MiR-26a and miR-191 are upregulated while PLAG1 and HIF2 are downregulated in pleomorphic adenomas of the salivary glands compared to Warthin tumors.

BACKGROUND: Salivary gland tumors (SGTs) are a heterogenous group of pathologies, which still represents a challenge regarding differential diagnosis and therapy. Although histological findings govern SGTs management, detection of molecular alterations is emerging as an effective additional tool. The aim of this study was to analyze the relative expression levels of three micro RNAs (miR-26a, miR-26b, and miR-191), and three pro-oncogenic molecular markers (PLAG1, MTDH, and HIF2) in SGTs and normal salivary gland (NSG) tissues and evaluate them as potential differential diagnosis markers. METHODS: This cross-sectional study included 58 patients with SGTs (23 pleomorphic adenomas, 27 Warthin tumors, and 8 malignant SGTs) and 10 controls (normal salivary gland tissues). Relative gene expression levels of all investigated molecules were determined by reverse transcriptase-real-time polymerase chain reaction. RESULTS: All three micro RNAs exhibited highest expression levels in benign SGTs, whereas miR-26a And miR-191 were significantly more expressed in PAs compared to WTs (p = 0.045 and p = 0.029, respectively). PLAG1 And HIF2 were both overexpressed in WTs compared to PAs (p = 0.048 and p = 0.053, respectively). Bioinformatic analysis suggested that all investigated micro RNAs function as negative regulators of MTDH. CONCLUSION: The results of this study suggest that all three micro RNAs have a considerable negative impact on MTDH oncogene expression in malignant tumors, while the differences between levels of miR-26a, miR-191, PLAG1, and HIF2 in PA and WT represent possible differential diagnosis markers.

Expression of miRNAs and proinflammatory cytokines in pregnant women with gestational diabetes mellitus.

Altered microRNAs (miRNAs1) and cytokines expression levels are associated with several pregnancy-induced complications. We evaluated the profile of circulating miRNAs (miR-17, miR-29a and miR-181a) and proinflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-17) in women with gestational diabetes mellitus (GDM2), as well as their potential use as GDM biomarkers. The case-control study included 65 pregnant women divided into 2 groups - GDM and control. Expression levels of miRNAs in plasma samples and cytokines mRNA isolated from peripheral blood buffy coat were analyzed by quantitative real-time PCR (qPCR3). Significant miR-29a downregulation was found in GDM compared to the control group, and was even more significant after adjustments for covariates. miR-17 and miR-181a expression levels did not differ between the examined groups. Expression levels of IL-1ß were significantly higher in GDM group compared to controls, while TNF-α, IL-6 and IL-17 did not show significant changes in expression between the two groups. As jugded from the ROC curve analysis, miR-29a and IL-1ß had a significant capacity to discriminate between CG and GDM. Additionally, a positive correlation was established between IL-1ß and TNF-α in the GDM group. GDM appeared to be associated with altered levels of miR-29a and IL-1ß making them markers of this condition.

Streptococcus mitis and Prevotella melaninogenica Influence Gene Expression Changes in Oral Mucosal Lesions in Periodontitis Patients.

Oral microbiome disruptions in periodontitis are related to the chronic inflammatory reactions that could in turn lead to the development of multiple oral diseases. The objective of the study was to assess the frequencies of Streptococcus mitis, Prevotella melaninogenica, and Prevotella intermedia in oral benign lesions, oral potentially malignant disorders (OPMDs), and oral squamous cell carcinomas (OSCCs) and investigate the impact of these bacteria on the expression patterns of the selected (potential) target genes (PI3CA/AKT2/mTOR, DUSP16/MAPK14, and COX2). After sample collection (25 benign lesions, 30 OPMDs, and 35 OSCCs) and DNA/RNA extraction, quantitative real-time polymerase chain reaction (qPCR) was performed to detect bacterial presence and assess relative gene expression levels in different lesion groups. Prevotella melaninogenica was the most prevalent of the three analyzed bacteria, with the frequency being 60% in benign lesions, 87% in OPMDs (p = 0.024), and 77% in OSCC. The OPMD tissues in which Prevotella melaninogenica was present exhibited a higher expression level of AKT2 (p = 0.042). Significantly lower expression of DUSP16 was observed in OSCC tissues containing Streptococcus mitis (p = 0.011). The obtained results indicate a substantial contribution of P. melaninogenica and Str. mitis in the pathogenesis of oral mucosal lesions, possibly via AKT2 upregulation and DUSP16 downregulation.

Red Light and 5% Aminolaevulinic Acid (5%) Inhibit Proliferation and Migration of Dysplastic Oral Keratinocytes via ROS Production: An In Vitro Study.

Undiagnosed and untreated oral precancerous lesions often progress into malignancies. Photodynamic therapy (PDT) might be a minimally invasive alternative to conventional treatments. 5-aminolevulinic acid (5-ALA) is one of the most commonly used photosensitizers in PDT, and it is effective on many cancer types. However, its hydrophilic characteristic limits cell membrane crossing. In the present study, the effect of a newly formulated gel containing 5% 5-ALA in combination with red light (ALAD-PDT) on a premalignant oral mucosa cell line was investigated. The dysplastic oral keratinocyte (DOK) cells were incubated with ALAD at different concentrations (0.1, 0.5, 1, and 2 mM) at two different times, 45 min or 4 h, and then irradiated for 7 min with a 630 nm LED (25 J/cm2). MTT assay, flow cytometry, wound healing assay, and quantitative PCR (qPCR) were performed. ALAD-PDT exerted inhibitory effects on the proliferation and migration of DOK cells by inducing ROS and necrosis. mRNA analysis showed modulation of apoptosis-related genes' expression (TP53, Bcl-2, survivin, caspase-3, and caspase-9). Furthermore, there was no difference between the shorter and longer incubation times. In conclusion, the inhibitory effect of the ALAD-PDT protocol observed in this study suggests that ALAD-PDT could be a promising novel treatment for oral precancerous lesions.

Clinical, microbiological and osteoimmunological findings in different peri-implant conditions - A cross-sectional study.

OBJECTIVES: The aim of this study was to assess the prevalence of certain microbiota and their potential correlation with clinical parameters, expression of proinflammatory cytokines, Notch signalling pathway molecules and bone remodelling mediators among different peri-implant conditions. MATERIALS AND METHODS: Included participants had at least one dental implant minimally 1 year in function. They were divided into peri-implantitis (PI), peri-implant mucositis (PM) and healthy implants (HIs) groups. Prevalence of P. ginigvalis, Fusobacterium spp., EBV and C. albicans was detected in participants' crevicular fluid (CF) using quantitative real-time polymerase chain reaction, different markers' expression, as well as clinical data, were correlated with the microbial presence. RESULTS: CF samples taken from one chosen implant from each of the 102 participants were analyzed. Significantly higher levels of P. gingivalis were found in PI compared with HI (p = .012) and PM (p = .026). Fusobacterium spp. was also more prevalent in PI (p = .041) and PM (0.008) than in HI. P. gingivalis was a predictor of PPDi (p = .011, R2 = 0.063) and CALi (p = .049, R2 = 0.038). A positive correlation was found in PI for the level of Fusobacterium spp. and TNFα expression (ρ = 0.419, p = .017) while in PM, P. gingivalis and Notch 2 expression were correlated (ρ = 0.316, p = .047). CONCLUSIONS: P. gingivalis appears to be involved in the osteolysis in patients with PI, while the positive correlation of its level with Notch 2 expression in patients with PM suggests a potential involvement of P. gingivalis in the progression of PM into PI.

Osteogenic and Adipogenic Differentiation Potential of Oral Cancer Stem Cells May Offer New Treatment Modalities.

(1) Treatment failure of oral squamous cell carcinoma (OSCC) is generally due to the development of therapeutic resistance caused by the existence of cancer stem cells (CSCs), a small cell subpopulation with marked self-renewal and differentiation capacity. Micro RNAs, notably miRNA-21, appear to play an important role in OSCC carcinogenesis. Our objectives were to explore the multipotency of oral CSCs by estimating their differentiation capacity and assessing the effects of differentiation on stemness, apoptosis, and several miRNAs' expression. (2) A commercially available OSCC cell line (SCC25) and five primary OSCC cultures generated from tumor tissues obtained from five OSCC patients were used in the experiments. Cells harboring CD44, a CSC marker, were magnetically separated from the heterogeneous tumor cell populations. The CD44+ cells were then subjected to osteogenic and adipogenic induction, and the specific staining was used for differentiation confirmation. The kinetics of the differentiation process was evaluated by qPCR analysis of osteogenic (Bone Morphogenetic Protein-BMP4, Runt-related Transcription Factor 2-RUNX2, Alkaline Phosphatase-ALP) and adipogenic (Fibroblast Activation Protein Alpha-FAP, LIPIN, Peroxisome Proliferator-activated Receptor Gamma-PPARG) markers on days 0, 7, 14, and 21. Embryonic markers (Octamer-binding Transcription Factor 4-OCT4, Sex Determining Region Y Box 2-SOX2, and NANOG) and micro RNAs (miRNA-21, miRNA-133, and miRNA-491) were also correspondingly evaluated by qPCR. An Annexin V assay was used to assess the potential cytotoxic effects of the differentiation process. (3) Following differentiation, the levels of markers for the osteo/adipo lineages showed a gradual increase from day 0 to day 21 in the CD44+ cultures, while stemness markers and cell viability decreased. The oncogenic miRNA-21 also followed the same pattern of gradual decrease along the differentiation process, while tumor suppressor miRNA-133 and miRNA-491 levels increased. (4) Following induction, the CSCs acquired the characteristics of the differentiated cells. This was accompanied by loss of stemness properties, a decrease of the oncogenic and concomitant, and an increase of tumor suppressor micro RNAs.

Notch signalling cascade and proinflammatory mediators in peri-implant lesions with different RANKL/OPG ratios-An observational study.

BACKGROUND & OBJECTIVE: Notch signaling pathway has been linked to bone loss in periodontitis and peri-implantitis. This research aimed to determine the Notch signaling molecules expression levels (Notch1, Notch2, Jagged1, Hes1, and Hey1), along with bone remodeling mediators (RANKL and OPG) and proinflammatory cytokines (TNF-α, IL-17, IL-1ß, and IL-6) in patients with peri-implant diseases. The aforementioned markers' expression was evaluated in patients with different RANKL/OPG ratios. METHODS: Fifty patients with peri-implantitis (PI group) and 45 patients with peri-implant mucositis (PM group) were enrolled. Relative gene expression levels of investigated molecules were determined by reverse transcriptase-real-time polymerase chain reaction. On the basis of RANKL/OPG ratio, all peri-implant lesions were divided into subgroups: RANKL-predominant (RANKL > OPG) and OPG-predominant (RANKL < OPG). Clinical periodontal parameters (probing depth-PD, bleeding on probing-BOP, clinical attachment level-CAL and plaque index-PLI), were recorded for each patient around every tooth, and around placed implants (PDi, BOPi, CALi, PLIi). RESULTS: RANKL-predominant PM patients exhibited higher expression levels of Notch2 (p = .044) and Hey1 (p = .005) compared to OPG-predominant lesions. In all RANKL-predominant cases, Hey1 (p = .001), IL-1ß (p = .005), IL-6 (p = .002) were overexpressed in PI comparing to PM, accompanied with significantly higher PDi, CALi and PLIi in PI than PM (p = .001, p = .001 and p = .009). CONCLUSIONS: Notch2 upregulation in RANKL-predominant PM lesions could be an important contributor to alveolar bone resorption and represent a predictor of PM to PI transition. Similarly, the overexpression of IL-1ß and IL-6 might provide an osteoclastogenic environment in PI RANKL-predominant lesions.

Analyses of P16INK4a gene promoter methylation relative to molecular, demographic and clinical parameters characteristics in non-small cell lung cancer patients: A pilot study.

BACKGROUND: The aim of this study was to examine the methylation status of p16INK4a promoter region in non small cell lung cancer (NSCLC) patients and their associations with single nucleotide polymorphisms (SNPs) of the epidermal growth factor receptor (EGFR) gene, as well as with demographic or clinical characteristics. METHODS: Formalin-fixed and paraffin-embedded (FFPE) DNA samples extracted from 22 NSCLC patients were analyzed with methylation-specific polymerase chain reaction (PCR) method to obtain promoter methylation profile. The same cohort was genotyped for - 216G > T, -191 C > A, and 181,946 C > T EGFR SNPs. RESULTS: There was a significant association between methylated p16INK4a in patients prior therapy (p = 0.017) since a significantly higher frequency of methylated p16INK4a was detected in these patients (40.9%) in comparison to frequency in patients after therapy (31.8%). Also, a higher frequency of methylated p16INK4a was detected among patients with leucopenia (p = 0.056). No associations were observed between the methylation status of the p16INK4a promoter region and EGFR SNPs or other clinical and demographic data in this cohort. CONCLUSION: High frequency of methylation of the p16INK4a gene promoter was observed in NSCLC patients prior therapy and with leucopenia that can indicate their significance related to advanced clinical stage.

Single nucleotide polymorphisms as a predisposing factor for the development of apical periodontitis-An umbrella review.

BACKGROUND: The interaction between heredity and different environmental factors in the modification of apical periodontitis (AP) susceptibility and prediction of its progression remain poorly elucidated. OBJECTIVES: This umbrella review aimed to (i) analyse the available relevant systematic reviews in an attempt to determine the association between genotype and allelic distribution of different single-nucleotide polymorphisms (SNPs) and the development of AP, (ii) report deficiencies and gaps in knowledge in this area and (iii) present recommendations to conduct future clinical studies and systematic reviews. METHODS: A literature search was conducted using Clarivate Analytics' Web of Science, Scopus, PubMed and Cochrane Database of Systematic Reviews, from inception to October 2021, with no language restrictions, including a grey literature search. Systematic reviews with/without meta-analysis evaluating genotype and allelic distribution of different SNPs between adult patients with/ without AP were included. All other type of studies were excluded. The methodological quality was assessed using the A MeaSurement Tool to Assess systematic Reviews (AMSTAR)-2 tool. Two independent reviewers were involved in study selection, data extraction and appraising the included reviews; disagreements were resolved by a third reviewer. RESULTS: The current study includes five systematic reviews. Three reviews performed meta-analysis. Three reviews were graded by AMSTAR 2 as 'critically low' quality, whereas the other two were graded as 'low' and 'moderate' quality. Two reviews indicated that carriers of specific genotypes and alleles of tumour necrosis factor-alpha (TNF-α) -308 G > A and interleukin 1-beta (IL-1ß) + 3954 C/T gene polymorphisms are more susceptible to an acute and persistent form of AP. However, high heterogeneity was observed. DISCUSSION: The statistical heterogeneity within included systematic reviews was a consequence of clinical and methodological diversity amongst primary studies. Although some of the included reviews suggested that carriers of specific genotype and/or allele of TNF-α -308 G > A and IL-1ß + 3954 C/T SNPs are more susceptible to AP, their conclusions should be interpreted with caution. CONCLUSIONS: No candidate genes could be identified as a definitive genetic risk or protective factor for the development and progression of AP, and further high-quality genome-wide association studies are warranted.

Methylation of tumour suppressor genes in benign and malignant salivary gland tumours: a systematic review and meta-analysis.

The aim of the present systematic review was to critically analyse the relationship between tumour suppressor genes (TSGs) promoter methylation, a potent mechanism of gene silencing, and the development of salivary gland tumours, as well as the possible effect on clinical/histological characteristics. Review protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) database (registration ID CRD42020218511). A comprehensive search of Web of Science, Scopus, PubMed, and Cochrane Central Register of Controlled Trials was performed utilizing relevant key terms, supplemented by a search of grey literature. Newcastle-Ottawa Quality Assessment Scale (NOQAS) was used for the quality assessment of included studies. Sixteen cross-sectional and 12 case-control studies were included in the review, predominantly dealing with methylation in TSGs related to DNA repair, cell cycle, and cell growth regulation and differentiation. Quantitative synthesis could be performed on P16 (inhibitor of cyclin-dependent kinase 4a), RASSF1A (Ras association domain family 1 isoform A) and MGMT (O6-methylguanine DNA methyltransferase) genes only. It showed that P16 and RASSF1A genes were more frequently methylated in salivary gland tumours compared to controls (P = .0002 and P < .0001, respectively), while no significant difference was observed for MGMT. Additionally, P16 did not appear to be related to malignant transformation of pleomorphic adenomas (P = .330). In conclusion, TSG methylation is involved in salivary gland tumour pathogenesis and several genes might play a considerable role. Further studies are needed for a better understanding of complex epigenetic deregulation during salivary gland tumour development and progression.

Gene polymorphisms in odontogenic keratocysts and ameloblastomas: A systematic review.

OBJECTIVES: The aim of this systematic review was to critically analyze available data on gene polymorphisms in odontogenic keratocysts (OKC) and ameloblastomas, including their possible relationship with clinical and histological features of these lesions. MATERIALS AND METHODS: A comprehensive search of Web of Science Scopus, PubMed, Cochrane Central Register of Controlled Trials and EMBASE was conducted using relevant key terms and supplemented by a gray literature search. Quality assessment of included studies was performed using criteria from the Strengthening the Reporting of Genetic Association (STREGA) statement. RESULTS: Ten studies were included in the final review. Survivin -31G/C, interleukin IL-1α -889 C/T, p53 codon 72 G/C, tumor necrosis factor TNF-α (-308G>A) and its receptor TNF-R1 (36A>G), glioma-associated oncogene homolog 1 rs2228224 and matrix metalloproteinase 2 rs243865 gene polymorphisms were reported to be associated with OKC. For ameloblastomas, p53 codon 72 G/C, X-ray repair cross-complementing protein 1-codons 194 and 399 and matrix metalloproteinase 9 rs3918242 gene polymorphisms were identified as risk factors. It was not possible to establish a relationship between specific polymorphisms and clinical and histological features of investigated lesions. CONCLUSIONS: Several gene polymorphisms might be considered as a risk factor for the development of these lesions. Future studies should investigate whether these polymorphisms might be used to identify patients with increased risk of recurrence or aggressive disease.

Periapical lesions in two inbred strains of rats differing in immunological reactivity.

AIM: To investigate the influence of strain differences in immune responses on the pathogenesis of experimental periapical lesions in Dark Agouti (DA) and Albino Oxford (AO) inbred strains of rats. METHODOLOGY: Periapical lesions were induced in male DA and AO rats by pulp exposure of the first mandibular right molars to the oral environment. Animals were killed 21 days after pulp exposure. The mandibular jaws were retrieved and prepared for radiographic, pathohistological, immunohistochemical analysis, real-time PCR and flow cytometry. Blood samples and the supernatant of periapical lesions were collected for measurement of cytokines and oxidative stress marker levels. Statistical analysis was performed using the Kruskal-Wallis H and Mann-Whitney U non-parametric tests or parametric One-Way anova and Independent Samples T-test to determine the differences between groups depending on the normality of the data. A significant difference was considered when p values were <.05. RESULTS: DA rats developed significantly larger (p < .05) periapical lesions compared to AO rats as confirmed by radiographic and pathohistological analysis. The immunohistochemical staining intensity for CD3 was significantly greater in periapical lesions of DA rats compared to AO rats (p < .05). In DA rats, periapical lesions had a significantly higher (p < .05) percentage of CD3+ cells compared to AO rats. Also, the percentage of INF-γ, IL-17 and IL-10 CD3+CD4+ cells was significantly higher in DA rats (p < .05). DA rats had a significantly higher Th17/Th10 ratio. RT-PCR expression of IL-1ß, INF-γ and IL-17 genes was significantly higher in periapical lesions of DA compared to AO rats (p < .05). The receptor activator of nuclear factor kappa-Β ligand/osteoprotegerin ratio was higher in DA compared to AO rats with periapical lesions (p < .05). Systemic levels of TNF-α and IL-6 were significantly higher in DA compared to AO rats (p < .05). Levels of lipid peroxidation measured as thiobarbituric acid reactive substances and reduced glutathione were significantly higher (p < .05) in the supernatant in the periapical lesions of DA rats. CONCLUSION: After pulp exposure, DA rats developed much larger periapical lesions compared to AO rats. Genetically determined differences in immunopathology have been demonstrated to be a significant element defining the severity of periapical lesions.

Notch down-regulation and inflammatory cytokines and RANKL overexpression involvement in peri-implant mucositis and peri-implantitis: A cross-sectional study.

OBJECTIVES: Notch signaling pathway, known to influence bone resorption in several oral diseases, has not been analyzed in peri-implantitis yet. Therefore, the aims of the present study were to determine the levels of Notch cascade, bone remodeling mediators, and pro-inflammatory cytokines, in conjunction with clinical parameters, in subjects with peri-implant mucositis and peri-implantitis. MATERIAL AND METHODS: Clinical parameters: peri-implant probing depth, bleeding on probing, suppuration on probing, and plaque index (PI) were recorded. Samples were collected from 130 participants, divided into peri-implantitis (PI), peri-implant mucositis (PM), and healthy implants (HI) group. Relative expression levels (REL) of Notch 1, Notch 2, Jagged 1, Hes 1, Hey 1, TNF-α, IL-17, IL-1ß, IL-6, RANKL, and OPG mRNA were determined by reverse transcriptase-real-time polymerase chain reaction. Quantitation of Notch 1, Il-17, and IL-6 proteins was performed using ELISA assays. RESULTS: All clinical parameters were significantly higher in PI compared to HI. Significant decrease of Notch 1, and higher REL of Hey 1, IL-1ß, IL-6, and RANKL were found in PI compared to HI. PM showed significant increase of IL-1ß REL in comparison with HI. In PI versus PM, significantly higher REL was found for Hey 1, TNF-α, IL-17, IL-1ß, IL-6, and RANKL. Additionally, higher protein concentrations of IL-6 and IL-17 were detected in PI versus PM and versus HI group. CONCLUSION: The combined effect of Notch 1 down-regulation and elevated expression of some key inflammation modulators might result in osteoclast activity increase and subsequent osteolysis in peri-implantitis.

Tumor Necrosis Factor Alpha -308 G/A Single-Nucleotide Polymorphism and Apical Periodontitis: An Updated Systematic Review and Meta-analysis.

INTRODUCTION: This study aimed to perform a more precise estimation of the association between tumor necrosis factor alpha (TNF-α) -308 G/A single-nucleotide polymorphism (SNP) and the risk of development of apical periodontitis (AP) and its phenotypes based on all available published studies. METHODS: The study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and is registered in PROSPERO (CRD42020176190). The literature search was conducted via Clarivate Analytics Web of Science, Scopus, PubMed, Cochrane Central Register of Controlled Trials, and China National Knowledge Infrastructure databases from inception to December 2020 with no language restrictions. Two reviewers were involved independently in the study selection, data extraction, and appraising the studies that were included. The quality of the included studies was evaluated using the Strengthening the Reporting of Genetic Association and the Grading of Recommendations Assessment, Development and Evaluation system. The frequencies of the genotypes and alleles of the TNF-α (G>A 308, rs1800629) gene with 95% odds ratio were used. RESULTS: Four studies met the inclusion criteria with moderate risk of bias. This study revealed no significant association between TNF-α -308 G/A SNP and AP and the risk of AP development. Moreover, there was no significant association between genotype or allele frequency distribution and clinical manifestations (acute vs chronic) of AP. The certainty of evidence per the Grading of Recommendations Assessment, Development and Evaluation system was very low. CONCLUSIONS: Because of very low certainty of evidence, whether there is an association between TNF-α -308 G/A SNP and AP warrants further well-designed multicentric studies to adjudicate a better understanding of the role of genetic factors in the etiopathogenesis of AP.

Impact of Notch signalling molecules and bone resorption regulators on clinical parameters in periodontitis.

BACKGROUND AND OBJECTIVE: Notch signalling cascade has recently been connected to alveolar bone resorption in periodontitis. Hence, the present cross-sectional study aimed to analyze the expression of Notch signalling pathway (Notch 1, Notch 2, Jagged 1, Hes 1, Hey 1) and periodontitis-related (tumor necrosis factor alpha- TNF-α, interleukin 17-IL-17, receptor activator of nuclear factor-kappa B ligand-RANKL, osteoprotegerin-OPG) molecules and correlate it with clinical parameters in aggressive (AP) and chronic (CP) periodontitis. Additionally, the aforementioned markers' expression was evaluated in periodontitis patients with different RANKL/OPG ratios. MATERIAL AND METHODS: Eighty patients were enrolled either in AP or CP group. Clinical attachment level (CAL), bleeding on probing (BOP), periodontal probing depth (PPD) and plaque index (PI) were recorded for each patient. Total RNA was extracted from gingival crevicular fluid samples. Relative gene expression of investigated markers was determined by reverse transcriptase-real-time polymerase chain reaction. RESULTS: Significantly higher values of PPD were observed in AP compared to CP (P = .010). Negative correlations between OPG and CAL, and OPG and PI, were found in AP (P = .045, P = .006, respectively), while Hey 1 and PI had a positive correlation (P = .049). In multivariate linear regression analysis, OPG and Notch 2 were predictors of CAL in AP group. TNF-α and IL-17 were higher in RANKL predominant than in OPG predominant cases (P = .007, P = .001, respectively). In RANKL predominant lesions Notch 1 and Jagged 1 were down-regulated in AP compared to CP patients (P = .010, P = .025, respectively). CONCLUSION: The present study demonstrated that changes in Notch 2 expression affected CAL in AP cases hence this molecule could be considered as a contributor to alveolar bone loss. In RANKL-activated settings, the down-regulation of Notch 1 might participate in more severe bone resorption in AP.

The association of tumor necrosis factor alpha, lymphotoxin alpha, tumor necrosis factor receptor 1 and tumor necrosis factor receptor 2 gene polymorphisms and serum levels with periodontitis and type 2 diabetes in Serbian population.

OBJECTIVES: Aiming to show that periodontitis (PD) and type 2 diabetes (T2D) are bidirectionally related and potentially linked by inflammatory cytokines, we searched for association between -308 G/A Tumor necrosis factor-alpha (TNFα), +252A/G Lymphotoxin-alpha (LTα), +36A/G Tumor necrosis factor receptor 1 (TNFR1) and +676 T/G tumor necrosis factor receptor 2 (TNFR2) single nucleotide polymorphisms (SNPs) and: risk of PD or PD + T2D; periodontitis parameters in PD and PD + T2D; serum levels of cytokines/their receptors. Relationship between periodontal inflammation and serum cytokine/receptor levels was also assessed. DESIGN: Subjects were stratified as: 57 healthy controls (HC); 58 PD; 65 PD + T2D. Sociodemographic, environmental, behavioral and periodontal clinical data were recorded. SNPs were genotyped using polymerase chain reaction-restriction fragment length polymorphism, while cytokines/receptors levels were quantified by enzyme-linked immunosorbent assay. Impact of periodontal inflammation was measured using periodontal inflamed surface area (PISA). RESULTS: TNFα AA genotype showed protective effect in T2D + PD compared to PD, even adjusted for behavioral/environmental factors (OR 0.18; 95 %CI 0.037-0.886; p = 0.035). LTα AG heterozygotes had increased risk of PD (OR 3.27; 95 %CI 1.35-7.96; p = 0.016), while TNFR2 TG genotype had protective effect (OR = 0.44; 95 %CI 0.954-0.9794; p = 0.043). TNFR1 AA was predictor of periodontal pocket depth and clinical attachment loss in PD. Correlation between TNFR2 concentration and PISA was negative in PD, positive in PD + T2D. CONCLUSIONS: None of the SNPs showed cross-susceptibility between PD and T2D. + 252A/G LTα and +676 T/G TNFR2 SNPs are associated with PD risk. Periodontal destruction in healthy individuals is influenced by TNFR1 genotype. Impact of periodontal on systemic inflammation is masked by T2D.

Endothelial nitric oxide synthase polymorphisms/haplotypes are strong modulators of oral cancer risk in Serbian population.

Oral carcinoma is the sixth most common malignancy worldwide, with survival rates of approximately 50%. The major type of oral cancer, present in 90% of the cases, is oral squamous cell carcinoma (OSCC). The genetic background predisposing an individual to OSCC is complex and largely unknown. Studies have suggested that endothelial nitric oxide synthase (eNOS) gene polymorphisms modulate the cancer risk, prompting us to assess the impact of three functional eNOS gene polymorphisms on OSCC risk. The present study included 50 patients with OSCC and 110 controls. Polymerase chain reaction and restriction fragment length polymorphism analysis were used for genotyping of single-nucleotide polymorphisms -786 T/C (rs2070744) and 894 G/T (rs1799983) and variable number of tandem repeats (VNTR) intron 4b/a polymorphism. Homozygous carriers of -786 T/C and intron 4b/a VNTR variant alleles paired with a significant increase of oral cancer risk [odds ratio (OR): 3.63, 95% confidence interval (CI): 1.08-12.21; P = 0.045 and OR: 11.29, 95% CI: 2.71-47.11; P < 0.001, respectively]. When combined, CC and 4b4a genotypes together led to a 21-fold OSCC risk increase (OR: 21, 95% CI: 2.07-213.29; P = 0.006). Haplotype analysis showed that the C-G-4b haplotype conferred an 11-fold increase in OSCC risk. In conclusion, eNOS polymorphisms considerably influence levels of OSCC risk in the Serbian population.

Comparison of four different suture materials in respect to oral wound healing, microbial colonization, tissue reaction and clinical features-randomized clinical study.

OBJECTIVES: Sutures are the most frequently used medical device for wound closure. They support tissue during the early phase of healing until it regains enough tensile strength. The aim of this study was to compare four different suture materials in terms of the influence on wound healing, microbial adherence, tissue reaction, and relevant clinical parameters which determine their clinical value. MATERIALS AND METHODS: Total number of 32 patients undergoing surgical extraction of four impacted third molars were involved in the study. Clinical parameters were estimated intraoperatively and during the control check-ups. Soft tissue healing around sutures were evaluated on the 3rd and 7th day postoperatively. Microbial colonization was assessed by means of qPCR. Also, histological analysis was done to assess inflammatory reaction. RESULTS: Significantly better soft tissue healing was found around monofilament and synthetic sutures compared to multifilament and natural ones respectively. Soft tissue healing was significantly better around all sutures on the 7th day than on the 3rd day postoperatively. CONCLUSIONS: Non-resorbable polypropylene suture showed superior clinical characteristics among all sutures. Moreover, the best healing of soft tissue and the least inflammatory reaction was found around this thread. The poorest soft tissue healing was found around non-resorbable silk suture. This suture elicited strongest inflammatory reaction and showed the greatest microbial adherence affinity compared to alternative sutures. CLINICAL RELEVANCE: Monofilament synthetic suture should be used in order to obtain the best soft tissue healing, reduce the risk of postoperative infection, and alleviate the suturing after oral surgery procedures.

Notch - a possible mediator between Epstein-Barr virus infection and bone resorption in apical periodontitis.

Objectives: This study aimed to investigate whether Epstein-Barr virus (EBV) positive periapical lesions exhibited higher mRNA levels of Notch signalling molecules (Notch2 and Jagged1), bone resorption regulators (receptor activator of nuclear factor kappa-ß ligand (RANKL) and osteoprotegerin (OPG)), and proinflammatory cytokines (tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and IL-6) compared to EBV negative lesions. Additionally, the potential correlation between investigated molecules in periapical lesions was analyzed.Materials and methods: Sixty-four apical periodontitis lesions were obtained subsequent to standard apicoectomy procedure. The presence of EBV was determined using nested PCR. Based on the presence of EBV all periapical lesions were divided into two groups, 29 EBV positive and 35 EBV negative lesions. A reverse transcriptase real-time PCR was used to determine mRNA levels of Notch2, Jagged1, RANKL, OPG, TNF-α, IL-1ß and IL-6.Results: Significantly higher mRNA levels of Notch2, Jagged1, RANKL and IL-1ß were observed in EBV positive compared to EBV negative lesions. Significant positive correlation was present between Notch2 and Jagged1, Jagged1 and RANKL, and IL-ß and TNF-α in EBV positive periapical lesions.Conclusions: Notch signalling pathway may be involved in alveolar bone resorption in apical periodontitis lesions infected by EBV.

Raman microspectroscopy: toward a better distinction and profiling of different populations of dental stem cells.

AIM: To characterize stem cells originating from different dental tissues (apical papilla [SCAP], dental follicle [DFSC], and pulp [DPSC]) and test the capacity of Raman microspectroscopy to distinguish between the three dental stem cell types. METHODS: SCAP, DFSC, and DPSC cultures were generated from three immature wisdom teeth originating from three patients. Cell stemness was confirmed by inducing neuro-, osteo-, chondro-, and adipo-differentiaton and by mesenchymal marker expression analysis by flow-cytometry and real-time polymerase chain reaction. Cellular components were then evaluated by Raman microspectroscopy. RESULTS: We found differences between SCAP, DFSC, and DPSC Raman spectra. The ratio between proteins and nucleic acids (748/770), a parameter for discriminating more differentiated from less differentiated cells, showed significant differences between the three cell types. All cells also displayed a fingerprint region in the 600-700 cm-1 range, and characteristic lipid peaks at positions 1440 cm-1 and 1650 cm-1. CONCLUSION: Although different dental stem cells exhibited similar Raman spectra, the method enabled us to make subtle distinction between them.