Development of a Specific Anti-capsid Antibody- and Magnetic Bead-Based Immunoassay to Detect Human Norovirus Particles in Stool Samples and Spiked Mussels via Flow Cytometry.

peer reviewedHuman noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 10(5) gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples

Unraveling clonal CD8 T cell expansion and identification of essential factors in γ-herpesvirus-induced lymphomagenesis

Alcelaphine gammaherpesvirus 1 (AlHV-1) asymptomatically persists in its natural host, the wildebeest. However, cross-species transmission to cattle results in the induction of an acute and lethal peripheral T cell lymphoma-like disease (PTCL), named malignant catarrhal fever (MCF). Our previous findings demonstrated an essential role for viral genome maintenance in infected CD8+ T lymphocytes but the exact mechanism(s) leading to lymphoproliferation and MCF remained unknown. To decipher how AlHV-1 dysregulates T lymphocytes, we first examined the global phenotypic changes in circulating CD8+ T cells after experimental infection of calves. T cell receptor repertoire together with transcriptomics and epigenomics analyses demonstrated an oligoclonal expansion of infected CD8+ T cells displaying effector and exhaustion gene signatures, including GZMA, GNLY, PD-1, and TOX2 expression. Then, among viral genes expressed in infected CD8+ T cells, we uncovered A10 that encodes a transmembrane signaling protein displaying multiple tyrosine residues, with predicted ITAM and SH3 motifs. Impaired A10 expression did not affect AlHV-1 replication in vitro but rendered AlHV-1 unable to induce MCF. Furthermore, A10 was phosphorylated in T lymphocytes in vitro and affected T cell signaling. Finally, while AlHV-1 mutants expressing mutated forms of A10 devoid of ITAM or SH3 motifs (or both) were able to induce MCF, a recombinant virus expressing a mutated form of A10 unable to phosphorylate its tyrosine residues resulted in the lack of MCF and protected against a wild-type virus challenge. Thus, we could characterize the nature of this γ-herpesvirus-induced PTCL-like disease and identify an essential mechanism explaining its development.</p

Dre-miR-2188 Targets Nrp2a and Mediates Proper Intersegmental Vessel Development in Zebrafish Embryos

BACKGROUND: MicroRNAs (miRNAs) are a class of small RNAs that are implicated in the control of eukaryotic gene expression by binding to the 3'UTR of target mRNAs. Several algorithms have been developed for miRNA target prediction however, experimental validation is still essential for the correct identification of miRNA targets. We have recently predicted that Neuropilin2a (Nrp2a), a vascular endothelial growth factor receptor which is essential for normal developmental angiogenesis in zebrafish, is a dre-miR-2188 target. METHODOLOGY: Here we show that dre-miR-2188 targets the 3'-untranslated region (3'UTR) of Nrp2a mRNA and is implicated in proper intersegmental vessel development in vivo. Over expression of miR-2188 in zebrafish embryos down regulates Nrp2a expression and results in intersegmental vessel disruption, while its silencing increases Nrp2a expression and intersegmental vessel sprouting. An in vivo GFP sensor assay based on a fusion between the GFP coding region and the Nrp2a 3'UTR confirms that miR-2188 binds to the 3'UTR of Nrp2a and inhibits protein translation. CONCLUSIONS: We demonstrate that miR-2188 targets Nrp2a and affects intersegmental vessel development in zebrafish embryos

Tumor microenvironment affects the composition of endothelial-derived exosomes: impact in tumor progression

The tumor microenvironment plays a crucial role in the progression of tumor growth and metastasis by deregulating various physiological processes including angiogenesis and inflammation. Several studies have previously demonstrated that tumor-derived exosomes are actively involved in the mediation of tumorigenesis by “reprogramming” target cells (e.g. endothelial cells (ECs)) through transfer of pro-angiogenic microRNAs. But the function of exosomes released by target cells is poorly studied. Consequently, we sought to determine the composition of exosomes released by ECs under tumor microenvironment, and to assess whether these vesicles present different functional properties. Using RNA-seq approaches, we demonstrated that exosomes released by ECs in tumor microenvironment context present a specific repertory of microRNAs associated to tumor angiogenesis and inflammatory pathways. Furthermore, we showed that these vesicles were able to optimize inflammatory response of immune cells by transferring several dysregulated microRNAs. Currently, we are identifying the molecular pathways modulated by endothelial-derived exosomes in tumor microenvironment

Tumor microenvironment affects the composition of endothelial-derived extracellular vesicles: Impact in tumor progression

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Sputum exosomes - promising biomarkers for idiopathic pulmonary fibrosis

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease of unknown etiology which leads rapidly to death. As diagnosis of IPF is complex, the development of novel molecular biomarkers is a central challenge for the future of translational research. Consequently, we sought to characterize microRNA (miR) content of exosomes from sputum of IPF patients compared to healthy donors in order to identify novel biomarkers of the disease. Methods: Exosomes were isolated from induced sputum samples of 14 IPF patients diagnosed following American Thoracic Society (ATS) /European Respiratory Society (ERS) recommendations and 11 healthy donors with standard ultracentrifugation protocol. Exosomal miR content was analysed by miR qPCR arrays, and diseases/biological processes associated to altered miRs were determined by bioinformatic analysis. Results: The presence of exosomes was confirmed in sputum from both IPF patients and healthy donors. The profiling of exosomal miRs revealed 21 differentially expressed miRs in the sputum of IPF patients compared to healthy donors. Further validation of miRs presenting an aberrant expression allowed us to identify for the first time an IPF-specific miR signature from sputum exosomes, among which miR-142-3p and miR-33a-5p present an upregulation (fold change (FC)>3, p < 0.01), whereas let-7d-5p a downregulation (FC < 0.5, p < 0.01). The bioinformatic analysis revealed that altered miRs are associated to inflammatory diseases, among which IPF is the most relevant one (p = 3.78E-10). Interestingly, most of the biological processes highlighted in this analysis are in agreement with IPF etiology, which confers to our candidates an evident role as IPF biomarkers. Based on these findings, functional tests with IPF-sputum exosomes and mimics of altered miRs are underway to test their impact on IPF progression. Summary/Conclusion: For the first time, we identified potential biomarkers for IPF from sputum exosomes. Our findings may thus lead to a better understanding about the roles of these miRs in the pathogenesis of IPF and thus open new avenues for therapeutic approaches. This study reinforced the concept that sputum exosomes might be a novel source of biomarkers for the diagnosis of pulmonary diseases

Extracellular Vesicles Mediate Communication between Endothelial and Vascular Smooth Muscle Cells.

The recruitment of pericytes and vascular smooth muscle cells (SMCs) that enwrap endothelial cells (ECs) is a crucial process for vascular maturation and stabilization. Communication between these two cell types is crucial during vascular development and in maintaining vessel homeostasis. Extracellular vesicles (EVs) have emerged as a new communication tool involving the exchange of microRNAs between cells. In the present study, we searched for microRNAs that could be transferred via EVs from ECs to SMCs and vice versa. Thanks to a microRNA profiling experiment, we found that two microRNAs are more exported in each cell type in coculture experiments: while miR-539 is more secreted by ECs, miR-582 is more present in EVs from SMCs. Functional assays revealed that both microRNAs can modulate both cell-type phenotypes. We further identified miR-539 and miR-582 targets, in agreement with their respective cell functions. The results obtained in vivo in the neovascularization model suggest that miR-539 and miR-582 might cooperate to trigger the process of blood vessel coverage by smooth muscle cells in a mature plexus. Taken together, these results are the first to highlight the role of miR-539 and miR-582 in angiogenesis and communication between ECs and SMCs