Valérie Lapierre

Updated Technology to Produce Highly Immunogenic Dendritic Cell-derived Exosomes of Clinical Grade: A Critical Role of Interferon-γ

Dendritic cell-derived exosomes (Dex) are nanovesicles bearing major histocompatibility complexes promoting T-cell−dependent antitumor effects in mice. Two phase I clinical trials aimed at vaccinating cancer patients with peptide-pulsed Dex have shown the feasibility and safety of inoculating clinical-grade Dex, but have failed to show their immunizing capacity. These low immunogenic capacities have led us to develop second-generation Dex with enhanced immunostimulatory properties. Here, we show that interferon-&ggr; is a key cytokine conditioning the dendritic cell to induce the expression of CD40, CD80, CD86, and CD54 on Dex, endowing them with direct and potent peptide-dependent CD8+ T-cell-triggering potential in vitro and in vivo. In this study, we describe the clinical grade process to manufacture large-scale interferon-&ggr;-Dex vaccines and their quality control parameters currently used in a phase II trial.

Occurrence and severity of adverse events after autologous hematopoietic progenitor cell infusion are related to the amount of granulocytes in the apheresis product

BACKGROUND: Adverse events (AEs) after hematopoietic progenitor cell (HPC) infusion are rare but might be life‐threatening. These reactions have traditionally been associated with the amount of infused cryoprotectant, but persistence of such events after dimethyl sulfoxide (DMSO) depletion has questioned this assumption.

Immune modulation and microchimerism after unmodified versus leukoreduced allogeneic red blood cell transfusion in cancer patients: results of a randomized study

BACKGROUND: Transfusion of red blood cells (RBCs) has been associated with immunomodulatory effects. Persistence of donor cells in the recipient may be contributive.

Enhanced activation of B cells in a granulocyte colony-stimulating factor-mobilized peripheral blood stem cell graft

In a randomized study that compared human leucocyte antigen‐identical allogeneic granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood stem cell (PBSC) versus bone marrow (BM) transplantation, the expression of activation markers, CD23, CD25 and CD45RO by B cells, was compared in blood before and after G‐CSF mobilization and in PBSC versus BM grafts. The fractions of CD23+ and CD25+ B cells were higher in PBSC than in BM grafts. Moreover, we observed a G‐CSF‐induced increase in B‐cell fractions in blood as well as in PBSC grafts when compared with BM grafts. Such an enhanced B‐cell activation could contribute to the accelerated kinetics of immuno‐haematological reconstitution, the occurrence of acute haemolysis in the ABO minor incompatibility setting, as well as the increased incidence of chronic graft‐versus‐host disease observed after PBSC transplantation.

Uncoupling of the Hippo and Rho pathways allows megakaryocytes to escape the tetraploid checkpoint.

Megakaryocytes are naturally polyploid cells that increase their ploidy by endomitosis. However, very little is known regarding the mechanism by which they escape the tetraploid checkpoint to become polyploid. Recently, it has been shown that the tetraploid checkpoint was regulated by the Hippo-p53 pathway in response to a downregulation of Rho activity. We therefore analyzed the role of Hippo-p53 pathway in the regulation of human megakaryocyte polyploidy. Our results revealed that Hippo-p53 signaling pathway proteins are present and are functional in megakaryocytes. Although this pathway responds to the genotoxic stress agent etoposide, it is not activated in tetraploid or polyploid megakaryocytes. Furthermore, Hippo pathway was observed to be uncoupled from Rho activity. Additionally, polyploid megakaryocytes showed increased expression of YAP target genes when compared to diploid and tetraploid megakaryocytes. Although p53 knockdown increased both modal ploidy and proplatelet formation in megakaryocytes, YAP knockdown caused no significant change in ploidy while moderately affecting proplatelet formation. Interestingly, YAP knockdown reduced the mitochondrial mass in polyploid megakaryocytes and decreased expression of PGC1α, an important mitochondrial biogenesis regulator. Thus, the Hippo pathway is functional in megakaryocytes, but is not induced by tetraploidy. Additionally, YAP regulates the mitochondrial mass in polyploid megakaryocytes.

Bacterial contamination of a cornea tissue bank: implications for the safety of graft engineering.

Purpose. To analyze the difficulties involved in managing an episode of bacterial contamination in a cornea bank. We describe (1) the circumstances of bacterial contamination discovery, (2) the methods used to investigate the outbreak, (3) the corrective measures adopted, and (4) the method introduced to improve the reaction capacity in case of bacterial contamination. Methods. All the samples collected were cultured in an attempt to identify the environmental reservoir of the contaminated epidemic clone. Bacteria were identified by Gram stain, oxidase test, and biochemical characteristics. The clonality of the strains was assessed by pulsed-field gel electrophoresis. Results. The bacterial contamination was confirmed for 28 corneas, and 70 additional corneas were discarded. The source of the contamination was identified 17 days after the beginning of the episode. It consisted of a clonal bacterial strain that was found in trypan blue, the dye, used to examine all the tissues. The contaminating bacterium was Burkholderia cepacia, a well-known nosocomial pathogen. A total of 169 grafted corneas had been checked with the contaminated reagent. No cases of post–graft infection were recorded. Conclusion. Trypan blue played a major role in this outbreak. The mode and chronology of contamination remain unresolved. This exceptional event emphasizes the risk of bacterial contamination in tissue/cell banks, the necessity to improve methods for its prevention, and procedures to limit its consequences.

Activity of nonmuscle myosin II isoforms determines localization at the cleavage furrow of megakaryocytes

Megakaryocyte polyploidy is characterized by cytokinesis failure resulting from defects in contractile forces at the cleavage furrow. Although immature megakaryocytes express 2 nonmuscle myosin II isoforms (MYH9 [NMIIA] and MYH10 [NMIIB]), only NMIIB localizes at the cleavage furrow, and its subsequent absence contributes to polyploidy. In this study, we tried to understand why the abundant NMIIA does not localize at the furrow by focusing on the RhoA/ROCK pathway that has a low activity in polyploid megakaryocytes. We observed that under low RhoA activity, NMII isoforms presented different activity that determined their localization. Inhibition of RhoA/ROCK signaling abolished the localization of NMIIB, whereas constitutively active RhoA induced NMIIA at the cleavage furrow. Thus, although high RhoA activity favored the localization of both the isoforms, only NMIIB could localize at the furrow at low RhoA activity. This was further confirmed in erythroblasts that have a higher basal RhoA activity than megakaryocytes and express both NMIIA and NMIIB at the cleavage furrow. Decreased RhoA activity in erythroblasts abolished localization of NMIIA but not of NMIIB from the furrow. This differential localization was related to differences in actin turnover. Megakaryocytes had a higher actin turnover compared with erythroblasts. Strikingly, inhibition of actin polymerization was found to be sufficient to recapitulate the effects of inhibition of RhoA/ROCK pathway on NMII isoform localization; thus, cytokinesis failure in megakaryocytes is the consequence of both the absence of NMIIB and a low RhoA activity that impairs NMIIA localization at the cleavage furrow through increased actin turnover.

Contrôle ultime pré-transfusionnel : évaluation de sept dispositifs

The transfusion unit of the Institut Gustave Roussy has tested seven pre-transfusion ABO control devices registered at the Agence francaise de securite sanitaire et des produits de sante. Determination of the optimal plan to replace the existing plan in our institution was the primary objective of this study. A significant heterogeneity was observed among tested devices. None of the tested plans fulfilled all the desired quality criteria.

Implementation of a specific approval process for blood-components prescription

We implemented a systematic computer-assisted validation process for transfusion prescriptions to improve transfusion safety. Assessment of this new approach indicates good adoption of validated transfusion guidelines and a reduction of exposure to blood products and overall costs.

Critical role of the HDAC6–cortactin axis in human megakaryocyte maturation leading to a proplatelet-formation defect

Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6–CTTN axis as a positive regulator of human but not mouse MK maturation.Histone deacetylase (HDAC) inhibitors, a class of cancer therapeutics, cause thrombocytopenia via an unknown mechanism. Here, the authors show that HDAC6 inhibition impairs proplatelet formation in human megakaryocytes, and show that this is linked to hyperacetylation of the actin-binding protein cortactin.