. TNF-α receptor type 1, which has an intracellular apoptotic pathway, was expressed in the salivary glands of rats. Western blot analysis of cultured rat primary salivary gland cells demonstrated that TNF-α induced cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 in a dose-dependent manner, indicating apoptosis induction. Additionally, we found increment of circulating lymphocytes in the model. Expression of mRNA and immunoreactive cells for the B lymphocyte marker CD19 were increased in the salivary gland in the model. Western blotting showed that coculture with extracted B cells from the periodontitis model increased cleaved PARP in salivary gland cells. CONCLUSIONS: Chronic periodontitis status leads to an increase in circulating TNF-α and B lymphocyte infiltration, resulting in apoptotic atrophy of the salivary gland as a periodontitis-induced systemic response.

Evaluation of PRF Efficiency in the Treatment of Infrabony Defects.

AIM: The present study aimed to investigate the effectiveness of PRF in the treatment of infrabony defects in patients with chronic periodontitis by evaluating the clinical outcome through periodontal depth, clinical attachment level at the baseline, 6 and 9 months post operatively. MATERIAL AND METHODS: Sixty infrabony defects with probing depth ≥ 5 mm were treated. The inclusion criterion was the necessity for surgical bilateral maxillary treatment. By using split-mouth study design, each patient had one side treated with conventional flap surgery and the other side with conventional flap surgery and PRF. Clinical parameters, such as probing depth (PD) and clinical attachment lost (CAL), were recorded in both groups at baseline, 6 and 9 months post operatively. RESULTS: Positive effects for all clinical and radiographic parameters were evident in the group with PRF. Mean PD reduction demonstrated statistically significant greater results in the test group (4.00±1.07 mm) compared to the control one (4.83±0.99 mm), p = 0.003 after 9 months postoperatively. After 9 months, there were better results in the test group compared to the control group for CAL (5.60±1.61 mm, 6.20±1.58 mm), but statistically not significant. CONCLUSION: Additional use of PRF in the conventional surgical treatment of infrabony defects demonstrated better parameters than the open flap debridement alone.

Emerging role of long non-coding RNAs in the pathogenesis of periodontitis.

Periodontitis is a bacteria-related chronic immune-associated condition that destructs bone and connective tissues around teeth. With a high incidence rate, it is regarded as a condition that impose substantial health burden. About half of the variance in the severity of periodontitis is attributed to genetic factors. Long non-coding RNAs (lncRNAs) have crucial roles in the development of several disorders such as periodontitis. A number of studies have reported dysregulation of lncRNAs such as UCA1, ANRIL, FGD5-AS1, NEAT1, FAS-AS1, Linc-RAM and NKILA in gingival tissues or blood samples of patients with periodontitis in comparison with healthy subjects. Moreover, several single nucleotide polymorphisms within lncRNAs have been associated with the susceptibility to this disorder. In the current review, we discuss the most recent articles about the role of lncRNAs in the pathogenesis of periodontitis.

Effect of VEGFC on lymph flow and inflammation-induced alveolar bone loss.

The lymphatic system plays a crucial role in the maintenance of tissue fluid homeostasis and the immunological response to inflammation. The effects of lymphatic drainage dysfunction on periodontitis have not been well studied. Here we show that lymphatic vessel endothelial receptor 1 (LYVE1)+ /podoplanin (PDPN)+ lymphatic vessels (LVs) are increased in the periodontal tissues, with accumulation close to the alveolar bone surface, in two murine periodontitis models: rheumatoid arthritis (RA)-associated periodontitis and ligature-induced periodontitis. Further, PDPN+ /alpha-smooth muscle actin (αSMA)- lymphatic capillaries are increased, whereas PDPN+ /αSMA+ collecting LVs are decreased significantly in the inflamed periodontal tissues. Both mouse models of periodontitis have delayed lymph flow in periodontal tissues, increased TRAP-positive osteoclasts, and significant alveolar bone loss. Importantly, the local administration of adeno-associated virus for vascular endothelial growth factor C, the major growth factor that promotes lymphangiogenesis, increases the area and number of PDPN+ /αSMA+ collecting LVs, promotes local lymphatic drainage, and reduces alveolar bone loss in both models of periodontitis. Lastly, LYVE1+ /αSMA- lymphatic capillaries are increased, whereas LYVE1+ /αSMA+ collecting LVs are decreased significantly in gingival tissues of patients with chronic periodontitis compared with those of clinically healthy controls. Thus, our findings reveal an important role of local lymphatic drainage in periodontal inflammation-mediated alveolar bone loss. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Systemic Th17 response in the presence of periodontal inflammation.

BACKGROUND: The relationship between periodontitis and the pathogenesis of other inflammatory diseases, such as diabetes, rheumatoid arthritis and obesity has been an important topic of study in recent decades. The Th17 pathway plays a significant role in how local inflammation can influence systemic inflammation in the absence of systemic pathology. OBJECTIVE: To determine Th17 biased-cells in systemically healthy patients in the presence of generalized chronic periodontitis. METHODOLOGY: A total of 28 patients were recruited without systemic inflammatory pathology, which was determined by clinical history, the Health Assessment Questionnaire (HAQ) and rheumatoid factor detection. Of these patients, 13 were diagnosed as healthy/gingivitis (H/G) and 15 as generalized chronic periodontitis (GCP). Th17 (CD4+CD161+) cells and Th17IL23R+ (CD4+CD161+IL-23R+) cells were quantified by flow cytometry, based on the total cells and on the lymphocyte region, termed the "enriched population" (50,000 events for each). RESULTS: The percentages of Th17 cells of the H/G and periodontitis groups were similar on total cells and enriched population (19 vs 21.8; p=4.134 and 19.6 vs 21.8; p=0.55). However, Th17IL23R+ cells differ significantly between periodontally healthy patients and generalized chronic periodontitis patients in both total cell (0.22% vs 0.65%; p=0.0004) and enriched populations (0.2% vs 0.75%; p=0.0266). CONCLUSIONS: GCP patients (otherwise systemically healthy) were characterized by increased Th17-proinflammatory cell phenotype positive for the IL-23 receptor in peripheral blood. The proportion of Th17 cells that are negative for the IL-23 receptor in the peripheral blood of systemically healthy patients seemed to be unaffected by the presence or absence of chronic periodontitis.

Integrin-α9 and Its Corresponding Ligands Play Regulatory Roles in Chronic Periodontitis.

Integrin-α9 (ITGA9) and its corresponding ligands are involved in inflammatory and immune responses. The present study aimed to investigate whether ITGA9 participates in the development of chronic periodontitis (ChP) and to explore the underlying mechanisms. We collected gingival tissue and gingival crevicular fluid in vivo from patients to determine the levels of ITGA9 and its ligands. We cultured primary periodontal ligament cells (PDLCs) in vitro and applied small interfering RNA to knock down ITGA9 in order to analyze the changes of inflammatory cytokines and explore the related cellular signaling pathways. The expression level of ITGA9 was significantly higher in the gingiva of patients with ChP than that of healthy individuals. ITGA9 knockdown in the PDLCs inhibited the secretion of interleukin (IL)-1ß, IL-6, and IL-8. Western blot analysis indicated that this change could be attributed to the regulation of the mitogen-activated protein kinase (MAPK) signaling pathway. ITGA9 plays a regulatory role in the homeostasis of ChP. The results of the present study provide potential insights into the treatment of periodontitis. Graphical abstract.

Butyric Acid in Saliva of Chronic Periodontitis Patients Induces Transcription of the EBV Lytic Switch Activator BZLF1: A Pilot Study.

BACKGROUND/AIM: Epstein-Barr virus (EBV) associates with human chronic periodontitis (CP) progression. We previously demonstrated that butyric acid (BA), produced by periodontopathic bacteria, induced EBV lytic switch activator BZLF1 expression. We investigated whether short chain fatty acids (SCFAs) in CP patients' saliva enabled EBV reactivation. MATERIALS AND METHODS: Saliva was collected from seven CP patients and five periodontally healthy individuals. SCFAs were quantified using HPLC. BZLF1 mRNA and its pertinent protein ZEBRA were determined with Real-time PCR and western blotting. Histone H3 acetylation (AcH3) was further examined. RESULTS: BZLF1 mRNA expression and transcriptional activity in EBV-infected Daudi cells were induced only when treated with the CP saliva. Among SCFAs, BA alone correlated significantly with the BZLF1 transcription (r=0.88; p<0.02). As expected, CP patients' saliva induced AcH3. CONCLUSION: BA in saliva may play a role in EBV reactivation and hence contribute to EBV-related disease progression in CP patients.

Exploring the putative interactions between chronic kidney disease and chronic periodontitis.

Chronic kidney disease (CKD) and chronic periodontitis (CP) are both common diseases, which are found disproportionately comorbid with each other and have been reported to have a detrimental effect on the progression of each respective disease. They have an overlap in risk factors and both are a source of systemic inflammation along with a wide selection of immunological and non-specific effects that can affect the body over the lifespan of the conditions. Previous studies have investigated the directionality of the relationship between these two diseases; however, there is a lack of literature that has examined how these diseases may be interacting at the localized and systemic level. This review discusses how oral microorganisms have the ability to translocate and have distal effects and provides evidence for microbial involvement in a systemic disease. Furthermore, it summarizes the reported local and systemic effects of CKD and CP and discusses how the interaction of these effects may be responsible for directionality associations reported.

Reduction of inflammation in a chronic periodontitis model in rats by TNF-α gene silencing with a topically applied siRNA-loaded calcium phosphate paste.

We developed a calcium phosphate-based paste containing siRNA against TNF-α and investigated its anti-inflammatory and bone-healing effects in vitro and in vivo in a rat periodontitis model. The bioactive spherical CaP/PEI/siRNA/SiO2 nanoparticles had a core diameter of 40-90 nm and a positive charge (+23 mV) that facilitated cellular uptake. The TNF- α gene silencing efficiency of the nanoparticles in J774.2 monocytes, gingival-derived cells, and bone marrow-derived cells was 12 ± 2%, 36 ± 8%, and 35 ± 22%, respectively. CaP/PEI/siRNA/SiO2 nanoparticles cancelled the suppression of alkaline phosphatase (ALP) activity in LPS-stimulated bone marrow-derived cells. In vivo, ALP mRNA was up-regulated, TNF-α mRNA was down-regulated, and the amount of released TNF-α was significantly reduced after topical application of the calcium phosphate-based paste containing siRNA-loaded nanoparticles. The number of TNF-α-positive cells in response to CaP/PEI/siRNA/SiO2 nanoparticle application was lower than that observed in the absence of siRNA. Elevated ALP activity and numerous TRAP-positive cells (osteoclasts) were observed in response to the application of all calcium phosphate pastes. These results demonstrate that local application of a paste consisting of siRNA-loaded calcium phosphate nanoparticles successfully induces TNF-α silencing in vitro and in vivo and removes the suppression of ALP activity stimulated by inflammation. STATEMENT OF SIGNIFICANCE: We developed a calcium phosphate-based paste containing nanoparticles loaded with siRNA against TNF-α. The nanoparticles had a core diameter of 40-90 nm and positive charge (+23 mV). The anti-inflammatory and osteoinductive effects of the paste were investigated in vitro and in vivo in a rat periodontitis model. In vitro, the TNF-α gene silencing efficiency of the nanoparticles in J774.2 monocytes, gingival-derived cells, and bone marrow-derived cells was 12 ± 2%, 36 ± 8%, and 35 ± 22%, respectively. The ALP activity of bone marrow-derived cells was recovered. In vivo, TNF-α mRNA was down-regulated and the amount of released TNF-α was significantly reduced, whereas the ALP mRNA was up-regulated. Elevated ALP activity and TRAP-positive cells were observed by immunohistochemistry.