Ana Pucar

Identification of senescence-inducing microRNAs in normal human keratinocytes

MicroRNAs (miRNAs) are epigenetic regulators of eukaryotic gene expression and play key roles in many cellular processes. However, the role of miRNAs for replicative senescence of normal human keratinocytes (NHKs) remains unknown. Thus, we examined the expression profiles of 847 miRNAs in exponentially replicating and senescent NHKs and identified 126 senescence-associated miRNAs (SA-miRs). Among SA-miRs, 117 miRNAs (93%) were upregulated and 9 miRNAs (7%) were downregulated in senescent NHKs compared to those of exponentially replicating cells. Among the above miRNAs, we selected two miRNAs, miR-137 and miR-668, for further investigation because they were consistently upregulated with replicative senescence of three independent NHK cultures. Ectopic overexpression of miR-137 or miR-668 induced senescence in rapidly proliferating NHKs; a notable increase in senescence-associated β-galactosidase activity, p16INK4A and p53 was observed, indicating that they are novel senescence-inducing miRNAs. In addition, these senescence-inducing miRNAs were gradually increased during organismal aging of normal human oral epithelia. We also detected downregulation of miR-137 and miR-668 in many tested human head and neck squamous cell carcinoma cell lines. Since senescence would be viewed as a potent tumor suppressive pathway, the newly identified senescence-inducing miRNAs deserve to be further investigated for their therapeutic application in cancer treatment.

Expression and mutation analysis of heterogeneous nuclear ribonucleoprotein G in human oral cancer

We previously reported that wild type (wt) hnRNP G exhibited tumor suppressive activity in human oral squamous cell carcinoma (HOSCC) cell lines lacking hnRNP G. Wt hnRNP G markedly inhibited the proliferation capacity, anchorage independency and in vivo tumorigenicity of HOSCC cells and notably enhanced the DNA repair capabilities of these cells. In the present study, we studied the genetic and expression states of hnRNP G in normal, premalignant and malignant human oral tissues to further understand the relationship between the hnRNP G alterations and the development of human oral cancer. To correlate the cancer development and the level of hnRNP G expression, we performed an immunohistochemistry staining of hnRNP G in normal, premalignant and malignant human oral tissues. Moreover, we examined the entire coding regions of hnRNP G from selected samples to understand the cause of the alterations of the gene expression. The expression of hnRNP G was notably decreased or completely abolished in 80% of premalignant-dysplastic and malignant oral epithelial tissues, whereas 100% of normal and 90% of hyperplastic non-dysplastic epithelium showed high level of hnRNP G in the nucleus of the basal cell layers. Approximately 80% of HOSCC lacking the expression of hnRNP G showed genetic alteration in hnRNP G, i.e., point mutation and exonic deletion. This study suggest that genetic alterations and aberrant expression of hnRNP G occurring during oral carcinogenesis might be useful markers for the early detection of human oral cancer.

Detection and sampling methods for isolation of Candidaspp. from oral cavities in diabetics and non-diabetics

The purpose of this study was to detect Candida spp. on the tongue and in the subgingival sites in healthy and type 2 diabetes (T2D) patients with chronic periodontitis (CP), and to compare the accuracy of sampling methods. This study included 131 patients divided into four groups: healthy control (group A), nondiabetics + CP (Group B), diabetics with good metabolic control + CP (group C) and diabetics with poor glycoregulation + CP (Group D). Cotton swab samples from tongue and subgingival samples were obtained from each patient with help of sterile paper points and a sterile curette. Swab cultures were made on Sabouraud dextrose agar. The number of CFUs was counted. The sampling methods for subgingival plaque were compared by Receiving Operator Curve (ROC). The presence of Candida spp. on the tongue was statistically significant among groups (group D vs. others three groups: χ(2): p < 0.005 for each group). Positive findings of subgingival Candida spp. did not differ among the groups. There were no significant differences in the quantification of Candida spp., neither on the tongue, nor in the subgingival samples. 17.2% of diabetic patients revealed the presence of Candida spp. in the subgingival samples, with negative finding on tongue. There was a significant difference in the sampling methods for subgingival plaque (p = 0.000). Candida spp. is more prevalent on the tongue of diabetics. The sampling of subgingival plaque by a sterile curette is more accurate than with paper points. Subgingival plaque may represent a reservoir of commensals. It is necessary to standardize the sampling of subgingival plaque.

Presence of Different Candida Species at Denture Wearers With Type 2 Diabetes and Clinically Healthy Oral Mucosa-Pilot Study

Summary Background/Aim: The aim of this study was to examine prevalence of different Candida spp. at diabetics and nondiabetics wearing dentures without clinical signs of Denture Stomatitis (DS) and to study if some local and systematic factors are confounders for harboring Candida at these subjects. Material and Methods: Total of 60 subjects wearing partial or complete upper acrylic denture having at least half of palatal mucosa covered by denture were selected and stratified into three experimental groups: systematically health subjects; patients with diagnosed Type 2 Diabetes (T2D) and good glycoregulation; and T2D subjects with poorly regulated blood sugar level. Cotton swab samples were obtained from each patient from hard palate mucosa and denture surface. Swab cultures were made on Sabouraud dextrose agar and ChromAgar Media for distinciton of various Candida spp. Density growth was also measured. Results: Frequency of Candida spp. findings were similar between groups. At healthy subjects, only C.albicans was detected. At diabetics, C.albicans was the most common isolated species, followed by C.glabrata and C.tropicalis. Negative finding of yeasts on palatal mucosa, but positive on denture surface were detected at all groups, with the highest frequency (33.4%) at diabetics with poor glycoregulation. Denture surface was heavier colonized than hard palate mucosa. Duration of diabetes in years were only independent predictors for harboring Candida spp. at denture surface (Exp B=1.186, CI=1.047-1.344, p=0.007). Conclusions: Prosthesis of denture wearers without DS may serve as reservoir of Candida spp. Presence of more pathogenic and resistant non-albicans species are related to diabetics, even without clinical signs of DS.

Relationship between serum tumor necrosis factor receptor-2 concentration and periodontal destruction in patients with type 2 diabetes: Cross-sectional study

Introduction The role of tumor necrosis factor-α (TNFα) is well documented in pathogenesis of chronic periodontitis (CP) and type 2 diabetes (T2D). Considering short half-life of TNFα, tumor necrosis factor receptor-2 (TNFR2) is used as prosperous surrogate marker of TNFα activity. Objective The aim was to detect TNFR2 serum concentration and correlate it with periodontal destruction in patients with diagnosed T2D and nondiabetics. Methods The study included 85 patients divided into three groups: T2D + CP (group T2D, n = 34); nondiabetics + CP (Group PD, n = 27); and healthy controls (group HC, n = 24). T2D was diagnosed according to WHO criteria (2013) and periodontitis was diagnosed using International Workshop for a Classification of Periodontal Diseases and Conditions criteria (1999). TNFR2 level was measured by enzyme-linked immunosorbent assay (ELISA). Results There was no difference in TNFR2 level among the groups (Kruskal–Wallis, p = 0.482). Significant correlation (Pearson’s correlation coefficient) was observed between clinical attachment loss (CAL) and TNFR2 concentration in PD group (rp = -0.460, p = 0.016). In T2D group, correlations were observed between TNFR2 concentration and CAL (rp = 0.363, p = 0.005) and periodontal inflamed surface area (PISA) (rp = 0.345, p = 0.046) and periodontal epithelial surface area (PESA) (rp = 0.578, p = 0.000). Conclusion Higher concentration of TNFR2 was associated with higher CAL, PESA, and PISA scores in T2D group. Contrary to that, nondiabetics with higher values of CAL exhibited lower concentration of TNFR2, presenting potential protective effect on periodontal destruction. These results imply that diabetes may alter TNFR2 secretion originated from periodontium.

Association of CXCL12 gene promoter methylation with periodontitis in patients with diabetes mellitus type 2

OBJECTIVES CXCL12 is widely expressed, constitutive chemokine involved in tissue repair and regeneration, while the extent of its expression is important in various chronic inflammatory conditions. Involvement of DNA methylation in CXCL12 gene suppression (CXCL12) has been shown in malignancy and some autoimmune diseases. The aim of this study was to investigate whether the alterations in DNA methylation of CXCL12 are also involved in progression of periodontitis in combination with diabetes, as these chronic inflammatory conditions are strongly interrelated. DESIGN Study included 72 subjects divided in three groups: healthy control (C, n=21), periodontitis (P, n=29) and diabetes/periodontitis group (D/P, n=22). DNA extracted from epithelial cells obtained by sterile cotton swabs from buccal mucosa was subjected to methylation specific polymerase chain reaction (MSP) to obtain DNA methylation pattern of CXCL12 promoter. RESULTS CXCL12 promoter was predominantly unmethylated in all groups. However, increase in the frequency of the methylated form and increase in percent of methylation of CXCL12 promoter in periodontitis and diabetes/periodontitis group compared to control group were found, although without statistical significance. However, statistically significant increase in Tm of MSP products in diabetes/periodontitis group was observed. Correlation analysis revealed statistically significant relationship between the extent of DNA methylation of the CXCL12 promoter and periodontal parameters, as well as between DNA methylation of CXCL12 and glycosylated hemoglobin. CONCLUSION Presented results suggest that chronic inflammation contributes to the change of CXCL12 DNA methylation in buccal cells and that DNA methylation profile of CXCL12 promoter plays important role in development and progression of periodontal disease.

Periodontal Inflammation as Risk Factor for Pancreatic Diseases

Jelena Milasin1, Natasa Nikolic Jakoba2, Dejan Stefanovic3, Jelena Sopta4, Ana Pucar2, Vojislav Lekovic2 and Barrie E. Kenney5 1Institute of Human Genetics, School of Dentistry, University of Belgrade, Belgrade 2Clinic for Periodontology and Oral Medicine, School of Dentistry, University of Belgrade 3Clinic for Gastroenterology, School of Medicine, University of Belgrade 4Institute of Pathology, School of Medicine, University of Belgrade, Belgrade 5Tarrson Family Endowed Chair in Periodontics, UCLA School of Dentistry, Los Angeles 1,2,3,4Serbia 5USA

The effect of chlorhexidine on the receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG) expression in chronic periodontitis in humans and companion animals

% OPG % RANKL KR nema AB Periodontal disease is a chronic, multi-factorial disease of the tissues supporting the teeth. Periodontitis in companion animals is an almost identical disease to that in humans in terms of disease course and clinical presentation. Receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG) are bioactive molecules that control bone resorption. This study aims to evaluate the effect of Chlorhexidine (CXD) on the RANKL and OPG expressions in gingival crevicular fluid (GCF) collected from subjects with chronic periodontitis. GCF was obtained from subjects with chronic periodontitis.10 subjects (CXD1) rinsed the mouth with 0.12% CXD, 10 subjects (CXD2) utilized 0.20% CXD and the last 10 (PL) used Placebo solution for 7 days. RANKL and OPG concentrations in GCF were measured by enzyme-linked immunosorbent assays ELIat baseline and after 7 days. Periodontal clinical variables: clinical attachment loss (CAL), probing pocket depth (PPD), papilla-bleeding index (PBI) were evaluated in all groups. After 7 days in CXD1 and CXD2 group RANKL/OPG ratio exhibited a significant decrease (p<0.05) in contrast to the PL group where results showed similar values of RANKL/OPG ratio at baseline and after the observation period. RANKL/OPG ratio was positively correlated with PPD, CAL and PBI before and after the observation period in both Chlorhexidine (CXD1, CXD2) groups. In an existing inflammatory response, chlorhexidine reduced the level of periodontal inflammation, which leads to reduction of RANKL/OPG relative ratio. Decrease of RANKL/OPG ratio will apparently induce maintenance of alveolar bone and slow down periodontal tissue breakdown. Parodontopatije su hronicna, multikauzalna oboljenja potpornog aparata zuba. Parodontalna oboljenja koja srecemo kod kucnih ljubimaca su prema toku i klinickoj slici skoro identicna onima koje se javljaju kod ljudi. RANKL i osteoprotegerin (OPG) su bioaktivni molekuli koji kontrolisu kostanu resorpciju. Cilj ove studije je evaluacija efekata hlorheksidina na ekspresiju RANKL-a i OPG-a u gingivalnoj tecnosti (GT) uzetoj od pacijenata sa hronicnom parodontopatijom. 10 pacijenata (CXD1) su ispirali usta sa 0.12% CXD, 10 pacijenata (CXD2) su koristili 0.20% CXD i poslednjih 10 pacijenata (PL) su koristili placebo rastvor 7 dana. RANKL i OPG koncentracije u GT su merene ELItestom na pocetku i posle sedam dana. Parodontalni klinicki parametri CAL, PPD i PBI su evaluirani u svim grupama. Posle 7 dana u CXD1 i CXD2 grupi RANKL/OPG odnos je pokazao signifikantno smanjenje (p<0.05) u poređenju sa PL grupom gde su zabaleženi slicni rezultati na pocetku i nakon opservacionog perioda. RANKL/OPG odnos je pokazao pozitivnu korelaciju sa vrednostima PPD-a, CAL-a i PBI-a pre i nakon observacionog perioda u obe eksperimentalne grupe (CXD1, CXD2). U prisutnom inflamatornom odgovoru hlorheksidin je redukovao nivo inflamacije, sto je uslovilo redukciju RANKL/OPG odnosa. Rezultati istraživanja dokazuju da koncentracija hlorheksidina ne utice statisticki znacajno na smanjenje RANKL/OPG odnosa. PR Projekat Ministarstva nauke Republike Srbije, br. P145042