Assessment of Three Mitochondrial Genes (16S, Cytb, CO1) for Identifying Species in the Praomyini Tribe (Rodentia: Muridae)

The Praomyini tribe is one of the most diverse and abundant groups of Old World rodents. Several species are known to be involved in crop damage and in the epidemiology of several human and cattle diseases. Due to the existence of sibling species their identification is often problematic. Thus an easy, fast and accurate species identification tool is needed for non-systematicians to correctly identify Praomyini species. In this study we compare the usefulness of three genes (16S, Cytb, CO1) for identifying species of this tribe. A total of 426 specimens representing 40 species (sampled across their geographical range) were sequenced for the three genes. Nearly all of the species included in our study are monophyletic in the neighbour joining trees. The degree of intra-specific variability tends to be lower than the divergence between species, but no barcoding gap is detected. The success rate of the statistical methods of species identification is excellent (up to 99% or 100% for statistical supervised classification methods as the k-Nearest Neighbour or Random Forest). The 16S gene is 2.5 less variable than the Cytb and CO1 genes. As a result its discriminatory power is smaller. To sum up, our results suggest that using DNA markers for identifying species in the Praomyini tribe is a largely valid approach, and that the CO1 and Cytb genes are better DNA markers than the 16S gene. Our results confirm the usefulness of statistical methods such as the Random Forest and the 1-NN methods to assign a sequence to a species, even when the number of species is relatively large. Based on our NJ trees and the distribution of all intraspecific and interspecific pairwise nucleotide distances, we highlight the presence of several potentially new species within the Praomyini tribe that should be subject to corroboration assessments

Molecular Sciences Tethering Innate Surface Receptors on Dendritic Cells: A New Avenue for Immune Tolerance Induction?

International audienceDendritic cells (DCs) play a key role in immunity and are highly potent at presenting antigens and orienting the immune response. Depending on the environmental signals, DCs could turn the immune response toward immunity or immune tolerance. Several subsets of DCs have been described, with each expressing various surface receptors and all participating in DC-associated immune functions according to their specific skills. DC subsets could also contribute to the vicious circle of inflammation in immune diseases and establishment of immune tolerance in cancer. They appear to be appropriate targets in the control of inflammatory diseases or regulation of autoimmune responses. For all these reasons, in situ DC targeting with therapeutic antibodies seems to be a suitable way of modulating the entire immune system. At present, the field of antibody-based therapies has mainly been developed in oncology, but it is undergoing remarkable expansion thanks to a wide variety of antibody formats and their related functions. Moreover, current knowledge of DC biology may open new avenues for targeting and modulating the different DC subsets. Based on an update of pathogen recognition receptor expression profiles in human DC subsets, this review evaluates the possibility of inducing tolerant DCs using antibody-based therapeutic agents

Monocytes differentiation upon treatment with a peptide corresponding to the C-terminus of activated T cell-expressed Tirc7 protein

International audienc

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

Infection of periodontal epithelial cells by EBV is associated with apoptotic cell death.

<p>TUNEL assay associated with IF LMP2 detection was used to identify apoptotic cells and EBV-infected cells in DS samples from 5 CP patients. (A) Representative double fluorescent staining with antibodies specific for LMP2 (upper right panel), TUNEL staining (below left panel), with the latter two merged (below right panel). The upper-left panel shows DAPI staining of nuclei in the same field (x20). (B) Quantitative evaluations of A. Cell counting of LMP2^pos (LMP2), TUNEL^pos (TUNEL) and double positive (LMP2 & TUNEL) pECs (n = 5). Data represent the frequency of positive pECs identified in each category. (C) Calculations from B showing the frequency of apoptotic pECs (TUNEL^pos) among EBV-infected pECs (LMP2) (left part) and the frequency of EBV-infected pECs (LMP2^pos) among apoptotic pECs (TUNEL) (right part). Mean and standard deviation is shown for each group.</p

Healthy donors and sample information.^a

a<p>Characteristics for the healthy donors (HDs) used in this study (n = 10).</p>b<p>For each patient, one sampling was performed in healthy gingival sulcus of the right mandibular molar. 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).</p>d<p>RNA samples used for EBNA1 and CCL20 gene expression analysis.</p>e<p>Overall mean values and standard deviations (SD).</p

EBV infection increases with disease severity.

<p>(A–B) EBER-ISH staining was performed on paired periodontal pocket DS and SS samples (n = 40) from CP patients (n = 20), and from HS samples from healthy gingival sulcus from HDs (n = 10). Two representative fields (x40) of EBER staining (EBER) is shown for (A) one selected CP patient and (B) for one selected HD. Negative controls (NEG) were processed using a random PNA probe, and for each individual, the same cell sampling was used for positive and negative staining. (C) EBER-ISH-based determination of the frequency of EBV-infected pECs (EBER^pos) in periodontal samples from healthy sites (HS), swallow sites (SS), and deep sites (DS). The graph (left part) shows the tendency curve of EBER^pos pECs and the clinical attachment level (CAL) in 40 paired-samples from 20 CP patients and 10 samples from healthy donors (same as in A and B). The dot-plot analysis (right part) shows the comparative analysis of the frequency of EBER^pos pECs in paired-samples (SS circles, DS triangles) collected from 20 CP patients (same samples as on graft). (D) The average levels of EBNA1 transcript were also compared between whole RNA from HS (n = 10) and 12 paired-samples (SS and DS, n = 6 for each) from 6 CP patients (same samples as in C). The 36B4 housekeeping gene was used for normalization, with results presented as relative to 36B4 using a LOG₁₀ scale. The p values were calculated using Wilcoxon signed-rank test.</p

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

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