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Favier B.,Institute of Emerging Diseases and Innovative Therapies iMETI | Charron D.,University Paris Diderot | Glotz D.,University Paris Diderot | Mooney N.,University Paris Diderot
Methods in Molecular Biology | Year: 2013

Rejection is still the major cause of allograft loss following organ transplantation and a more complete comprehension of the alloimmune response is required in order to develop new therapeutic approaches. Allogenicity is primarily generated by the expression of major histocompatibility complex (MHC) molecules in the donor organ. Graft microvascular endothelial cells express both HLA class I and class II molecules. They are the fi rst target of the allogeneic response because of their vascular localization and are also able to present antigen to recipient T cells. The endothelium can therefore be considered as both a stimulator of and a target for alloimmune responses and both aspects require further study. We have established a model of constitutive expression of HLA-DR by human microvascular endothelial cells following transduction with a lentiviral vector. This model was employed in a study demonstrating that endothelial cells can induce allogeneic expansion of regulatory and pro-in fl ammatory CD4+ T lymphocyte subsets. Because microvascular endothelial cells rapidly lose their expression of HLA-DR ex vivo, this experimental system of lentiviral-mediated expression of HLA-DR allows the study of alloantigen presentation without requiring addition of in fl ammatory cytokines and thus provides a model for the study of the intra-graft allogeneic CD4+ T cell response at the single-cell level. © Springer Science+Business Media, LLC 2013. Source


Deschaseaux F.,French National Center for Scientific Research | Deschaseaux F.,Center Atlantique | Gaillard J.,Center Atlantique | Gaillard J.,University of Tours | And 13 more authors.
FASEB Journal | Year: 2013

Bone-marrow mesenchymal stem cells (MSCs) are the origin of bone-forming cells with immunomodulation potential. HLA-G5 is among the generated immunosuppressive molecules. HLA-G proteins play a crucial role in promoting the acceptance of allografts. However, the mechanisms regulating the expression of HLA-G5 in human MSCs are unknown. We induced differentiation of MSCs and found that HLA-G5 was greatly up-regulated only in osteoblastic cells (+63% for mRNA). Growth plates and bone callus postfracture in adults showed that only bone-lining cells and mesenchymal progenitors were positive for HLA-G5. Use of gene silencing and dominant-negative factors revealed that HLA-G5 depends on the expression and function of the skeletogenesis master genes RUNX2 and DLX5. In addition, HLA-G5 could directly inhibit osteoclastogenesis by acting on monocytes through SHP1. However, in mature osteoblasts, the expression of HLA-G5 protein was greatly suppressed whereas the proosteoclastogenic factor, RANKL, was concomitantly increased. Down-regulation of HLA-G5 expression during the maturation of osteoblasts was due to binding of the repressor GLI3, a signal transducer of the Hedgehog pathway, to the GLI binding element within the HLA-G promoter. Our findings show that mesenchymal progenitors and osteoblastic cells specifically express HLA-G5 during osteogenesis, with a key role in bone homeostasis. © FASEB. Source


Grosselin J.,Institute of Emerging Diseases and Innovative Therapies iMETI | Grosselin J.,University Paris - Sud | Sii-Felice K.,Institute of Emerging Diseases and Innovative Therapies iMETI | Sii-Felice K.,University Paris - Sud | And 9 more authors.
Stem Cells | Year: 2013

Our understanding of system dynamics of mixed cell populations in whole organisms has benefited from the advent of individual cell marking by nonarrayed DNA barcodes subsequently analyzed by high-throughput DNA sequencing. However, key limitations include statistical biases compromising quantification and the lack of applicability to decon-volute individual cell fate in vivo after pooling single cells differentially exposed to different conditions ex vivo. Here, we have derived an arrayed lentiviral library of DNA barcodes and obtained a proof-of-concept of its resolving capacity by quantifying hematopoietic regeneration after engraftment of mice with genetically modified autologous cells. This method has helped clarify and bridge the seemingly opposed clonal-succession and continuous-recruitment models of he-matopoietic stem cell behavior and revealed that myeloid-lymphoid biases are common occurrences in steady-state hematopoiesis. Arrayed lentiviral barcoding should prove a versatile and powerful approach to deconvolute cell dynamics in vivo with applications in hematology, embryology, and cancer biology. © AlphaMed Press. Source


McDowell K.L.,U.S. Food and Drug Administration | Nag N.,U.S. Food and Drug Administration | Franco Z.,U.S. Food and Drug Administration | Bu M.,U.S. Food and Drug Administration | And 6 more authors.
Transfusion | Year: 2015

Background: Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative infection that can be transmitted by blood and blood products from donors in the latent phase of the disease. Currently, there is no validated antemortem vCJD blood screening test. Several blood tests are under development. Any useful test must be validated with disease-relevant blood reference panels. Study Design and Methods: To generate blood reference materials, we infected four cynomolgus macaques with macaque-adapted vCJD brain homogenates. Blood was collected throughout the preclinical and clinical phases of infection. In parallel, equivalent blood was collected from one uninfected macaque. For each blood collection, an aliquot was stored as whole blood and the remainder was separated into components. Aliquots of plasma from terminally ill macaques were assayed for the presence of PrPTSE with the protein misfolding cyclic amplification (PMCA) method. Infectivity of the macaque brain homogenate used to infect macaques was titrated in C57BL/6 and RIII J/S inbred wild-type mice. Results: We sampled blood 19 times from the inoculated monkeys at various stages of the disease over a period of 29 months, generating liters of vCJD-infected macaque blood. vCJD was confirmed in all inoculated macaques. After PMCA, PrPTSE was detected in plasma from infected monkeys, but not from uninfected animals. Both mouse models were more sensitive to infection with macaque-adapted vCJD agent than to primary human vCJD agent. Conclusion: The macaque vCJD blood panels generated in this study provide a unique resource to support vCJD assay development and to characterize vCJD infectivity in blood. © 2014, Wiley-Blackwell. All rights reserved. Source


Vieillard V.,Institute National Of La Sante | Vieillard V.,University Pierre and Marie Curie | Dereuddre-Bosquet N.,Institute of Emerging Diseases and Innovative Therapies iMETI | Dereuddre-Bosquet N.,University Paris - Sud | And 6 more authors.
Vaccine | Year: 2012

Background: The immunodeficiency defining AIDS results from a progressive decline in the number of CD4+ T cells. Although no single viral protein is likely to be the sole effector of immune impairment, the gp41 envelope protein is believed to contribute significantly to AIDS pathogenesis. We have shown that 3S, a unique motif of gp41, is highly conserved in HIV-1 strains and specifically induces NKp44L, a ligand of the natural cytotoxicity receptor NKp44, on CD4+ T cells, rendering them sensitive to NK lysis. We therefore hypothesized that a 3S/gp41 vaccine strategy designed to modulate the NK cell compartment might improve CD4+ T cell resistance, independently of any effect on viral load. Methods: Nine macaques were chronically infected with the SHIV163P3; four were then immunized with the 3S/gp41 peptide and five with the carrier alone. Frequency of CD4+ T cell subsets, proliferation, cell activation and apoptosis were analyzed in the periphery and the lymph nodes, spleen and rectum by flow cytometry. Results: The anti-3S antibodies prevented NKp44L expression on CD4+ T cells in vivo and subsequently preserved the CD4+ central memory T cells in 3S/gp41-vaccinated animals. They also limited the NK cytotoxic activity against autologous CD4+ T cells, the cell activation, the proliferation, and the apoptosis in peripheral blood and secondary lymphoid tissues remained intact. Conclusion: These data suggest a new paradigm for AIDS vaccine development, aimed at generating specific responses to protect a specific subset of CD4+ T cells from attack by activated NK cells. © 2012 Elsevier Ltd. Source

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