技术资料

Ewing sarcoma gene EWS encodes a putative RNA-binding protein with proposed roles in transcription and splicing,but its physiological role in vivo remains undefined. Here,we have generated Ews-deficient mice and demonstrated that EWS is required for the completion of B cell development and meiosis. Analysis of Ews(-/-) lymphocytes revealed a cell-autonomous defect in precursor B lymphocyte (pre-B lymphocyte) development. During meiosis,Ews-null spermatocytes were deficient in XY bivalent formation and showed reduced meiotic recombination,resulting in massive apoptosis and complete arrest in gamete maturation. Inactivation of Ews in mouse embryonic fibroblasts resulted in premature cellular senescence,and the mutant animals showed hypersensitivity to ionizing radiation. Finally,we showed that EWS interacts with lamin A/C and that loss of EWS results in a reduced lamin A/C expression. Our findings reveal essential functions for EWS in pre-B cell development and meiosis,with proposed roles in DNA pairing and recombination/repair mechanisms. Furthermore,we demonstrate a novel role of EWS in cellular senescence,possibly through its interaction and modulation of lamin A/C. View Publication

BACKGROUND The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated. RESULTS Exposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4),hepatocyte growth factor (HGF),insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4,followed by HGF,followed by HGF+ITS+dexamethasone),however,resembling the order of secretion during liver embryogenesis,induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation,as evidenced by acquisition of an epithelial morphology,chronological expression of hepatic proteins,including hepatocyte-nuclear factor (HNF)-3beta,alpha-fetoprotein (AFP),CK18,albumin (ALB),HNF1alpha,multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)alpha,and functional maturation,i.e. upregulated ALB secretion,urea production and inducible cytochrome P450 (CYP)-dependent activity. CONCLUSION hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells. View Publication

View Publication

Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response,as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells,we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast,rescue from AICD was not associated with a significant change of Bcl-2,an inhibitor of apoptosis induced by cell stress. Accordingly,MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis,a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall,MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state,providing a model of their activity inside the HSC niche. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Umbilical cord blood (UCB) has been used as a potential source of various kinds of stem cells,including hematopoietic stem cells,mesenchymal stem cells,and endothelial progenitor cells (EPCs),for a variety of cell therapies. Recently,EPCs were introduced for restoring vascularization in ischemic tissues. An appropriate procedure for isolating EPCs from UCB is a key issue for improving therapeutic efficacy and eliminating the unexpected expansion of nonessential cells. Here we report a novel method for isolating EPCs from UCB by a combination of negative immunoselection and cell culture techniques. In addition,we divided EPCs into 2 subpopulations according to the aldehyde dehydrogenase (ALDH) activity. We found that EPCs with low ALDH activity (Alde-Low) possess a greater ability to proliferate and migrate compared to those with high ALDH activity (Alde-High). Moreover,hypoxia-inducible factor proteins are up-regulated and VEGF,CXCR4,and GLUT-1 mRNAs are increased in Alde-Low EPCs under hypoxic conditions,while the response was not significant in Alde-High EPCs. In fact,the introduction of Alde-Low EPCs significantly reduced tissue damage in ischemia in a mouse flap model. Thus,the introduction of Alde-Low EPCs may be a potential strategy for inducing rapid neovascularization and subsequent regeneration of ischemic tissues. View Publication

Wortmannin (Wm),a steroid-like molecule of 428.4 Da,appears to be unstable in biological fluids (apparent chemical instability),yet it exhibits an antiproliferative activity in assays employing a 48 hr incubation period (prolonged bioactivity),a situation we refer to as the wortmannin paradox." Under physiological conditions View Publication

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling,we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C),R-848,or CpG),in the presence or absence of cytokine (TNF-alpha or IL-3),and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons,the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast,PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation,and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection. View Publication

Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer,including acute myeloid leukemia (AML),and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML,we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins,including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells,and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis,and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles. View Publication

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56(superbright)CD16(-)CD9(+)KIR(+) distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16(+)CD9(-) peripheral blood NK cells in medium containing TGFbeta1 resulted in a transition to CD16(-)CD9(+) NK cells resembling decidual NK cells. Decidual stromal cells,when isolated and cultured in vitro,were found to produce TGFbeta1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFbeta1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFbeta. In addition,Lin(-)CD34(+)CD45(+) hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor. View Publication

The kinase inhibitor imatinib mesylate targeting the oncoprotein Bcr-Abl has revolutionized the treatment of chronic myeloid leukemia (CML). However,even though imatinib successfully controls the leukemia in chronic phase,it seems not to be able to cure the disease,potentially necessitating lifelong treatment with the inhibitor under constant risk of relapse. On a molecular level,the cause of disease persistence is not well understood. Initial studies implied that innate features of primitive progenitor cancer stem cells may be responsible for the phenomenon. Here,we describe an assay using retroviral insertional mutagenesis (RIM) to identify genes contributing to disease persistence in vivo. We transplanted mice with bone marrow cells retrovirally infected with the Bcr-Abl oncogene and subsequently treated the animals with imatinib to select for leukemic cells in which the proviral integration had affected genes modulating the imatinib response. Southern blot analysis demonstrated clonal outgrowth of cells carrying similar integration sites. Candidate genes located near the proviral insertion sites were identified,among them the transcription factor RUNX3. Proviral integration near the RUNX3 promoter induced RUNX3 expression,and Bcr-Abl-positive cell lines with stable or inducible expression of RUNX1 or RUNX3 were protected from imatinib-induced apoptosis. Furthermore,imatinib treatment selected for RUNX1-expressing cells in vitro and in vivo after infection of primary bone marrow cells with Bcr-Abl and RUNX1. Our results demonstrate the utility of RIM for probing molecular modulators of targeted therapies and suggest a role for members of the RUNX transcription factor family in disease persistence in CML patients. View Publication

IL-12 is an immunoregulatory cytokine,which promotes Th1 cell differentiation and is a major inducer of IFN-gamma. IFN-beta,a Type I IFN used in the treatment of multiple sclerosis,has been shown to significantly increase the expression of the anti-inflammatory cytokine IL-10,a major suppressor of Th1 cytokines. The beneficial immunomodulatory effects of IFN-beta may in part be a result of its ability to suppress IL-12. However,IL-12 and IFN-beta signal via the STAT4 pathway. Our aim was to investigate the relationship between IL-12 and IFN-beta by observing the effect of prior exposure to IL-12 or IFN-beta on the ability of T cells to subsequently respond to the other cytokine. We report that IFN-beta increases IL-12-induced STAT4 phosphorylation and up-regulates IL-12 receptor beta1 and beta2 expression. However,despite this up-regulation,IFN-beta suppressed IL-12-induced IFN-gamma expression. Our results suggest that this may be a result of the parallel induction of IL-10 by IFN-beta. View Publication

Microbial products serving as superantigens (SAgs) have been implicated in triggering various T cell-mediated chronic inflammatory disorders,including severe asthma. Given earlier evidence demonstrating that airway smooth muscle (ASM) cells express MHC class II molecules,we investigated whether ASM can present SAg to resting CD4(+) T cells,and further examined whether this action reciprocally elicits proasthmatic changes in ASM responsiveness. Coincubation of CD4(+) T cells with human ASM cells pulsed with the SAg,staphylococcal enterotoxin A (SEA),elicited adherence and clustering of class II and CD3 molecules at the ASM/T cell interface,indicative of immunological synapse formation,in association with T cell activation. This ASM/T cell interaction evoked up-regulated mRNA expression and pronounced release of the Th2-type cytokine,IL-13,into the coculture medium,which was MHC class II dependent. Moreover,when administering the conditioned medium from the SEA-stimulated ASM/T cell cocultures to isolated naive rabbit ASM tissues,the latter exhibited proasthmatic-like changes in their constrictor and relaxation responsiveness that were prevented by pretreating the tissues with an anti-IL-13 neutralizing Ab. Collectively,these observations are the first to demonstrate that ASM can present SAg to CD4(+) T cells,and that this MHC class II-mediated cooperative ASM/T cell interaction elicits release of IL-13 that,in turn,evokes proasthmatic changes in ASM constrictor and relaxant responsiveness. Thus,a new immuno-regulatory role for ASM is identified that potentially contributes to the pathogenesis of nonallergic (intrinsic) asthma and,accordingly,may underlie the reported association between microbial SAg exposure,T cell activation,and severe asthma. View Publication