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Browsing ES- Artigos by Author "Barreto, Vasco M."
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- In Vivo Approaches Reveal a Key Role for DCs in CD4+ T Cell Activation and Parasite Clearance during the Acute Phase of Experimental Blood-Stage MalariaPublication . Borges da Silva, Henrique; Fonseca, Raíssa; Cassado, Alexandra dos Anjos; Machado de Salles, Érika; de Menezes, Maria Nogueira; Langhorne, Jean; Perez, Katia Regina; Cuccovia, Iolanda Midea; Ryffel, Bernhard; Barreto, Vasco M.; Marinho, Cláudio Romero Farias; Boscardin, Silvia Beatriz; Álvarez, José Maria; D’Império-Lima, Maria Regina; Tadokoro, Carlos EduardoDendritic cells (DCs) are phagocytes that are highly specialized for antigen presentation. Heterogeneous populations of macrophages and DCs form a phagocyte network inside the red pulp (RP) of the spleen, which is a major site for the control of blood-borne infections such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, limiting our knowledge regarding their protective role in malaria. Here, we used in vivo experimental approaches that enabled us to deplete or visualize DCs in order to clarify these issues. To elucidate the roles of DCs and marginal zone macrophages in the protection against blood-stage malaria, we infected DTx (diphtheria toxin)-treated C57BL/6.CD11c-DTR mice, as well as C57BL/6 mice treated with low doses of clodronate liposomes (ClLip), with Plasmodium chabaudi AS (Pc) parasites. The first evidence suggesting that DCs could contribute directly to parasite clearance was an early effect of the DTx treatment, but not of the ClLip treatment, in parasitemia control. DCs were also required for CD4+ T cell responses during infection. The phagocytosis of infected red blood cells (iRBCs) by splenic DCs was analyzed by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, at three distinct phases of Pc malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis when the parasitemia decline coincides with spleen closure. In vivo and ex vivo imaging of the spleen revealed that DCs actively phagocytize iRBCs and interact with CD4+ T cells both in T cell-rich areas and in the RP. Subcapsular RP DCs were highly efficient in the recognition and capture of iRBCs during pre-crisis, while complete DC maturation was only achieved during crisis. These findings indicate that, beyond their classical role in antigen presentation, DCs also contribute to the direct elimination of iRBCs during acute Plasmodium infection.
- Independent recruitment of Igh alleles in V(D)J recombinationPublication . Alves-Pereira, Clara F.; de Freitas, Raquel; Lopes, Telma; Gardner, Rui; Marta, Filipa; Vieira, Paulo; Barreto, Vasco M.How the vast majority of B cells express only one of the two alleles at their immunoglobulin loci remains a biological puzzle. Here, in mice reconstituted with a single haematopoietic stem cell, we demonstrate that each of the two immunoglobulin heavy chain (Igh) alleles has a similar probability to be the first to undergo V(H) to DJ(H) rearrangement. We also observe this similar probability in clones from multipotent and common lymphoid precursors. The extreme biases in the expression of the alleles that we find in more differentiated subsets are mostly due to constraints imposed by early rearrangements. Our data demonstrate that each of the two Igh alleles in a B cell behaves independently of the other, up to the moment when a successful rearrangement in one allele triggers a feedback mechanism that prevents further recombination.
- A Novel Quantitative Fluorescent Reporter Assay for RAG Targets and RAG ActivityPublication . Trancoso, Inês; Bonnet, Marie; Gardner, Rui; Carneiro, Jorge; Barreto, Vasco M.; Demengeot, Jocelyne; Sarmento, Leonor M.Recombination-Activating Genes (RAG) 1 and 2 form the site specific recombinase that mediates V(D)J recombination, a process of DNA editing required for lymphocyte development and responsible for their diverse repertoire of antigen receptors. Mistargeted RAG activity associates with genome alteration and is responsible for various lymphoid tumors. Moreover several non-lymphoid tumors express RAG ectopically. A practical and powerful tool to perform quantitative assessment of RAG activity and to score putative RAG-Recognition signal sequences (RSS) is required in the fields of immunology, oncology, gene therapy, and development. Here we report the detailed characterization of a novel fluorescence-based reporter of RAG activity, named GFPi, a tool that allows measuring recombination efficiency (RE) by simple flow cytometry analysis. GFPi can be produced both as a plasmid for transient transfection experiments in cell lines or as a retrovirus for stable integration in the genome, thus supporting ex vivo and in vivo studies. The GFPi assay faithfully quantified endogenous and ectopic RAG activity as tested in genetically modified fibroblasts, tumor derived cell lines, developing pre-B cells, and hematopoietic cells. The GFPi assay also successfully ranked the RE of various RSS pairs, including bona fide RSS associated with V(D)J segments, artificial consensus sequences modified or not at specific nucleotides known to affect their efficiencies, or cryptic RSS involved in RAG-dependent activation of oncogenes. Our work validates the GFPi reporter as a practical quantitative tool for the study of RAG activity and RSS efficiencies. It should turn useful for the study of RAG-mediated V(D)J and aberrant rearrangements, lineage commitment, and vertebrate evolution.