Pathogenesis Of Chronic Periodontitis Research Articles

The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function.

This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

Novel transcriptional regulator OxtR1 regulates potential ferrodoxin in response to oxygen stress in Treponema denticola

ObjectiveTreponema denticola has been strongly implicated in the pathogenesis of chronic periodontitis. Previously, we reported that the potential transcriptional regulator TDE_0259 (oxtR1) is upregulated in the bacteriocin ABC transporter gene-deficient mutant. OxtR1 may regulate genes to adapt to environmental conditions during colonization; however, the exact role of the gene in T. denticola has not been reported. Therefore, we investigated its function using an oxtR1-deficient mutant. MethodsThe growth rates of the wild-type and oxtR1 mutant were monitored under anaerobic conditions; their antibacterial agent susceptibility and gene expression were assessed using a liquid dilution assay and DNA microarray, respectively. An electrophoretic mobility shift assay was performed to investigate the binding of OxtR1 to promoter regions. ResultsThe growth rate of the bacterium was accelerated by the inactivation of oxtR1, and the mutant exhibited an increased minimum inhibitory concentration against ofloxacin. We observed a relative increase in the expression of genes associated with potential ferrodoxin (TDE_0260), flavodoxin, ABC transporters, heat-shock proteins, DNA helicase, iron compounds, and lipoproteins in the mutant. OxtR1 expression increased upon oxygen exposure, and oxtR1 complementation suppressed the expression of potential ferrodoxin. Our findings also suggested that OxtR1 binds to a potential promoter region of the TDE_0259–260 operon. Moreover, the mutant showed a marginal yet significantly faster growth rate than the wild-type strain under H2O2 exposure. ConclusionThe oxygen-sensing regulator OxtR1 plays a role in regulating the expression of a potential ferrodoxin, which may contribute to the response of T. denticola to oxygen-induced stress.

DETEСTION OF miRNA IN CHRONIC PERIODONTITIS CLINICAL STUDIES: A SYSTEMATIC REVIEW

There is a list of limitations for accurate clinical assessment of chronic periodontitis (CP) activity. The search for new mechanisms of pathogenesis leads to the selection of appropriate test substrates and research methods. Today, a number of miRNAs are known, which are involved in maintaining a healthy state or periodontal diseases. The aim of this systematic review was to analyze researches devoted to the role of miRNA in chronic periodontitis and various research methods to clarify the further study. Materials and methods. We followed PRISMA guidelines to conduct this systematic review. To identify the types of miRNAs, and their research methods in CP, PubMed database were systematically screened for medical literature. The articles published from 2019 up to November 27, 2023, were included. The articles were screened by using the search strategy as "chronic periodontitis miRNA". Results. We identified 35 miRNAs whose expression was significantly altered in chronic periodontitis (CP) compared to healthy gingiva. Fifteen of these miRNAs were correlated with the clinical manifestations of CP. The studies differed in the RNA-containing substrates, which included crevicular fluid, saliva, blood plasma, or exosomes from these substrates, as well as gingival biopsies. Real-time reverse transcription-PCR was the final method used for miRNA identification in all the studies. miRNAs are involved in almost all cellular processes and play a crucial role in development, differentiation, and homeostasis. They achieve these functions indirectly by suppressing gene expression, particularly through the inhibition of translation at the initiation stage. Target genes associated with the pathogenesis of chronic periodontitis (CP) have been previously identified for some miRNAs. Conclusion. Among the studied miRNAs, there are promising candidates to become targets for correction to optimize treatment, especially considering their predicted or known target genes involved in the pathogenesis of chronic periodontitis. However, no study has been devoted to pharmacological corrections of altered miRNA expression in chronic periodontitis.

Proteomic analysis of salivary inflammatory biomarkers of developmental gingival enlargements in patients with West and Noonan syndromes: a preliminary pilot single-center retrospective study.

The aim of the preliminary pilot single-center retrospective cross-sectional study was to analyze and compare the presence of non-secretory salivary inflammatory biomarkers in pediatric patients with West syndrome, Noonan syndrome, and a healthy control group. A total of 60 saliva samples were collected during dental check-ups. The saliva samples collected were analyzed by liquid chromatography. The results were analyzed with a t-test, and the statistical significance was given by a p-value lower than 0.05. We found statistical significance for defensin α1 (p=0.006) and thymosin β4 (p=0.025) in the Noonan syndrome. In the West syndrome, only the defensin α1 had a statistically significant difference with the other groups (p=0.022). Proteomic analysis revealed an overexpression of peptides related to the innate (thymosin β4) and acquired (defensin α1, α3) immunity. West and Noonan's syndromes showed the overexpression of molecular biomarkers involved in the pathogenesis of chronic periodontitis. The inflammatory status is triggered and amplified by the abnormal overgrowth of gingival tissues, the amplified release of proinflammatory cytokines from the immune cells, and the poor cooperation in maintaining adequate oral hygiene.

Microvesicles and neutrophil aggression factors in the pathogenesis of chronic periodontitis

Background. It is known that the number of polymorphonuclear leukocytes increases with the development of periodontitis. However, their role in the pathogenesis of chronic periodontitis has not been fully defined.
 Aim. To study the pathogenetic role of polymorphonuclear leukocytes in the development of chronic periodontitis, realized by microvesicles and aggression factors (metalloproteinases, myeloperoxidase, calprotectin, lipocaine 2).
 Material and methods. 30 patients aged 30 to 50 years with a diagnosis of chronic generalized periodontitis of moderate severity (K05.3) and no severe somatic pathology were under observation. The comparison group included 20 people with no inflammatory diseases in the oral cavity, comparable with the main group in terms of gender, age, nationality, and the presence of bad habits. Using a standard set of indices, the state of periodontal tissues was assessed, and all examined patients underwent X-ray examination. In the oral fluid of the examined, the total number of microvesicles and their phenotype, the concentration of matrix metalloproteinases-2 and -9, myeloperoxidase, calprotectin, lipocaine 2 were determined. The parameters were determined using a flow cytometer. Descriptive statistics are represented by the median and interquartile range (25th and 75th percentiles). Two independent sample sets were compared using the MannWhitney test. Correlation analysis was carried out according to the Spearman method.
 Results. The total number of microvesicles in the oral fluid in patients with chronic periodontitis was 11 times higher than in healthy volunteers (p=0.00001). More than a third of them had neutrophil markers. The levels of neutrophil aggression factors were also higher in patients with periodontitis and correlated with the value of the periodontal index, the depth of the periodontal pocket, bleeding index, indicators of periodontal health. Thus, a strong positive relationship was found between the total number of microvesicles and the value of the periodontal index (r=0.675), the depth of the periodontal pocket (r=0.763), the bleeding index (r=0.704); the number of neutrophilic microvesicles and indicators of periodontal health r=0.816, r=0.837 and r=0.776, respectively. The content of metalloproteinase-2 correlated with the depth of the periodontal pocket (r=0.754), bleeding index (r=0.811), papillary-marginal-alveolar index (r=0.675).
 Conclusion. Neutrophils contribute to the pathogenesis of chronic periodontitis through the secretion of aggression factors: metalloproteinases, myeloperoxidase, calprotectin, lipocaine 2, and microvesicles.

Screening of interleukin 17F gene polymorphisms and eight subgingival pathogens in chronic periodontitis in Libyan patients

ABSTRACT Background: Chronic periodontitis (CP) is triggered by periodontal pathogens influenced by genetic and environmental factors. Recent studies have suggested that anti-inflammatory cytokines such as interleukin 17 (IL−17) play a prominent role in the pathogenesis of CP. Aim:This study aimed to investigate the association between eight sub-gingival pathogens and interleukin 17F (IL−17F) gene single nucleotide polymorphisms with CP among Libyans. Materials and Methods: A case–control study was conducted on 100 individuals between the ages of 25–65 years. Species-specific 16S rRNA primers for each pathogen were used in a multiplex PCR reaction to detect sub-gingival pathogens from a paper point sample. DNA was also extracted from buccal swab samples and IL−17F polymorphisms were detected by Sanger sequencing. Results: A highly significant association between the seven sub-gingival pathogens and CP, (p-value 0.0001) and a high prevalence of P. intermedia (100%), T. forsythia (96%), T. denticola and E. corrodens (92%), P. gingivalis (82%), C. rectus (74%), P. nigrescens (72%), A. actinomvcetcmcomitans (40%) were found in the case group compared with control group. A novel variant in the c. *34 G>A in IL−17F gene caused a change in glutamic amino acid to lysine amino acid, position on chromosome number (6) in the third exon, mRNA/genomic position 597, found in 14.6% of CP patients (p-value = 0.010) while the IL−17F (rs763780) SNP showed no association with CP (p-value = 0.334). Conclusion: P. intermedia appear as keystone pathogen for CP in the Libyan population. A novel variant in the IL−7F gene may be related to the severity of CP.

Bestatin as a treatment modality in experimental periodontitis.

Chronic periodontitis (CP), the most prevalent dysbiotic bacteria-driven chronic inflammatory disease, is an underestimated global health problem in itself, and due to a causative relationship with other disorders such as cardiovascular diseases or Alzheimer disease. The CP pathogenesis is primarily driven by Porphyromonas gingivalis in humans, and Porphyromonas gulae in dogs. These microorganisms initiate a pathogenic shift in the composition of the tooth-surface microflora. Our objective was to evaluate antimicrobial effects of bestatin, a potential CP drug candidate. We evaluated bestatin bacteriostatic efficiency against periodontopathogens in planktonic cultures via microplate assay, and mono- and multispecies oral biofilm models. Neutrophil bactericidal activities, such as phagocytosis, were investigated in vitro using granulocytes isolated from the peripheral blood. The therapeutic efficacy and the immunomodulatory function of bestatin was assessed in a murine model of CP. Bestatin exhibited bacteriostatic activity against both P. gingivalis and P. gulae, and controlled the formation and species composition of the biofilm. We demonstrated that bestatin promotes the phagocytosis of periodontopathogens by neutrophils. Finally, we found that providing bestatin in the animal feed prevented alveolar bone resorption. We show that in a murine model of CP bestatin not only shifted the biofilm species composition from pathogenic to a commensal one, but also promoted bacteria clearance by immune cells and alleviated inflammation. Taken together, these results suggest that bestatin is a promising drug choice for the treatment and/or prevention of periodontitis and clinical trials are required to fully evaluate its potency.

Cyclic di-adenosine monophosphate regulates the osteogenic and adipogenic differentiation of hPDLSCs via MAPK and NF-κB signaling.

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that can be recognized by infected host cells and activate the immunoinflammatory response. The purpose of this study is to demonstrate the effect of c-di-AMP on the differentiation of human periodontal ligament stem cells (hPDLSCs) and its underlying mechanisms. In the present study, we find that the gingival crevicular fluid (GCF) of patients with chronic periodontitis has a higher expression level of c-di-AMP than that of healthy people. In vitro, c-di-AMP influences the differentiation of hPDLSCs by upregulating Toll-like receptors (TLRs); specifically, it inhibits osteogenic differentiation by activating NF-κB and ERK/MAPK and promotes adipogenic differentiation through the NF-κB and p38/MAPK signaling pathways. Inhibitors of TLRs or activated pathways reduce the changes induced by c-di-AMP. Our results establish the potential correlation among bacterial c-di-AMP, periodontal tissue homeostasis and chronic periodontitis pathogenesis.

Porphyromonas gingivalis HmuY and Its Possible Effect on the Pathogenesis of Periodontitis

Periodontitis, one of the most common inflammatory oral diseases in human beings, threatens the health of teeth and mouth and is closely associated with the development of many systemic diseases. Existing research about the pathogenesis of periodontitis mainly focuses on the oral microbial homeostasis and its complex interaction with the immune system. Among all the oral microorganisms, Porphyromonas gingivalis ( P. gingivalis) is considered to be the main pathogen causing chronic periodontitis. Recent studies have shown that P. gingivalis poesseses HmuY, a special heme binding protein, which binds with heme to provide essential nutrition for P. gingivalis and activates the host immune system. Therefore, HmuY plays an important role in the growth, proliferation, invasion, and pathogenesis of P. gingivalis and is a potential virulence factor of the bacteria. Existing studies on HmuY are limited to the host immune response that HmuY triggers, and there are still no conclusive findings on whether HmuY participates in the pathogenesis of periodontitis through other ways, such as influencing periodontal bone metabolism. Herein, we reviewed the latest research findings on the biological characteristics and physiological functions of HmuY and its relationship with chronic periodontitis, so as to provide new ideas for in-depth research and further explorations into the pathogenesis of chronic periodontitis.

Imipramine prevents Porphyromonas gingivalis lipopolysaccharide-induced microglial neurotoxicity

Porphyromonas gingivalis (P. gingivalis) is a Gram-negative anaerobe involved in the pathogenesis of chronic periodontitis, including local inflammation of the oral cavity. However, periodontal disease has recently been identified as a significant factor in the pathogenesis of neural diseases, including Alzheimer's disease. A virulence factor, P. gingivalis-lipopolysaccharide (LPS-PG), is involved in pro-inflammatory responses, not only in peripheral tissues but also in the brain. In this study, we examined whether P. gingivalis-induced brain inflammation could be ameliorated by pharmacotherapy, using in vivo and in vitro studies. In an animal experiment, peripheral administration of LPS-PG induced inflammation in the hippocampus via microglial activation, which was inhibited by pre-treatment with the antidepressant imipramine. Similarly, LPS-PG-induced inflammation in MG-6 cells, a mouse microglial cell line, was inhibited by pre-treatment with imipramine, which caused imipramine-induced inhibition of NF-κB signaling. Culture media obtained from LPS-PG-treated MG-6 cells induced neuronal cell death in Neuro-2A cells, a mouse neuroblastoma cell line, which was prevented by pre-treatment of MG-6 cells with imipramine. These results indicate that imipramine inhibits LPS-PG-induced inflammatory responses in microglia and ameliorates periodontal disease-related neural damage.

Immunoexpression of interferon-gamma in the interdental gingiva of chronic periodontitis patients with interferon-gamma (+874A/T) rs62559044 polymorphism.

Interferon-gamma (IFNγ), which is involved in inflammation, has an essential role in the pathogenesis of chronic periodontitis (CP). The association of IFNγ +874A/T gene polymorphisms with a higher incidence of CP has been reported previously. We investigated the immunoexpression of IFNγ in human gingiva from CP patients with IFNγ +874A/T gene polymorphisms compared to a healthy group. Biopsies from interdental gingiva (n=60) were obtained from CP patients (n=38) and individuals who had clinically healthy gingiva (n=22). The specimens were classified according to genotypes of IFNγ +874A/T gene polymorphism using amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) (10AA, 18AT, and 10TT). After tissue processing and slide preparation, we evaluated the expression levels of IFNγ in the interdental gingiva using the immunohistochemistry technique. Values were compared between two study groups by Mann-Whitney and Kruskal-Wallis analysis. P<0.05 was considered the level of significance. There was a noticeable increase in the immunoexpression of IFNγ in the gingival epithelium, fibroblasts, and capillaries of gingival tissue from CP patients compared to the control group. We found different levels of IFNγ expression in different parts of the gingival epithelium. The highest levels were found in the middle regions of the gingival epithelium in patients with TT genotypes. Immune cells and vascular endothelium of gingiva in the AA group also showed increased expression of IFNγ. Results suggest that IFNγ +874A/T gene polymorphism may change the expression level of IFNγ and subsequently the progression of CP.

Effect of Non-surgical Periodontal Therapy on Platelet-to-Lymphocyte Ratio and Neutrophil-to-Lymphocyte Ratio in Chronic Periodontitis

Published on:June 2022 Journal of Young Pharmacists, 2022; 14(2):227-230 Original Article | doi:10.5530/jyp.2022.14.42 Authors: Nida Malik1,*, Veena Kalburgi2, Saba Malik3, Sumedha Srivastava4,Harshita Verma5, Kalpana Sharma Rai61 Department of Periodontics, People’s College of Dental Sciences and Research Centre, Bhopal, Madhya Pradesh, INDIA.2Department of Periodontology, People’s College of Dental Sciences and Research Centre, Bhopal, Madhya Pradesh, INDIA.3Private Practitioner, Bhopal, Madhya Pradesh, INDIA.4Department of Periodontology, People’s College of Dental Sciences and Research Centre, Bhopal, Madhya Pradesh, INDIA.5Department of Periodontics, Bharati Vidhyapeeth Dental College, Pune, Maharashtra, INDIA.6Department of Endodontics, Peoples College of Dental Science and Research Center, Bhopal, Madhya Pradesh, INDIA. Abstract: Introduction: Haematological investigations have for long being considered as potential diagnostic and prognostic markers in the pathogenesis of Chronic Periodontitis. An exaggerated understanding of these markers with the consideration of relatively new markers Neutrophil to Lymphocyte Ratio (NLR) and Platelet to Lymphocyte Ratio (PLR) obtained from the peripheral blood count may serve as a potential marker in the understanding of the correlation of Chronic Periodontitis and Systemic Inflammatory Response. These markers haven’t been extensively studied in context to Periodontitis. Materials and Methods: 60 Patients were enrolled and 90 samples were collected. Group A-Experimental (30 Patients) who were assessed at baseline and Post-treatment after 4 weeks and Group B-Controls (30 Patients). GI, PI, PPD, CAL, Neutrophil Count, Lymphocyte Count and Platelet counts were obtained. NLR was calculated as the ratio to Neutrophil to Lymphocyte and PLR was calculated as the ratio of Platelet to Lymphocyte. Statistical analysis of the data was performed. Results: The results varied significantly in both the groups and in the diseased group at baseline and post-treatment. The ROC cut off values for NLR and PLR was found to be 1.5 and 92.5 respectively which may be used as prognostic values for Chronic Periodontitis. Conclusion: Both the novel markers, NLR and PLR can successfully be used in the assessment of Chronic Periodontitis with the ease of applicability and can also be used further to rule out any ongoing systemic inflammatory challenge. Keywords: Chronic Periodontitis, Neutrophil, Lymphocyte, Platelet, Systemic Inflammatory response, Inflammatory markers.

Effect of Green Tea on the Level of Salivary Interleukin-1 Beta in Patients with Chronic Periodontitis: A Randomized Clinical Trial.

Aim Interleukin-1 beta (IL-1β) is one of the major biomarkers involved in the pathogenesis of chronic periodontitis. The aim of this study was to evaluate the changes in salivary IL-1β concentration in patients with chronic periodontitis following daily consumption of green tea. Methods and Materials Thirty patients with an average age of 45.8 years suffering from chronic periodontitis were randomly assigned into 2 groups (i.e., experimental and control groups). Besides receiving phase 1 periodontal treatment (scaling and root planning (SRP)), the experimental group drank green tea for a period of 6 weeks. To measure the concentration of salivary IL-1β, saliva samples were taken from both groups at 2 time points, i.e., prior to SRP (time point 1 (T0)) and after 6 weeks (time point 2 (T1)). The nonparametric Wilcoxon test was used to examine and compare the changes in the concentration of salivary IL-1β in each group relevant to the 2 time points (T0 and T1). Data were submitted to statistical analysis. Results At the end of the study period, a significant reduction (P=0.0001) in the concentration of salivary IL-1β was observed in the experimental group (A). As for the control group (B), however, there was no significant change (P=0.307) in the concentration of salivary IL-1β after 6 weeks following phase 1 periodontal treatment. Conclusion Green tea supplementation, in addition to SRP, may reduce salivary IL-1β levels in patients with chronic periodontitis for a period of 6 weeks.

Quantity (Number) and Quality (Morphology) Comparison of Mast Cells in Healthy and Diseased Gingiva

Introduction: Comprehending the pathogenesis of chronic periodontitis is helpful in the prevention of this disease. Since there are conflicting results regarding the role of mast cells in periodontitis, the aim of this study was to compare mast cell counts and morphology in periodontitis with clinically healthy gingiva.&#x0D; Materials and Methods: This cross sectional case-control study was performed on 36 gingival samples (obtained from 14 patients with chronic periodontitis and 22 cases with clinically healthy gingiva among those referred to a specialized medical center in 2013. Toluidine blue and hematoxylin -eosin staining was used to identify mast cells. The total number of mast cells (intact and degranulated) was counted under the light microscope in 5 consecutive high power fields (10×40) with no overlap. Data were recorded and analyzed statistically by SPSS 22 and Independent T-test. All of the data with a statistical significance (p value &lt; 0.05) were analyzed.&#x0D; Results: According to Independent t-test, mean number of degranulated mast cells in periodontitis group was 17.78 and in healthy gingiva group was 29.72 which was not statistically significant(p value = 0.071). Mean number of mast cells in periodontitis group was 20.57 and in healthy gingiva was 36.54 and was statistically significant (p value = 0.042). Mast cell degranulation proportion in periodontitis group was 0.857 and in healthy gingiva group was 0.866, not statistically significant (p value = 0.891).&#x0D; Conclusion: According to results of present study it can be concluded that total number of mast cells and proportion of degranulated mast cell to the total number showed no significant relationship but the total number of mast cell was significantly increased in healthy group.

Correlations between periodontal indices and osteoporosis.

In the pathogenesis of chronic periodontitis, there are general systemic factors which play a major role, such as osteoporosis, with menopause as the most common etiological factor, and other pathological determining conditions for osteoporosis as well (ovary, thyroid and malignant tumors). The aim of the present study was to assess the correlations between periodontal indices and osteoporosis. The study was performed on 35 patients with periodontal disease aged between 45 and 79 years. These patients were divided into two groups: a study group with osteoporosis and periodontal disease (n=25) and a control group with periodontal disease (n=10) only. The periodontal assessment included community periodontal index (CPI), gingival inflammation index (GI), plaque index (PI), body mass index (BMI), bone mineral density (BMD), tooth mobility and tooth loss. Osteoporosis was assessed by dual-energy X-ray absorptiometry. Results were statistically analyzed with Microsoft Excel software and XLSTAT. The results showed that patients in the study group had higher values of periodontal indices, and a highly significant inverse correlation was observed between the CPI and the tooth loss. Inverse correlations between BMI and tooth mobility, as well as BMI and CPI were determined for the study group. In conclusion, the positive association between BMD and GI shows that the gingival index can be a predictive factor in the occurrence of osteoporosis.

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-β(TGF-β), 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.

Evaluation of Salivary Level of Heat Shock Protein 70 in Patients with Chronic Periodontitis.

Statement of the Problem: Traditional clinical criteria are usually not sufficient for determining the sites of active periodontal disease, monitoring the response to treatment, or measuring the susceptibility to future disease development. Past studies have shown that heat shock protein 70 (HSP70) are involved in the etiology of periodontal disease.Purpose: The aim of this study was to evaluate the level of HSP70 in saliva of patients with chronic periodontitis (CP).Materials and Method: In our case-control study, the saliva samples of 45 patients with CP and 45 age- and sex-matched healthy subjects were collected. Salivary HSP70 was measured by enzyme-linked immunosorbent assay method. The results were analyzed by statistical tests using SPSS 16 and the statistically significant difference was set at p< 0.05.Results: In this study, the mean salivary HSP70 level was 2.81±0.61ng/ml in the patient group and 1.96±0.77ng/ml in the healthy group, with a significant difference (p< 0.05). In addition, the results of spearman correlation analysis showed a positive correlation between salivary HSP 70 and clinical periodontal index.Conclusion: The results of this study showed that the salivary HSP70 level in patients with CP is higher than that in healthy subjects. As a result, salivary HSP70 might be considered as a marker in the pathogenesis of CP.

Genetic association and epistatic interaction analysis of cluster of differentiation 14 and mannan-binding lectin 2 gene polymorphic variants in susceptibility to chronic periodontitis

ObjectiveTo study the association and epistatic interactions of cluster of differentiation 14 (CD14) and mannan-binding lectin 2 (MBL2) gene polymorphic variants in Indian patients with chronic periodontitis (CP). DesignWe enrolled a total of 242 individuals (121 patients and 121 control subjects), age 35 to 60 years both irrespective of gender and identified CD14 (−159C > T, NC_000005.10:g.2569190:C > T) and MBL2 (codon 52, C > T, NM_000242:c.52C > T) polymorphic variants in peripheral blood samples. We also performed epistatic interaction analysis using multifactor dimensionality reduction (MDR) approach and predicted ribonucleic acid (RNA) structure of MBL2 gene using Genebee online RNA tool. ResultsSignificantly increased frequency of ‘CT' heterozygote and variant allele ‘T' in chronic periodontitis patients was observed for both CD14 and MBL2 gene polymorphisms. MDR analysis showed approximately two-fold increased risk of CP. In silico analysis showed lack of transcription factor ETF (TEA domain family member 2) binding-site in presence of ‘T' allele ‘in CD14 (-159C > T) polymorphism. The secondary RNA structure prediction of MBL2 (codon 52, C > T) polymorphism showed structural variations having approximately similar free energies. ConclusionsA dominant effect of genotype ‘CT' heterozygote and variant ‘T' allele observed for both CD14 and MBL2 gene polymorphisms in patients with CP. The presence of ‘T' allele also results in lack of transcription factor binding site at CD14 (-159C > T) and changes in arrangements of RNA molecules that may further affect expression of CD14 and MBL2 genes leading to increased susceptibility to CP pathogenesis.

Influence of the new formulation based on silver alginate microcapsules loaded with tannic acid on the microcirculation of the experimental periodontitis in rats

The microvascular changes caused by disorders of host immune response to oral microorganisms resulting in long-lasting inflammation of gums play a critical role in the periodontal lesion in the pathogenesis of chronic periodontitis. Current strategies of non-surgical periodontal therapy are aimed at the attainment of anti-inflammatory effects. We hypothesized that the usage of the microencapsulated form of anti-inflammatory substances with vasoactive effects could enhance the efficiency of the therapy by the prolonged release of active components. The prepared suspension of silver-alginate microcapsules loaded with tannic acid in the hydrogel was applied in vivo to the experimental model of periodontitis in rats induced by a ligature. The effect of this formulation was assessed by monitoring changes in local microcirculation performed by the Laser Doppler Flowmetry (1 and 24 h after application of hydrogel on intact gums and 21-days after the start of periodontitis' modeling). Application of the hydrogel containing multicomponent microcapsules to the affected area of gums allows correction of inflammatory microcirculatory disorders in model periodontitis. Immobilization of tannic acid into microcapsules allows increasing the correction of the following parameters: perfusion disorders, neurogenic tone of arterioles, myogenic tone of precapillary sphincters, as well as a venous outflow in the microvasculature of the gums. The hydrogel containing multicomponent microcapsules reduces the vascular inflammatory response in the model of periodontitis. Loading of silver-alginate microcapsules with tannic acid enhances the efficiency of microvascular disorders' correction in the model of periodontitis that suggests the prospects for application of this drug delivery system for non-surgical treatment of periodontitis.

TPCA-1 negatively regulates inflammation mediated by NF-κB pathway in mouse chronic periodontitis model.

The dysregulation of immune system plays a crucial function in periodontitis development. Pro-inflammatory cytokines are thought to be critical for the generation and development of periodontitis. The enhanced activity of osteoclasts contributes to periodontitis pathogenesis. Nuclear factor-κB (NF-κB) signaling pathway directly enhances osteoclast differentiation and maturation. 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) is a IκB kinases (IKK) inhibitor. This research aimed to investigate whether TPCA-1 had influence on the pathogenesis of chronic periodontitis. Mouse chronic periodontitis was induced by an in vivo ligature-induced periodontitis model. TPCA-1 was intravenously injected into mice after chronic periodontitis induction. Bone marrow-derived macrophages were cultured in macrophage colony-stimulating factor (M-CSF)-conditioned media with receptor activator of nuclear factor-kappa B ligand (RANKL) induce in vitro osteoclast differentiation. Western blot was used to analyze protein levels and mRNA levels were analyzed through qRT-PCR. TPCA-1 promoted osteoclastogenesis and osteoclast-related gene expression in vitro. The production of pro-inflammatory cytokines in osteoclasts induced by lipopolysaccharides was inhibited by TPCA-1 in vitro. In vitro TPCA-1 treatment inhibited Aggregatibacter actinomycetemcomitans (A.a)-induced expression of pro-inflammatory cytokines and NF-κB signal activation in osteoclasts. The induction of chronic periodontitis was inhibited by the absence of IKKb in mice. This research demonstrates that the treatment of TPCA-1 negatively regulates inflammation response and inhibits the osteoclastogenesis through the inactivation of NF-κB pathway in mouse chronic periodontitis model.