Vamsi Lavu

Differential expression of microRNAs let-7a, miR-125b, miR-100, and miR-21 and interaction with NF-kB pathway genes in periodontitis pathogenesis

Periodontitis is a chronic inflammatory disease which is caused by destruction of the tissues that surrounds and supports the tooth. Deregulation of microRNAs has been reported to cause several inflammatory diseases such as autoimmune disease, chronic periodontitis, and cancer. In the present study, we have investigated the expression pattern of microRNAs let‐7a, miR‐125b, miR‐100, miR‐21, and RNA‐binding protein LIN‐28A among healthy individuals and chronic periodontitis patients. Total RNA was isolated from gingival tissue samples collected from 100 healthy individuals and 100 chronic periodontitis patients. The expression of microRNAs and LIN‐28 was performed by qPCR. Target prediction for the microRNAs was done using miRWalk and miRTarbase online databases and the experimentally validated targets were analyzed for their molecular function, biological processes, and related pathways using gProfiler software. The expression analysis revealed that let‐7a and miR‐21 were upregulated, whereas, miR‐100, miR‐125b, and LIN‐28 were down regulated. The age dependent expression analysis revealed that the expression levels of all the microRNAs and LIN‐28 were found to increase with age (more than 50 years), thereby suggesting an increased risk to chronic periodontitis. Among the various targets predicted using miRWalk and miRTarbase databases, NFKB was found to be a common target among all the four microRNAs. gProfiler revealed several functions such as NF‐ĸB signaling pathway, cytokine‐cytokine receptor interaction, osteoclast differentiation, etc., all of which specific to inflammation and periodontitis.

Comparison of the Proportion of Non-Classic (CD14+CD16+) Monocytes/Macrophages in Peripheral Blood and Gingiva of Healthy Individuals and Patients With Chronic Periodontitis

BACKGROUND Monocyte subsets with low CD14 expression that coexpress CD16 (CD14+CD16+) are called non-classic or hyperinflammatory monocytes. Previous studies have reported an increase in the percentage of CD14+CD16+ monocytes in the peripheral blood of patients with chronic periodontitis (CP). To our knowledge, there are no reports demonstrating the presence of CD14+CD16+ monocyte-derived macrophages (MDMs) in the gingival tissue. The objective of this study is to identify the proportion of non-classic (CD14+CD16+) monocytes/macrophages in peripheral blood and gingiva of healthy individuals and patients with CP. METHODS A total of 60 individuals (n = 30 per group) were recruited for the study. Group 1 included 30 individuals with healthy gingiva, and group 2 included 30 patients with CP. Direct immunofluorescent staining was done in 200 μL whole-blood and single-cell suspensions obtained from gingival tissue, with fluorochrome-conjugated monoclonal antibodies against CD14, CD16, and human leukocyte antigen-DR (HLA-DR), and subjected to flow cytometric analysis. RESULTS The mean percentage of CD14+CD16+ monocytes in the peripheral blood of healthy individuals was 9.10% ± 1.39%, and for patients with CP it was 14.18% ± 2.69% (P <0.05). The mean percentage of CD14+CD16+ MDMs in the gingival tissue of healthy individuals was found to be 0.93% ± 0.33%, whereas in patients with CP, it was 1.92% ± 0.78% (P <0.01). Non-classic monocytes/macrophages showed a high median fluorescent intensity for HLA-DR (DR++). CONCLUSION This study demonstrates an increased proportion of CD14+CD16+HLA-DR++ monocytes/macrophages in the peripheral blood and gingiva of patients with CP.

Elevated levels of cyclooxygenase 1 and 2 in human cyclosporine induced gingival overgrowth

OBJECTIVE This study was carried out to immuno-localize and estimate the levels of cyclooxygenase 1 and 2 in human gingival tissue samples from healthy individuals, chronic periodontitis patients and patients with cyclosporine induced gingival overgrowth. METHODS Group I consisted of individuals with healthy gingiva (n=6), Group II - cyclosporine induced gingival overgrowth (n=9) and Group III - chronic periodontitis patients (n=6). Gingival tissue samples were collected from subjects of all the three groups. COX-1, COX-2 levels were estimated in tissue homogenates by enzyme activity assay. Immuno-localization for COX-1 and COX-2 was also done in sections of gingival tissue. RESULTS The study results demonstrated a significantly higher mean levels of COX-1 and 2 in drug induced gingival overgrowth samples (p<0.05). COX-1 and COX-2 was localized to epithelium and connective tissue in human gingival tissue sections from cyclosporine induced gingival overgrowth. CONCLUSION Cyclooxygenase enzymes appear to be potential mediators involved in the pathogenesis of cyclosporine induced gingival overgrowth.

Polymorphic Regions in Fc Gamma Receptor and Tumor Necrosis Factor-α Genes and Susceptibility to Chronic Periodontitis in a Cohort From South India

BACKGROUND Polymorphisms in the immunoglobulin G Fc receptor II (FcGR) and tumor necrosis factor-α (TNFA) genes are known to influence pathogenesis and severity of several inflammatory conditions. Association of FcGR and TNFA gene polymorphisms with chronic periodontitis (CP) susceptibility has been found to be diverse among different ethnic populations. Objectives of the present study are to determine association of functional single nucleotide polymorphisms (SNPs) in FcGR and TNF-α genes with CP susceptibility in a cohort from South India. METHODS Polymorphisms of: 1) FCGR2A 131His/Arg (rs1801274); 2) FCGR2B 232Ile/Thr (rs1050501); 3) TNFA -1031T/C (rs1799964); and 4) TNFA -863C/A (rs1800630) were analyzed among patients with healthy gingiva (n = 176) and patients with CP (n = 177). Genotyping was performed using allele-specific real-time polymerase chain reaction assay. Association between CP and SNPs was examined by multivariable logistic regression analysis with adjustment for: 1) age; 2) sex; and 3) oral hygiene index (OHI). Epistatic interaction between FcGR polymorphisms and interleukin 1B (IL1B) +3954C/T (rs1143634) was assessed using multifactorial dimensionality reduction analysis. RESULTS Among four SNPs analyzed, only FCGR2A 131His/Arg showed significant association with CP in a dominant model (odds ratio: 1.6; 95% confidence interval: 1.028 to 2.530). This significance disappeared after correcting for multiple comparisons using Bonferroni analysis, or after adjusting for age, sex, and OHI. A significant redundant interaction between IL1B +3954 C/T and FCGR2A 131His/Arg was observed. CONCLUSION Study results suggest the variant form of the SNP in FCGR2A 131His/Arg, FCGR2B 232Ile/Thr, TNFA -1031T/C, and TNFA -863C/A are not associated with CP susceptibility in the selected cohort from South India.

Evaluation of antimicrobial properties of bioactive glass used in regenerative periodontal therapy

Context: Bone grafting materials which have an inherent anti-microbial property against initial colonizers of plaque bacteria would be useful in regenerative periodontal surgical procedures. Aims: This study was performed to analyze the antibacterial property of a Perioglas™ against a common oral commensal Streptococcus salivarius (early colonizer). Settings and Design: In vitro observational study. Materials and Methods: Perioglas™ (in various concentrations) was assessed for its antibacterial property against the ATCC 13419 strain of S. salivarius. The anti-microbial activity was analyzed in terms of reduction in colony-forming units in culture plates and smear following a 24 h incubation at 37°C. Statistical Analysis Used: Observational study - No statistical analysis applicable. Results: The bioactive glass (BAG) exerted an antibacterial effect against the S. salivarius in the suspending media and smear. The antibacterial activity of BAG increased in proportion with its concentration. Conclusions: Perioglas™ demonstrated a considerable antibacterial effect against S. salivarius at 50 mg/mL concentration.

Immuno-localization of glucose transporter 4 in healthy human gingiva.

BACKGROUND The gingiva has been shown to be a target tissue for several hormones. Insulin induces uptake of glucose in the peripheral tissues by upregulating the Glucose transporter 4 expression. Little information is available on the expression of Glucose transporter 4 in human gingiva. AIM In this regard, a pilot study was performed with the aim of determining the distribution pattern of Glucose transporter 4 in healthy human gingiva. MATERIALS AND METHODS Immuno-histochemistry was performed on 10 mounted sections of healthy human gingiva with the primary antibody Glucose Transporter 4 (GLUT 4). Appropriate positive and negative controls were used. RESULTS Glucose transporter 4 expression was observed in the basal and suprabasal layers of the gingival epithelium and fibroblasts of the gingival connective tissue. CONCLUSION This may be the first study to demonstrate the expression of GLUT 4 in the healthy human gingiva. The results of this study raise the possibility that gingiva may serve as a target tissue for insulin action.

Efficacy of a root conditioning agent on fibrin network formation in periodontal regeneration: A SEM evaluation.

Background: Even though numerous biomaterials have been devised and employed for periodontal regeneration, it should be well understood that the root surface receptiveness to clot formation and maintenance during initial periodontal wound healing, decides the nature of the connective tissue attachment. So this study was carried out with the prime objective of assessing the initial wound healing events occurring in vivo after the application of citric acid on to the root surfaces during periodontal regenerative therapy. Materials and Methods: Thirty-two human teeth were used for this in vitro study. Two dentin blocks each measuring 4 × 2 × 1 mm were made from each tooth. These dentin blocks were planed and treated differently with Phosphate Buffered Saline (PBS), citric acid, PBS and fresh human blood, citric acid and fresh human blood and were segregated into eight groups. Finally all the dentin blocks were processed and subjected to a scanning electron microscope study. Results: In PBS-treated samples, the dentin surface was irregular corresponding to smear layer and the dentinal tubule openings were obscured. Whereas, in those treated with citric acid revealed a smooth dentin surface devoid of smear layer and the dentinal tubular openings were clear. Further samples that were treated with PBS plus blood showed little or no fibrin network formation whereas with those citric acid plus blood showed a fine thick fibrin network formation adhered to dentinal surface. Conclusion: The results of this present in vitro study suggests that use of citric acid as a root conditioning agent has a beneficial effect on initial wound healing events, which are critical for periodontal regenerative therapies.

Chronic periodontitis prevalence and the inflammatory burden in a sample population from South India

Context: Periodontal diseases are among the most prevalent oral diseases in the world. Apart from repercussions in the oral cavity, there is evidence that periodontitis contributes to systemic damage in chronic diseases such as cardiovascular disease, diabetes, and preterm low birth weight. Aims: The aims of this study were to estimate the prevalence of chronic periodontitis in a sample urban population (<18 years) in Tamil Nadu and to estimate the inflammatory burden posed by chronic periodontitis by calculating the periodontal inflammatory surface area. Settings and Design: This was a population-based study and cross-sectional design. Subjects and Methods: A total of 1000 individuals (<18 years) were selected and screened for their periodontal status, oral hygiene status (OHI), and the periodontal inflamed surface area (PISA) in an outreach center located in Chennai, India. Statistical Analysis Used: The proportion of individuals with different periodontal states (health, gingivitis, and periodontitis) was determined. A multivariate logistic regression analysis was performed to assess the influence of the individual risk factors such as habits (tobacco use), systemic conditions (diabetes), and oral hygiene maintenance on periodontitis prevalence in the sample population. Results: A high prevalence of periodontal disease was observed in the study population (42.3%). Among the urban participants, age, cigarette smoking, pan chewing, decayed, missing, and filled teeth scores, OHI scores, and PISA scores were found to be significantly associated with periodontitis (P < 0.05). Conclusions: Periodontitis prevalence appears to be high even in areas with adequate access to oral health care and an inflammatory burden risk exists in a definitive manner.

Natural T Regulatory Cells (n Treg) in the Peripheral Blood of Healthy Subjects and Subjects with Chronic Periodontitis - A Pilot Study.

INTRODUCTION The T cells play a central role in the aetiopathogenesis of periodontal disease. Natural T regulatory cells (nTreg) are the key stone immunoregulatory elements having an anergic phenotype and play an important role in the suppression of exaggerated immune responses thereby maintaining homeostasis. There are increasing evidences for the role of nTreg in the periodontal disease pathogenesis. AIM To identify the proportion of natural T regulatory cells in the peripheral blood of periodontally healthy subjects and subjects with chronic periodontitis. MATERIALS AND METHODS A total of 15 subjects (7 with healthy gingiva and 8 with chronic periodontitis) were recruited for this pilot study. Baseline periodontal parameters were recorded and 5 ml of peripheral blood was collected. The samples from both the groups were analysed for the relative proportion of nTreg (identified by the expression CD45RB+CD4+CD25+FOXP3+) using flow cytometry. RESULTS The mean percentages of the CD45RB+CD4+CD25+ cells expressing FOXP3 in control and chronic periodontitis group were found to be 14.75±5.04 and 43.13±11.17 respectively. The mean proportion of nTreg were compared between the control and chronic periodontitis sample using Mann-Whitney Test and was found to be statistically significant with (p<0.001). CONCLUSION A higher proportion of nTreg in the peripheral blood sample of chronic periodontitis subjects were observed as compared to that of healthy individuals.