Bulletin du GIRSO

Epigenetic modifications in salivary glands from patients with Sjögren's syndrome affect cytokeratin 19 expression

O.D. Konsta — 2016-06-28

Sjögren’s syndrome (SS) is a chronic autoimmune epithelitis, and several lines of experiments indicate that multifactorial factors contribute to salivary gland epithelial cells (SGEC) dysfunctions including a combination of environmental factors, lymphocytic infiltrations, genetic predispositions as well as epigenetic defects. Such statement is reinforced by the observation that global DNA methylation (5MeCyt) is altered in minor salivary glands from pSS patients and that such defect is associated cytokeratin 19 (KRT19) overexpression. An epigenetic deregulation of the KRT19 gene was further tested by treating the human salivary gland (HSG) cell line with the DNA demethylating agent 5-azacytidin, and with the histone acetylase inhibitor trichostatin A. Blocking DNA methylation, but not histone acetylation, with 5-azacytidin was associated with KRT19 overexpression at both transcriptional and protein level. Next, analysis of the CpG genome-wide methylome array in the KTR19 locus from long term cultured SGEC obtained from 8 pSS patients revealed a more reduced DNA methylation level in those patients with defective global DNA methylation. Altogether, our data, therefore, suggest that alteration of DNA methylation in SGEC may contribute to pSS pathophysiology in part by controlling the expression of KRT19.

Intraplaque hemorrhage, a potential consequence of periodontal bacteria gathering in human carotid atherothrombosis

Adrian Brun — 2016-06-28

Periodontal diseases are multifactorial inflammatory diseases, caused by a bacterial biofilm involving both innate and adaptative immunity, characterized by the destruction of tooth-supporting tissues. In the context of periodontitis, the spread of weak pathogenic bacteria into the bloodstream has been described. These bacteria will preferentially localize to existing clot within the circulation. Atherothrombosis of the carotid arteries is a local pathology and a common cause of cerebral infarction. Intraplaque hemorrhages render the lesion more prone to clinical complications such as stroke. The main objective of this study is to explore the biological relationship between carotid intraplaque hemorrhage and periodontal diseases.

This study included consecutive patients with symptomatic or asymptomatic carotid stenosis, admitted for endarterectomy surgical procedure (n=41). In conditioned media of the carotid samples collected, markers of neutrophil activation (myeloperoxidase or MPO, DNA-MPO complexes) and hemoglobin were quantified. To investigate the presence of DNA from periodontal bacteria in atherosclerotic plaque, PCR analysis using specific primers was performed.

Our preliminary results indicate an association between neutrophil activation and intraplaque hemorrhages, reflected by the release of MPO (p<0,01) and MPO-DNA complexes (p<0,05). Presence of DNA from periodontitis-associated bacteria was found in 32/41 (78%) atheromatous plaque samples. More specifically, DNA from Pg, Tf, Pi, Aa was found in 46%, 24%, 34% and 68% of the samples, respectively. Hemoglobin levels were higher in conditioned media in carotid samples where the bacteria were found, but this was not statistically significant.

Our data confirm the relationship between intraplaque hemorrhage and neutrophil activation. In addition, the presence of periodontal bacteria DNA in carotid atheromatous plaque, may contribute to this activation. Further analysis is needed to fully explore the raw data and specimens.

Expression of phosphate transporters in optimized cell culture models for dental cells biomineralization

Laure Merametdjian — 2016-06-28

Phosphate is a key component of dental mineral composition. The physiological role of membrane proteins of dental cells is suspected to be crucial for mineralization mechanisms. Contrary to published data related to calcium, data on regulation of phosphate flux through membrane of mineralizing cells are scarce. To address this lack of data, we studied the expression of six membranous phosphate transporters in two dental cell lines: a rat odontoblastic cell line (M2H4) and a mouse ameloblastic cell line (ALC) for which we optimized the mineralizing culture conditions.

Impact of three endocrine disruptors, Bisphenol A, Genistein and Vinclozolin on female rat enamel

Katia Jedeon — 2016-06-28

Concerns about the potential adverse effects of endocrine disruptors (EDs) have been increasing over the last three decades. Bisphenol A (BPA), genistein (G) and vinclozolin (V) are three widely used EDs sharing similar effects.
Since populations are exposed to many diverse EDs simultaneously, we demonstrated recently their impact alone or combined on male rat tooth enamel. The purpose of this study was therefore to assess their effects on female rat tooth enamel in order to understand why they are differentially sensitive. Rats were exposed daily in utero and after birth to low doses of EDs during the critical fetal and suckling periods when amelogenesis takes place. Enamel of rats exposed to EDs presented opaque areas of hypomineralization. The proportion of affected rats was the highest in the groups of rats treated with BPA alone and higher in males than in females (in all the groups). Comparison of enamel key gene expression levels showed modulations of Klk4 and Enamelin in males but no significant variations in females. These findings show that female rats are less affected than males by the three EDs chosen in this study and suggest that enamel hypomineralization may differ between males and females

Mineral density of hypomineralised and sound enamel

Elsa Garot — 2016-06-28

Molar Incisor Hypomineralisation (MIH) is a structural anomaly that affects the quality of tooth enamel and has important consequences for oral health. The developmentally hypomineralised enamel has normal thickness and can range in colour from white to yellow or brown. The purpose of the present study is to compare the mineral density of hypomineralised and normal enamel. The sample included eight MIH teeth from seven patients. MIH teeth were scanned using high resolution microtomography. Non-parametric statistical tests (Wilcoxon test for paired samples) were carried out. Hypomineralised enamel has decreased mineral density (mean 19%; p < 0.0001) compared to normal enamel. This weak enamel has implications in clinical management of MIH lesions.

A standardized procedure to obtain mesenchymal stem/stromal cells from minimally manipulated dental pulp and Wharton’s jelly samples

Maxime Ducret — 2016-06-28

Transplantation of mesenchymal stem/stromal cells (MSCs) has emerged as an effective method to treat diseased or damaged organs and tissues, and hundreds of clinical trials using MSCs are currently under way to demonstrate the validity of such a therapeutic approach. However, most MSCs used for clinical trials are prepared in research laboratories with insufficient manufacturing quality control.
In particular, laboratories lack standardized procedures for in vitro isolation of MSCs from tissue samples, resulting in heterogeneous populations of cells and variable experimental and clinical results. MSCs are now referred to as Human Cellular Tissue-based Products or Advanced Therapy Medicinal Products, and guidelines from the American Code of Federal Regulation of the Food and Drug Administration (21 CFR Part 1271) and from the European Medicines Agency (European Directive 1394/2007) define requirements for appropriate production of these cells. These guidelines, commonly called “Good Manufacturing Practices” (GMP), include recommendations about laboratory cell culture procedures to ensure optimal reproducibility, efficacy and safety of the final medicinal product. In particular, the Food and Drug Administration divides ex vivo cultured cells into “minimally” and “more than minimally” manipulated samples, in function of the use or not of procedures “that might alter the biological features of the cells”. Today, minimal manipulation conditions have not been defined for the collection and isolation of MSCs (Torre et al. 2015)(Ducret et al. 2015).
Most if not all culture protocols that have been reported so far are unsatisfactory, because of the use of xeno- or allogeneic cell culture media, enzymatic treatment and long-term cell amplification that are known to alter the quality of MSCs. The aim of this study was to describe a standardized procedure for recovering MSCs with minimal handling from two promising sources, the dental pulp (DP) and the Wharton’s jelly (WJ) of the umbilical cord. The quality and homogeneity of the expanded cell populations were assessed by using flow cytometry with criteria that go beyond the International Society of Cellular Therapy (ISCT) guidelines for MSC characterization.