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EBV infects and immortalizes B cells in vitro and in vivo. It is the causative agent of most immune deficiency–related lymphoproliferative disorders and is associated with various lymphomas. EBV latency III–transformed B cells are known to express two immunosuppressive molecules, IL-10 and PD-L1, two characteristics of regulatory B cells (Bregs). In this study, we show that, in addition to secretion of the Breg immunosuppressive cytokines IL-10, IL-35, and TGF-β1, EBV latency III–transformed B cells were able to repress proliferation of their autologous T cells preactivated by CD2, CD3, and CD28. This inhibitory effect was likely caused by CD4+ T cells because EBV latency III–transformed B cells induced a strong proliferation of isolated autologous CD8 T cells. Indeed, EBV was able to promote expansion of autologous FOXP3+ CD39high CTLA4+, Helios+, GITR+, LAG3+ CD4 T cells (i.e., regulatory T cells [Tregs]). Two types of Tregs were induced: unconventional CD25neg and conventional CD25pos Tregs. These Tregs expressed both the latency-associated peptide (LAP) and the PD-1 receptor, two markers of functional Tregs. Expansion of both Treg subtypes depended on PD-L1, whose expression was under the control of LMP1, the main EBV oncogene. These results demonstrate that, like Bregs, EBV latency III–transformed B cells exhibit strong immunoregulatory properties. These data provide clues to the understanding of how after EBV primo-infection, EBV-proliferating B cells can survive in an aggressive immunological environment and later emerge to give rise to EBV-associated B cell lymphomas such as in elderly patients.

Designing multitarget drugs have raised considerable interest due to their advantages in the treatment of complex diseases such as cancer. Their design constitutes a challenge in antitumor drug discovery. The present study reports a dual inhibition of tubulin polymerization and HDAC activity. On the basis of 1,1-diarylethylenes ( isoCA-4) and belinostat, a series of hybrid molecules was successfully designed and synthesized. In particular compounds, 5f and 5h were proven to be potent inhibitors of both tubulin polymerization and HDAC8 leading to excellent antiproliferative activity.

International audience; Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

EBV-immortalized B cells induce a complex immune response such that the virus persists as a clinically silent infection for the lifetime of the infected host. B7-H1, also called PD-L1, is a cosignaling molecule of the B7 family that can inhibit activated T cell effectors by interaction with its receptor PD-1. In this work, we have studied the dependence of B7-H1 on NF-κB and c-Myc, the two main transcription factors in EBV latency III proliferating B cells, on various lymphoblastoid and Burkitt lymphoma cell lines, some of them being inducible or not for the EBV latency III program and/or for c-Myc. We found that B7-H1 repressed killing of EBV-immortalized B cells by their autologous T and NK cells. At the mRNA level, NF-κB was a weak inducer whereas c-Myc was a strong repressor of B7-H1 expression, an effect mediated by STAT1 inhibition. At the protein level, B7-H1 molecules were stored in both degradative and unconventional secretory lysosomes. Surface membrane B7-H1 molecules were constitutively internalized and proteolyzed in lysosomes. The EBV latency III program increased the amounts of B7-H1–containing secretory lysosomes and their export to the surface membrane. By repressing actin polymerization, c-Myc blocked secretory lysosome migration and B7-H1 surface membrane export. In addition to B7-H1, various immunoregulatory molecules participating in the immunological synapse are stored in secretory lysosomes. By playing on actin polymerization, c-Myc could thus globally regulate the immunogenicity of transformed B cells, acting on export of secretory lysosomes to plasma membrane.

International audience; Fluorescent labeled monoclonal antibodies (mAbs) against CD36 are routinely used as monocyte, erythroid, or platelet markers in clinical cytometry. CD36 has recently been proposed by various authors as a valuable marker helping to enumerate leukocyte's subpopulations by flow cytometry. However, it is known that binding of CD36 may induce platelet activation and formation of platelet's rosettes on leukocytes, resulting in false expression of platelet markers on white blood cells. To study this phenomenon, we have combined classical flow cytometry and a new quantitative flow imaging technique with the ImageStream(®) analyzer. We show that CD36 ligation induces activation of platelets with CD62 expression and their adhesion on leukocytes due to CD62 and CD162 interactions. Preincubation of whole blood samples with either anti-CD62 or anti-CD162 antibodies could prevent formation of these rosettes. Our approach also emphasizes the fact that immunomorphological analysis of cell events with ImageStream technology is a useful tool to validate the specificity of marker's labeling or to elucidate incoherent results obtained with classical flow cytometry. We thus propose to prevent false platelet labeling on leukocytes by preincubation with either anti-CD62 or anti-CD162 antibodies when using CD36 mAbs.

International audience; B-cell fate and responses are modulated by soluble mediators and direct cellular interactions. Migration properties also vary during differentiation, commitment and activation. In many cells, modulation of responses to stimuli involves cell surface glycans, whose architecture depends on the simultaneous expression of multiple enzymes. By looking at the glycosylation-related gene expression patterns among B cell populations, we determined in this study that the strongest variations were observed for CSGalNAcT-1 and EXTL1. These are enzymes involved in the biosynthesis of alternative forms of glycosaminoglycans, namely chondroitin sulphate and heparan sulphate respectively. These two enzymes showed inverse fluctuations in progenitors, resting B cells and activated B cells, suggesting a developmentally regulated switch between chondroitin and heparan sulphate synthesis. To explore whether these variations contributed to optimal B cell differentiation, we over-expressed EXTL1 in the B-cell lineage of transgenic mice, yielding a partial differentiation blockade at the pro-B to pre-B transition. In the periphery, this defect was almost fully compensated for in vivo, with normal-size B-cell compartments and normal serum immunoglobulin levels in the transgenic EXTL1 mice. The peripheral B cells from EXTL1 transgenics were only affected with regard to their in vitro responses to polyclonal activation, showing reduced proliferation. Together the data suggest that, despite their low amounts in lymphocytes, the heparan sulphate chains decorating the endogenous glycosaminoglycans appear to be regulators of B-cell physiology.

International audience; A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrPC, into a pathogenic isoform, PrPSc. The molecular requirements for efficient PrP conversion remain unknown. Altered glycosylation has been linked to various pathologies and the N-glycans harbored by two prion protein isoforms are different. In order to search for glycosylation-related genes that could mark prion infection, we used a glycosylation-dedicated microarray that allowed the simultaneous analysis of the expression of 165 glycosylation-related genes encoding proteins of the glycosyltransferase, glycosidase, lectin, and sulfotransferase families to compare the gene expression profiles of normal and scrapie-infected mouse brain and spleen. Eight genes were found upregulated in "scrapie brain" at the final state of the disease. In the spleen, five genes presented a modified expression. Three genes were also upregulated in the spleen of infected mice, and two (Pigq and St3gal5) downregulated. All changes were confirmed by qPCR and biochemical analyses applied to Pigq and St3gal5 proteins.

The Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1) is thought to act as the major transforming protein in various cell types, by rerouting the tumor necrosis factor receptor family signaling pathway. Despite this implication in EBV-associated transformation of cells, LMP1 toxicity is a well-known but poorly studied feature, perhaps because it contradicts its role in transformation. We show that LMP1 physiological levels are very heterogeneous and that the highest levels of LMP1 correlate with Fas overexpression and spontaneous apoptosis in lymphoblastoid cell lines (LCLs). To understand the cytotoxic effect of LMP1 in LCLs, we cloned wild-type LMP1 into a doxycycline double-inducible episomal vector pRT-1, with a truncated version of NGFR as a surrogate marker of inducibility. We found that LMP1 overexpression induced apoptosis in LCL B cells, as shown by annexin V labeling, sub-G1peak, and poly(ADP ribose) polymerase cleavage. Knocking down Fas expression by small interfering RNA abolished LMP1-induced apoptosis. The absence of detectable levels of Fas ligand mRNA suggested a ligand-independent activation of Fas. LMP1 induced Fas overexpression with its relocalization in lipid raft microdomains of the membrane. Fas immunoprecipitation detected FADD (Fas-associated death domain protein) and caspase 8, suggesting a Fas-dependent formation of the death-inducing signaling complex. Caspases 8, 9, 3, and 7 were activated by LMP1. Caspase 8 activation was associated with BID cleavage and truncated-BID mitochondrial relocalization, consistent with type II apoptosis. Therefore, our results are in agreement with a model where LMP1-dependent NF-κB activation induces Fas overexpression and autoactivation that could overwhelm the antiapoptotic effect of NF-κB, revealing an ambivalent function of LMP1 in cell survival and programmed cell death.

Ursolic acid (UA) is a pentacyclic triterpenoid compound which exists widely in nature and is known to have a pleitropic biological activity profile. For the last few decades, extensive work has been carried out to establish its biological activities and pharmacological actions. It is described as a promising chemopreventive agent with an antiproliferative effect on cancer cells that stems from its ability to induce apoptosis. We investigated and compared the role played by mitochondria during the apoptotic process induced by UA in human HaCaT-derived keratinotic cells and M4Beu human melanoma cells. In both cell lines, UA induced significant caspase-3 activation, the downstream central effector of apoptosis. Subsequent JC-1/TOTO-3 double staining clearly demonstrated that UA induces strong mitochondrial-transmembrane potential collapse in M4Beu cells, while mitochondria from HaCaT-treated cells remain largely unstimulated. This was confirmed by Western blot analysis, which revealed a Bax/Bcl-2-balance change in favor of Bax, the proapoptotic member, in UA-treated M4Beu cells. It can be concluded that UA induces apoptosis in M4Beu through the mitochondrial pathway, while other mechanisms are activated in the case of HaCaT cells.

It is now well-established that P-glycoprotein 170 (P-gp), an efflux pump involved in multidrug resistance (MDR) is overexpressed at the plasma membrane of doxorubicin-resistant K562 leukemia cells. Nevertheless, several results suggested: (i) that P-gp-mediated drug efflux was not the only mechanism involved in resistance; (ii) that intracellular compartments could accumulate the drug, preventing it from reaching its nuclear targets; (iii) that agents able to reverse multidrug resistance may lead to intracellular drug redistribution. We have studied the localization of P-gp in mitochondria as well as its functional properties in this compartment. Using several monoclonal antibodies (MoAbs) directed against different P-gp epitopes, a protein was detected in the cytoplasm of two doxorubicin-resistant K562 sublines and, by confocal laser scanning microscopy, this protein was shown to co-localize in the Golgi apparatus and in mitochondria, in equivalent proportions. Purified mitochondria were isolated from K562 cell variants; the presence of a protein of about 170 kDa and reacting with several anti-P-gp antibodies was assessed in MDR cells by Western blotting and flow cytometry. Functional assays have shown that mitochondrial P-gp was involved in doxorubicin accumulation inside the organelle but not in its efflux, suggesting an orientation of P-gp in the mitochondrial membrane inverse to that observed in the plasma membrane. A potential role for mitochondrial P-gp in MDR cells would be to protect the nucleus from doxorubicin. This is the first demonstration of the presence and functional activity of P-gp in mitochondria of MDR cells.

Background: Age-related changes in the antibody response have been classically associated with alterations in T-cell help, but increasing evidence shows that intrinsic B-cell defects exist. This article analyzes the apoptotic susceptibility of peripheral B-cells in aged and young control mice. Materials and Methods: Freshly isolated lymphocytes from spleen and Peyer's patches (PPs) were labeled for B-cell lineage (B220+ cells) and germinal center B subset (GCs, B220+/PNA+ cells). Alternatively, splenic B-cells purified by MACS were used. Apoptosis was monitored by the Annexin V binding, incorporation of 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)), propidium iodide (PI) staining, and morphological changes. Moreover, intracellular Bcl-2 expression and Bad phosphorylation status were also analyzed in B-cells. Results: We showed in aging mice an enhanced Annexin V+/PI− cell percentage in splenic B-lymphocytes, which was correlated with a lower ΔΨm. By contrast, no change in apoptosis was observed in compartments known to be enriched in activated B-cells (GCs and PPs). Analysis of Bcl-2 levels revealed no modification. When using B-cells purified by MACS, we strongly confirm data obtained on staining cells. Moreover, enhanced spontaneous apoptosis of splenic B-cells in aged mice was found to be correlated with a reduced phosphorylated Bad expression. Conclusion: Increased apoptosis of resting B-cells in old mice may be determined by an altered Bad phosphorylation, which in turn contributes to cell death by lowering the mitochondrial threshold for apoptosis. © 2006 International Society for Analytical Cytology

hal-01211869; International audience; Calpastatin is a specific endogenous protein inhibitor of the ubiquitous calcium dependent proteinases mu- and m-calpain. The calpain-calpastatin system is involved in various physiological and pathological processes. In the present study, we determined the bovine calpastatin gene structure and demonstrated that four promoters direct its expression. The gene harbours 35 exons spanning at least 130kb on genomic DNA. Its structure is similar to that of mouse, pig, and human gene. Transient transfection assays in both C2C12 and COS7 cell lines demonstrated that the putative promoter regions situated 5' to exon 1xa, 1xb, 1u, and 14t were functional. We also established that the region situated upstream exon 14t is subjected to a tissue specific regulation. The implication of numerous high-scoring cis acting transcriptional motifs which are present in these regions will need to be determined. The existence of four promoters suggests differential expression patterns which must have a physiological significance.

Ursolic acid (UA) is a pentacyclic triterpene compound isolated from many kinds of medicinal plants and present in human diet. In this study, we investigated the pro-apoptotic effect of UA on HaCat derived keratinocyte cell line. Treatment with UA decreased the viability of HaCat cells in a concentration- and time-dependent manner. In addition, cell cycle analysis revealed that UA treated HaCat cells were blocked predominantly in G1 phase. Moreover, expression of p21WAF1, a cell cycle regulator, was increased by UA, indicating that UA-induced cell cycle arrest could be mediated through p21WAF1. During UA treatment, we also demonstrated that p53 was phosphorylated at serine 392 and translocated to the nucleus. It is well established that p53 achieves its tumor suppressor activity by inducing apoptosis on cells. To define the apoptotic process in our system, we examined effect of UA on caspase activities, and demonstrated caspase-3 activation. In conclusion, our results suggest that UA induces: i) cell cycle arrest concomitantly with the apparition of the apoptotic sub group G1 peak, and ii) cell death through apoptosis, which is mediated by caspase-3.

Eighteen generations of divergent selection for residual feed intake have been completed in two Rhode Island Red lines of domestic fowl. The high intake R+ line and the low intake R- line cocks used to sire Generation 19 of the selection experiment have been compared for associated responses on fertility, hatching, and sperm quality. Evaluations of sperm samples were based on volume, cell concentration, biochemical parameters (pH, uric acid and protein concentrations), and motility and morphology of spermatozoa. Finally, individual spermatozoa were analyzed by flow-cytometry (FCM) using Rhodamine 123 (Rh123) and nonyl-acrydine-orange (NAO) specific fluorochromes to assess, respectively, overall mitochondrial activity and overall mitochondrial content. Hatchability of incubated eggs was 20 points higher for the R- line, mainly because unfertilized eggs were only 6 vs 30% in the R+ line. Early embryo mortality was also twice as high in the R+ line (21%). The ratio of Rh123 to NAO fluorescence was identical for both lines. This result suggests that there was no difference in the energy producing potential of the individual mitochondria. Therefore, the difference seen for both dyes between the two lines might be attributed to a difference in the quantity of mitochondrial inner membranes present in the cell (with 17% less for the R+ line). In the R+ line, the poor performance at fertilization and during early embryonic development was associated with lower production of motile spermatozoa, possibly in relation to a lower quantity of mitochondria in spermatozoa from R+ cocks. Although the female contribution to the differences between lines was not explored separately, results suggest that selection for residual feed intake may have altered some cellular function related to the production of energy in the R+ line.