[Influence of epigenetic in Sjögren's syndrome]. / Épigénome et syndrome de Gougerot-Sjögren.

Sjögren's syndrome (SS) is a systemic autoimmune epithelitis with a major female incidence, and characterized by a dry syndrome, impaired quality of life, visceral involvement, and lymphoma for the most aggressive cases. During this process, epithelial cells acquire the capacity to produce cytokines, chemokines, and autoantigens which can in turn be presented to the immune system. Consequently, this epithelitis is accompanied by lymphocytic infiltrations leading to the formation of pseudo-follicles in which self-reactive B lymphocytes are present. The recent integration of genomic and especially of epigenomic data, which make it possible to analyze the different cellular partners, opens new perspectives and allows to a better understanding of this complex and still incurable disease.

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

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.

Defective DNA methylation in salivary gland epithelial acini from patients with Sjögren's syndrome is associated with SSB gene expression, anti-SSB/LA detection, and lymphocyte infiltration.

The pathogenesis of primary Sjögren's syndrome (pSS) is complex, in part due to DNA methylation abnormalities. This study was undertaken to evaluate the importance of global DNA methylation ((5m)C) as determined in minor salivary glands (MSG) from well characterized pSS patients. Twenty-two pSS patients and ten controls were selected, and MSG were stained with anti-(5m)C, anti-(5m)C/anti-cytokeratin (KRT)19, or with anti-SSB/La antibodies (Ab). The DNA methylation status at the SSB gene promoter P1 and P1' was evaluated by methylation-sensitive restriction enzymes (MSRE) coupled with PCR. The effect of the DNA demethylating drug 5 azacytidine (5-Aza) was tested in the human salivary gland (HSG) cell line. In pSS, the reduction of global DNA methylation ((5m)C) was associated with lymphocyte infiltration, the emergence of (5m)C(low) and KRT19(high) acini, and the detection of circulating anti-SSB/La Ab, but not with disease activity (ESSDAI). Next, treating HSG cells with 5-Aza was effective in inducing SSB expression. Finally in pSS patients positive for anti-SSB/La Ab, we further observed DNA demethylation at the SSB gene promoter P1 with consequent SSB overexpression at both the transcriptional and protein levels in salivary gland epithelial cells. In conclusion, our results highlight the importance of DNA methylation in the pathophysiology of pSS and to the emergence of anti-SSB/La Ab.

Identification of genes regulating TRAIL-induced apoptosis in rheumatoid arthritis fibroblasts-like synoviocytes.

We previously described that sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis varied in rheumatoid arthritis fibroblasts-like synoviocytes (RAFLS) from one patient to another and was correlated with disease severity. Therefore, we screened for genes differentially expressed in RAFLS sensitive and resistant to TRAIL-induced apoptosis. The sensitivity of RAFLS was defined based on the percentage of TRAIL-induced apoptosis: 0-10% for resistant cells and >25% for sensitive RAFLS. We performed transcriptomic comparison between RAFLS-S (n=6) and RAFLS-R (n=6) and then examined the implication of identified candidates in the regulation of apoptosis using small interference RNA (siRNA). Microarray analysis revealed 10 functional genes differentially expressed according to TRAIL sensitivity. These factors are implicated in different functions, such as the respiratory chain (ND3), the transport of lipids (OSBP2, PLTP), the regulation of signaling linked to extracellular factors (SULF2, GALNT1, SIAE) or the regulation of gene expression (TET2 and LARP6). We confirmed differential expression for GALNT1 and LARP6 by quantitative reverse transcriptase-PCR. Using siRNA extinction, we demonstrated the implication of GALNT1, SULF2 and LARP6 in the control of TRAIL-induced responses. These results are of particular interest as GALNT1 and LARP6 have been implicated in the regulation of cell death and may represent interesting targets to induce apoptosis of RAFLS.

Social immunity in honeybees (Apis mellifera): transcriptome analysis of varroa-hygienic behaviour.

Honeybees have evolved a social immunity consisting of the cooperation of individuals to decrease disease in the hive. We identified a set of genes involved in this social immunity by analysing the brain transcriptome of highly varroa-hygienic bees, who efficiently detect and remove brood infected with the Varroa destructor mite. The function of these candidate genes does not seem to support a higher olfactory sensitivity in hygienic bees, as previously hypothesized. However, comparing their genomic profile with those from other behaviours suggests a link with brood care and the highly varroa-hygienic Africanized honeybees. These results represent a first step toward the identification of genes involved in social immunity and thus provide first insights into the evolution of social immunity.

Sensitivity of N170 and late positive components to social categorization and emotional valence.

Previous experiments have shown that categorization of people into two distinct fictive groups has an impact on cognitive processes. The main objective of the present study was to examine whether this mere categorization improves information processing speed and alters early and late onset ERPs during a social judgment task. In a group membership situation, in-group evaluation enhanced information processing speed and occipito-temporal N170 amplitudes, associated with orthographic processing, compared to out-group evaluation, more so for positive than negative attributes. Moreover, negative adjectives elicited larger N170 amplitudes and faster information processing speed than positive adjectives. In contrast, positive adjectives in a non-membership context enhanced a late positive component in prefrontal regions. These results reflect the existence of a motivational top-down influence due to social categorization in early perceptual stages of word processing. These findings are also in accord with the existence of two distinct systems of evaluation, the first implicating an automatic processing represented in occipito-temporal neocortex and the other a more controlled processing represented in PFC.

Characterization of the human CD5 endogenous retrovirus-E in B lymphocytes.

All T lymphocytes and some B lymphocytes express CD5. This coreceptor is encoded by one gene that consists of 11 exons. We have previously described a B cell-specific alternative exon 1, leading to the synthesis of a protein, devoid of leader peptide, and, therefore, retained in the cytoplasm. The novel exon 1 originates from a human endogenous retrovirus (HERV) at a time interval between the divergence of New World monkeys from Old World monkeys, and prior to the divergence of humans from Old World monkeys. Based on sequence similarity to gamma-retroviruses, it was categorized as class I: based on the specificity of its primer binding site, it was allotted to the subclass E, and based on its location within the cd5 gene, named HERV-E.CD5. Alternative transcripts were detected in lymphoid organs including fetal liver (not adult liver), more particularly in CD5-negative cell surface B-1b than in CD5-positive cell surface B-1a, and not at all in B-2 cells. By alignment of 5' long terminal repeats, HERV-E.CD5 was distinguished from similar proviruses. This could be central to the regulation of membrane expression of CD5 in human B lymphocytes, and, thereby, to the strength of the B-cell antigen receptor signaling.

Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: identification, characterization and immunolocalization of a putative taproot storage glycoprotein.

In taproot of oilseed rape (Brassica napus L.), a 23 kDa polypeptide has been recently identified as a putative vegetative storage protein (VSP) because of its accumulation during flowering and its specific mobilization to sustain grain filling when N uptake is strongly reduced. The objectives were to characterize this protein more precisely and to study the effect of environmental factors (N availability, daylength, temperature, water deficit, wounding) or endogenous signals (methyl jasmonate, abscisic acid) that might change the N source/sink relationships within the plant, and may therefore trigger its accumulation. The 23 kDa putative VSP has two isoforms, is glycosylated and both isoforms share the same N-terminal sequence which had been used to produce specific polyclonal antibodies. Low levels of an immunoreactive protein of 24 kDa were found in leaves and flowers. In taproot, the 23 kDa putative VSP seems to accumulate only in the vacuoles of peripheral cortical parenchyma cells, around the phloem vessels. Among all treatments tested, the accumulation of this protein could only be induced by abscisic acid and methyl jasmonate. When compared to control plants, application of methyl jasmonate reduced N uptake by 89% after 15 d, induced a strong remobilization of N from senescing leaves and a concomitant accumulation of the 23 kDa putative VSP. These results suggested that, in rape, the 23 kDa protein is used as a storage buffer between N losses from senescing leaves promoted by methyl jasmonate and grain filling.

Cloning of a genetically unstable cytochrome P-450 gene cluster involved in degradation of the pollutant ethyl tert-butyl ether by Rhodococcus ruber.

Rhodococcus ruber (formerly Gordonia terrae) IFP 2001 is one of a few bacterial strains able to degrade ethyl tert-butyl ether (ETBE), which is a major pollutant from gasoline. This strain was found to undergo a spontaneous 14.3-kbp chromosomal deletion, which results in the loss of the ability to degrade ETBE. Sequence analysis of the region corresponding to the deletion revealed the presence of a gene cluster, ethABCD, encoding a ferredoxin reductase, a cytochrome P-450, a ferredoxin, and a 10-kDa protein of unknown function, respectively. The EthB and EthD proteins could be easily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were induced by ETBE in the wild-type strain. Upstream of ethABCD lies ethR, which codes for a putative positive transcriptional regulator of the AraC/XylS family. Transformation of the ETBE-negative mutant by a plasmid carrying the ethRABCD genes restored the ability to degrade ETBE. Complementation was abolished if the plasmid carried ethRABC only. The eth genes are located in a DNA fragment flanked by two identical direct repeats of 5.6 kbp. The ETBE-negative mutants carry a single copy of this 5.6-kbp repeat, suggesting that the 14.3-kbp chromosomal deletion resulted from a recombination between the two identical sequences. The 5.6-kbp repeat is a class II transposon carrying a TnpA transposase, a truncated form of the recombinase TnpR, and a terminal inverted repeat of 38 bp. The truncated TnpR is encoded by an IS3-interrupted tnpR gene.

Genomic sequence and transcriptional analysis of a 23-kilobase mycobacterial linear plasmid: evidence for horizontal transfer and identification of plasmid maintenance systems.

Linear plasmids were unknown in mycobacteria until recently. Here, we report the complete nucleotide sequence of 23-kb linear plasmid pCLP from Mycobacterium celatum, an opportunistic pathogen. The sequence of pCLP revealed at least 19 putative open reading frames (ORFs). Expression of pCLP genes in exponential-phase cultures was determined by reverse transcriptase PCR (RT-PCR). Twelve ORFs were expressed, whereas no transcription of the 7 other ORFs of pCLP was detected. Five of the 12 transcribed ORFs detected by RT-PCR are of unknown function. Sequence analysis revealed similar loci in both M. celatum pCLP and the Mycobacterium tuberculosis chromosome, including transposase-related sequences. This result suggests horizontal transfer between these two organisms. pCLP also contains ORFs that are similar to genes of bacterial circular plasmids involved in partition (par operon) and postsegregational (pem operon) mechanisms. Functional analysis of these ORFs suggests that they probably carry out similar maintenance roles in pCLP.

Impaired endothelium-dependent relaxation by adrenomedullin in monocrotaline-treated rat arteries.

The effects of adrenomedullin were evaluated in isolated vascular rings from rats treated with monocrotaline (60 mg/kg, s.c.) causing pulmonary hypertension and right ventricular hypertrophy within 3 to 4 weeks. Sham animals (NaCl-treated rats) were used for comparison. The relaxing effects of adrenomedullin (10(-8) M) and acetylcholine (10(-6) M) were determined in thoracic aorta and pulmonary artery rings precontracted with phenylephrine (10(-7) M). In sham animals, adrenomedullin caused significant vasorelaxation of aorta and pulmonary artery although of different amplitude (24 +/- 3% and 40 +/- 2%, respectively). A greater relaxation was observed in response to acetylcholine. Monocrotaline-treated rats exhibited a reduction in adrenomedullin relaxation in pulmonary artery (54 and 68% loss of effect, at 3 and 4 weeks, respectively, P < 0.01 vs. sham) and comparable reductions in acetylcholine responses. The decrease in adrenomedullin relaxing effect was less pronounced in aorta than in pulmonary artery, suggesting a distinct tissue sensitivity to monocrotaline. In contrast, the relaxing effect of acetylcholine on aorta was decreased at 4 weeks (36% reduction, P < 0.01 vs. sham). In this model, the adrenomedullin-induced relaxation of the pulmonary artery was impaired due to a severe endothelial dysfunction which may contribute partly to the evolving pathophysiological process.