EBV infection is common in gingival epithelial cells of the periodontium and worsens during chronic periodontitis.

An amplifying role for oral epithelial cells (ECs) in Epstein-Barr Virus (EBV) infection has been postulated to explain oral viral shedding. However, while lytic or latent EBV infections of oro/nasopharyngeal ECs are commonly detected under pathological conditions, detection of EBV-infected ECs in healthy conditions is very rare. In this study, a simple non-surgical tissue sampling procedure was used to investigate EBV infection in the periodontal epithelium that surrounds and attaches teeth to the gingiva. Surprisingly, we observed that the gingival ECs of the periodontium (pECs) are commonly infected with EBV and may serve as an important oral reservoir of latently EBV-infected cells. We also found that the basal level of epithelial EBV-infection is significantly increased in chronic periodontitis, a common inflammatory disease that undermines the integrity of tooth-supporting tissues. Moreover, the level of EBV infection was found to correlate with disease severity. In inflamed tissues, EBV-infected pECs appear to be prone to apoptosis and to produce larger amounts of CCL20, a pivotal inflammatory chemokine that controls tissue infiltration by immune cells. Our discovery that the periodontal epithelium is a major site of latent EBV infection sheds a new light on EBV persistence in healthy carriers and on the role of this ubiquitous virus in periodontitis. Moreover, the identification of this easily accessible site of latent infection may encourage new approaches to investigate and monitor other EBV-associated disorders

Joint effect of smoking and NQO1 C609T polymorphism on undifferentiated nasopharyngeal carcinoma risk in a North African population.

International audienceBACKGROUND:Nasopharyngeal carcinoma (NPC) has a higher incidence in North Africa than in most parts of the world. In addition to environmental factors such as Epstein-Barr virus infection and chemical carcinogen exposure, genetic susceptibility has been reported to play a key role in the development of NPC. NAD(P)H: quinone oxidoreductase 1 is a cytosolic enzyme that protects cells from oxidative damage. A C to T transition at position 609 in the NQO1 gene (OMIM: 125860) has been shown to alter the enzymatic activity of the enzyme and has been associated with increased risk to several cancers. This study investigates for the first time the effect of this polymorphism on NPC susceptibility in a North African population.METHODS:The NQO1 C609T polymorphism was genotyped using PCR-RFLP in 392 NPC cases and 365 controls from Morocco, Algeria, and Tunisia.RESULTS:The allele frequencies and distributions of genotypes did not differ between cases and controls (p > 0.05). When stratifying according to smoking status, we observed two-fold higher NPC risk in ever-smokers carrying the CT or TT genotype. Multiple logistic regression analysis revealed that there was a significant interaction between T allele and smoking status (OR = 1.95, 95% CI = 1.20-3.19; interaction p = 0.007).CONCLUSION:In this North African population, the functional NQO1 polymorphism was associated with a significantly higher risk of NPC among smokers and did not affect the risk among nonsmokers

The Dynamic Change in Plasma Epstein–Barr Virus DNA Load over a Long-Term Follow-Up Period Predicts Prognosis in Nasopharyngeal Carcinoma

The current study was designed to investigate the changes in the circulating Epstein–Barr virus DNA load (EBV DNA) at various time points before and after treatment and its clinical significance in nasopharyngeal carcinoma (NPC). A total of 142 patients with NPC were prospectively enrolled in this study. The plasma EBV DNA concentration was measured before and after treatment using qPCR. The prognostic values of the EBV DNA load were analyzed using the Kaplan–Meier and Cox regression tests. Following multivariate analysis, our data showed that high pre-EBV DNA loads were associated with significantly poorer distant metastasis free survival (DMFS) and progression free survival (PFS); detectable end-EBV DNA loads were associated with significantly worse loco-regional recurrence free survival (LRRFS) and PFS, and the detecTable 6 months-post-EBV DNA loads were associated with significantly poorer overall survival (OS), DMFS and PFS (p < 0.05). Additionally, combining the pre-EBV DNA load and the stage of the disease, our results showed that patients at stage III-IVA with a low pre-EBV DNA load had similar survival rates as patients at stage II with a low or high pre-EBV DNA load, but had better survival rates than those at stage III-IVA with a high pre-EBV DNA load. Taken together, we showed that the change of the EBV DNA load measured at several time points was more valuable than at any single time point for predicting patients’ survival for NPC. Furthermore, combining the pre-EBV DNA load and the TNM classification could help to formulate an improved prognostic model for this cancer

EBER-ISH staining of liquid-based periodontal samples reveals EBV-Infected epithelial cells.

<p>(A) Representative MGG staining of cytospin cells collected from PP samples (n = 10). Large spread epithelial-like cells (EC), polymorphonuclear leucocytes (PMN), mononuclear cells (MNC) as well as traces of dental plaque (DP) are indicated with arrows. Size bar represents 15 µM. (B) Representative CK19 staining of pECs from 5 CP patients. Coverslips were processed for IHC with DAB chromogen staining, CK19 specific staining was assessed by comparing with background staining observed using non specific mouse IgGs (not shown). Size bar represents 15 µM. (C) Nuclear EBER-ISH staining in pECs and palECs. EBER-ISH was used to detect EBER in periodontal and palatal cells sampled from 3 patients with chronic periodontitis. Two representative fields (x20) of EBER staining (EBER) are shown for the same selected CP patient with pECs (left panel) and palECs (right panel).</p

Specific real-time RT-PCR quantification of EBV gene expression in periodontal material.

<p>Whole RNA was isolated from 12 periodontal paired-samples (PP) from 6 CP patients, 3 palatal sites from 3 additional CP patients (pal), 4 EBV-infected cell lines (B95-8, L591, LCL1, C666-1) and an EBV-negative cell line (HepG2). The average levels of EBNA1, EBNA2, LMP1, LMP2, and BZLF1 EBV transcripts in clinical samples (shown as mean and SD) were compared with the expression of these EBV-genes in the different cell lines. Values obtained with an EBV-negative cell line were used to determine background level (HDLM2). The average levels of the B-cell marker CD20 transcript were also compared between LCL1, pal, and PP samples. The 36B4 housekeeping gene was used for normalization, with results presented as relative to 36B4 using a LOG₁₀ scale. The results shown are from one representative experiment of two RT-PCR experiments performed, each in triplicate.</p

Additional file 1: Table S1. of No association between TGF-β1 polymorphisms and risk of nasopharyngeal carcinoma in a large North African case-control study

Association of TGF-β1 gene polymorphisms with TNM stage. (DOCX 21 kb

Production of the inflammatory chemokine CCL20 by EBV-infected periodontal epithelial cells.

<p>(A) Left panel shows representative CCL20 staining of pECs from 5 CP patients (DS samples) (HIS, x40) of EBV-infected cells (EBER-ISH) (solid arrows) and of EBV-negative pECs (dotted arrows). Right panel shows background staining observed using nonspecific goat IgGs. (B) Quantitation of (A) (n = 5). The frequency (%) of CCL20^pos pECs among EBV-infected pECs (EBER, left part) and of EBER^pos pECs among CCL20-producing pECs (CCL20, right part) is shown. Mean and standard deviations are shown for each group. (C) Real-time RT-PCR quantification of CCL20 transcripts in whole RNA isolated from 12-paired RNA samples from 6 CP patients (SS n = 6, DS n = 6) and 2 RNA samples from healthy donors. (D) Real-time RT-PCR quantification of EBNA1 and CCL20 transcripts in whole RNA isolated from CP patients (n = 8) and from healthy donors (n = 2). Graph shows the linear tendency curve of CCL20 related to EBNA1. Simple linear regression analysis showed a positive correlation. Data are representative of 2 independent experiments, each performed in triplicate. The 36B4 housekeeping gene was used for normalization. The results are presented as relative to 36B4 (×10⁻⁶).</p

Patients and sample information.^a

a<p>Characteristics for the main CP patient cohort used in this study (n = 20).</p>b<p>For each patient, paired-sampling was performed, and upper and lower values shown correspond to shallow and deep periodontal sites (SS and DS), respectively. Normal clinical attachment level and gingival index values are considered to be less than 3 mm and 1, respectively.</p>c<p>Frequency (%) of EBV-infected periodontal epithelial cells (pECs). The mean frequency (and standard deviation) of EBV-infected cells was 18.5% (+/−5.12) for SS, and 26.45% (+/−6.8) for DS.</p>d<p>RNA samples used for EBV and CCL20 gene expression analysis.</p>e<p>Samples used for TUNEL experiments.</p>f<p>Overall mean values and standard deviations (SD).</p