14 research outputs found

    Identification of the neutralizing epitopes of Merkel cell polyomavirus major capsid protein within the BC and EF surface loops.

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    Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops

    The corepressors GPS2 and SMRT control enhancer and silencer remodeling via eRNA transcription during inflammatory activation of macrophages

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    International audienceWhile the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates in vivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases

    MCPyV VP1 insertional mutants BC, DE, EF and HI.

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    <p><b>A)</b> MCPyV surface exposed loops model generated using the MKT21 sequence with the VP1 structure information of MCPyV w162 strain (4FMG pdb file) by Swiss-Model. The StreptagII motif (WSHPQFEK) coding sequence was inserted into each predicted surface exposed loop, after S88 of BC, after H150 of DE, after T189 of EF and after T296 of HI to generate four insertional mutants, BC, DE, EF and HI, respectively. <b>B)</b> MCPyV VP1 mutant particles observed by transmission electron microscopy after recombinant baculovirus expression and CsCl gradient purification.</p

    ELISA reactivity of MCPyV monoclonal antibodies against MCPyV insertional mutants BC, DE, EF and HI.

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    <p>In order to characterize the epitopes of the MCPyV VP1, the reactivity of mAbs was analyzed using the MCPyV wt VP1 VLPs and the four mutants with insertion within the BC, DE, EF and HI VP1 loops. ELISAs were performed using hybridoma culture supernatants diluted 1:3. The results are presented as relative binding defined as the reactivity of mAb to mutant VLPs divided by the reactivity of the same mAb observed with wild-type VLPs. The data presented are the means of three determinations (+/- SEM).</p

    Monoclonal antibodies neutralization mechanism after pre (black) or post (white) MCPyV pseudovirions attachment.

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    <p>For the detection of neutralizing antibodies, COS-7 cells (10<sup>4</sup>/well) and MCPyV luciferase pseudovirions (0.2 RLU) were used. For the pre attachment determination, pseudovirions were mixed with monoclonal antibodies supernatants diluted 1:3 during 1 h and then added to the cells for 3 h at 37°C. The mixture was removed and 100 μl of DMEM-FCS were added. For investigation of post-attachment neutralization, pseudovirions were bound to cells for 1 h at 4°C. Unbound virions were removed and then antibodies diluted 1:3 were added during 1h. The antibodies were removed and 100 μl of DMEM-FCS were added. After incubation for 48 h at 37°C the luciferase activity was measure. The results were expressed as the percentage of inhibition of luciferase activity. The data presented are the means of three determinations performed in duplicate (+/- SEM).</p

    ELISA reactivity of 10 neutralizing anti-MCPyV positive human sera (MCC patients) against MCPyV insertional mutants BC, DE, EF and HI.

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    <p>In order to characterize the epitopes of the MCPyV VP1, the reactivity of the human sera was analyzed from ten patients using the MCPyV wt VP1 VLPs and the four mutants with insertion within the BC, DE, EF and HI VP1 loops. ELISAs were performed using neutralizing anti-MCPyV positive human sera diluted 1:1000. The results are presented as relative binding defined as the reactivity of human serum to mutant VLPs divided by the reactivity of the same human serum observed with wild-type VLPs. The data presented are the means of three determinations (+/- SEM).</p

    Blood Monocyte Phenotype Is A Marker of Cardiovascular Risk in Type 2 Diabetes

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    International audienceBACKGROUND: Diabetes is a major risk factor for atherosclerotic cardiovascular diseases with a 2-fold higher risk of cardiovascular events in people with diabetes compared with those without. Circulating monocytes are inflammatory effector cells involved in both type 2 diabetes (T2D) and atherogenesis. METHODS: We investigated the relationship between circulating monocytes and cardiovascular risk progression in people with T2D, using phenotypic, transcriptomic, and metabolomic analyses. cardiovascular risk progression was estimated with coronary artery calcium score in a cohort of 672 people with T2D. RESULTS: Coronary artery calcium score was positively correlated with blood monocyte count and frequency of the classical monocyte subtype. Unsupervised k-means clustering based on monocyte subtype profiles revealed 3 main endotypes of people with T2D at varying risk of cardiovascular events. These observations were confirmed in a validation cohort of 279 T2D participants. The predictive association between monocyte count and major adverse cardiovascular events was validated through an independent prospective cohort of 757 patients with T2D. Integration of monocyte transcriptome analyses and plasma metabolomes showed a disruption of mitochondrial pathways (tricarboxylic acid cycle, oxidative phosphorylation pathway) that underlined a proatherogenic phenotype. CONCLUSIONS: In this study, we provide evidence that frequency and monocyte phenotypic profile are closely linked to cardiovascular risk in patients with T2D. The assessment of monocyte frequency and count is a valuable predictive marker for risk of cardiovascular events in patients with T2D. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT0435386
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