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Hematopoietic stem cells (HSCs) are defined by their ability to repopulate all of the hematopoietic lineages in vivo and sustain the production of these cells for the life span of the individual. In the absence of reliable direct markers for HSCs,their identification and enumeration depends on functional long-term,multilineage,in vivo repopulation assays. The extremely low frequency of HSCs in any tissue and the absence of a specific HSC phenotype have made their purification and characterization a highly challenging goal. HSCs and primitive hematopoietic cells can be distinguished from mature blood cells by their lack of lineage-specific markers and presence of certain other cell-surface antigens,such as CD133 (for human cells) and c-kit and Sca-1 (for murine cells). Functional analyses of purified subpopulations of primitive hematopoietic cells have led to the development of several procedures for isolating cell populations that are highly enriched in cells with in vivo stem cell activity. Simplified methods for obtaining these cells at high yield have been important to the practical exploitation of such advances. This article reviews recent progress in identifying human and mouse HSCs and current techniques for their purification. View Publication

We used primary peripheral blood T cells,a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously,to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly,T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that,in the face of chronic nicotine exposure,selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases. View Publication

NK cells protect hosts against viral pathogens and transformed cells,and dendritic cells (DCs) play important roles in activating NK cells. We now find that murine IL-15Ralpha-deficient DCs fail to support NK cell cytolytic activity and elaboration of IFN-gamma,despite the fact that these DCs express normal levels of costimulatory molecules and IL-12. By contrast,IL-15Ralpha expression on NK cells is entirely dispensable for their activation by DCs. In addition,blockade with anti-IL-15Ralpha and anti-IL-2Rbeta but not anti-IL-2Ralpha-specific Abs prevents NK cell activation by wild-type DCs. Finally,presentation of IL-15 by purified IL-15Ralpha/Fc in trans synergizes with IL-12 to support NK cell priming. These findings suggest that murine DCs require IL-15Ralpha to present IL-15 in trans to NK cells during NK cell priming. View Publication

Hematopoietic progenitor/stem cell homing to the bone marrow requires the concerted action of several adhesion molecules. Endothelial P- and E-selectins play an important role in this process,but their ligands on a large subset of neonate-derived human CD34+ cells are absent,leading to a reduced ability to interact with the bone marrow (BM) microvasculature. We report here that this deficiency results from reduced alpha1,3-fucosyltransferase (FucT) expression and activity in these CD34+ cells. Incubation of CD34+ cells with recombinant human FucTVI rapidly corrected the deficiency in nonbinding CD34+ cells and further increased the density of ligands for both P- and E-selectins on all cord blood-derived CD34+ cells. Intravital microscopy studies revealed that these FucTVI-treated CD34+ cells displayed a marked enhancement in their initial interactions with the BM microvasculature,but unexpectedly,homing into the BM was not improved by FucTVI treatment. These data indicate that,although exogenous FucT enzyme activity can rapidly modulate selectin binding avidity of cord blood CD34+ cells,further studies are needed to understand how to translate a positive effect on progenitor cell adhesion in bone marrow microvessels into one that significantly influences migration and lodgement into the parenchyma. View Publication

Hereditary hemochromatosis (HH),an iron overload disease associated with mutations in the HFE gene,is characterized by increased intestinal iron absorption and consequent deposition of excess iron,primarily in the liver. Patients with HH and Hfe-deficient (Hfe-/-) mice manifest inappropriate expression of the iron absorption regulator hepcidin,a peptide hormone produced by the liver in response to iron loading. In this study,we investigated the contribution of Hfe expression in macrophages to the regulation of liver hepcidin levels and iron loading. We used bone marrow transplantation to generate wild-type (wt) and Hfe-/- mice chimeric for macrophage Hfe gene expression. Reconstitution of Hfe-deficient mice with wt bone marrow resulted in augmented capacity of the spleen to store iron and in significantly decreased liver iron loading,accompanied by a significant increase of hepatic hepcidin mRNA levels. Conversely,wt mice reconstituted with Hfe-deficient bone marrow had a diminished capacity to store iron in the spleen but no significant alterations of liver iron stores or hepcidin mRNA levels. Our results suggest that macrophage Hfe participates in the regulation of splenic and liver iron concentrations and liver hepcidin expression. View Publication

PURPOSE: The propensity of tumor cells to escape immune elimination could limit,if not defeat,the long-term benefits of effective immunotherapeutic protocols. Immunologic targeting of tumor stroma could significantly reduce the ability of tumors to evade immune elimination. Murine studies have shown that inducing immunity against angiogenesis-associated products engenders potent antitumor immunity without significant pathology. It is,however,not known whether T cells corresponding to stromal products are present in humans. In this study,we describe a method to screen for human stromal products that have not triggered significant tolerance and could therefore serve as candidate antigens for cancer immunotherapy. EXPERIMENTAL DESIGN: To identify candidates for human stromal antigens,we used an in vitro-screening method to determine whether dendritic cells transfected with mRNA encoding products,which are overexpressed in the tumor stroma,are capable of stimulating cytotoxic CD8(+) (CTL) responses from human peripheral blood mononuclear cells. RESULTS: CTL responses could be consistently generated against fibroblast activation protein (FAP) but not against matrix metalloproteinase-9 (MMP-9) or MMP-14. To enhance the immunogenicity of the mRNA-translated FAP product,a lysosomal targeting signal derived from lysosome-associated membrane protein-1 (LAMP-1) was fused to the COOH terminus of FAP to redirect the translated product into the class II presentation pathway. Dendritic cells transfected with mRNA encoding the FAP-LAMP fusion product stimulated enhanced CD4(+) and CD8(+) T-cell responses. CONCLUSION: This study identifies FAP,a protease preferentially expressed in tumor-associated fibroblasts,as a candidate human stromal antigen to target in the setting of cancer immunotherapy,and shows that differential expression of stromal products is not a sufficient criteria to indicate its immunogenicity in a vaccination setting. View Publication

Transplantation-associated stress can compromise the hematopoietic potential of hematopoietic stem cells (HSCs). As a consequence,HSCs may undergo exhaustion" in serial transplant recipients� View Publication

To evaluate the role of the TCR in the alphabeta/gammadelta lineage choice during human thymocyte development,molecular analyses of the TCRbeta locus in gammadelta cells and the TCRgamma and delta loci in alphabeta cells were undertaken. TCRbeta variable gene segments remained largely in germline configuration in gammadelta cells,indicating that commitment to the gammadelta lineage occurred before complete TCRbeta rearrangements in most cases. The few TCRbeta rearrangements detected were primarily out-of-frame,suggesting that productive TCRbeta rearrangements diverted cells away from the gammadelta lineage. In contrast,in alphabeta cells,the TCRgamma locus was almost completely rearranged with a random productivity profile; the TCRdelta locus contained primarily nonproductive rearrangements. Productive gamma rearrangements were,however,depleted compared with preselected cells. Productive TCRgamma and delta rearrangements rarely occurred in the same cell,suggesting that alphabeta cells developed from cells unable to produce a functional gammadelta TCR. Intracellular TCRbeta expression correlated with the up-regulation of CD4 and concomitant down-regulation of CD34,and plateaued at the early double positive stage. Surprisingly,however,some early double positive thymocytes retained gammadelta potential in culture. We present a model for human thymopoiesis which includes gammadelta development as a default pathway,an instructional role for the TCR in the alphabeta/gammadelta lineage choice,and a prolonged developmental window for beta selection and gammadelta lineage commitment. Aspects that differ from the mouse are the status of TCR gene rearrangements at the nonexpressed loci,the timing of beta selection,and maintenance of gammadelta potential through the early double positive stage of development. View Publication

Mice lacking CD137L (4-1BBL) show normal primary expansion and contraction of the CD8+ T cell response to influenza virus,but exhibit a defect in Ag-specific CD8+ T cell numbers at 3-6 wk postinfection. Previous results showed that the decrease in CD8+ T cell numbers in this model is not due to a programming defect during primary expansion. Thus,it appears that 4-1BB/4-1BBL interactions control the number of surviving CD8+ effector memory cells,late in the primary response. In this report,we asked how 4-1BB on T cells could play a role after Ag has apparently been cleared from the host. We show that IL-15,a cytokine involved in regulation of CD8+ memory T cell survival,induces the expression of 4-1BB on CD8+CD44(high) memory phenotype T cells,but not on CD4+ T cells. The Ag-independent induction of 4-1BB by IL-15 was dependent on MAPK p38 and ERK activation. Transfer of in vitro-generated OT-I CD8+ memory T cells into unimmunized wild-type or 4-1BBL-deficient hosts revealed a 2- to 3-fold survival advantage when 4-1BBL was present,recapitulating the effect seen in the endogenous response to influenza in mice. Decreases in the overall number of memory CD8+ T cells were also observed in the bone marrow of unmanipulated 4-1BBL-deficient mice. These data suggest a model whereby 4-1BB expression on memory CD8+ T cells,perhaps due to encounter with IL-15 in the bone marrow,allows 4-1BB/4-1BBL interactions to maintain memory CD8 T cell survival in the absence of Ag. View Publication

Current strategies for the production of therapeutic mAbs include the use of mammalian cell systems to recombinantly produce Abs derived from mice bearing human Ig transgenes,humanization of rodent Abs,or phage libraries. Generation of hybridomas secreting human mAbs has been previously reported; however,this approach has not been fully exploited for immunotherapy development. We previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g.,affinity and titers) of mAb-producing cell lines,including hybridomas. We reasoned that this process,named morphogenics,could be used to improve suboptimal hybridoma cells generated by means of ex vivo immunization and immortalization of antigen-specific human B cells for therapeutic Ab development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human mAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralizing activity against human granulocyte-macrophage colony-stimulating factor was identified that is now considered for preclinical development for autoimmune disease indications. Moreover,these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large-scale manufacturing. View Publication

The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However,the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study,we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow,in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells,NK progenitors,and NKG2A-negative NK cells),and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore,we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally,we show that acetylated histones are associated with the CpG-rich region in NKG2A positive,but not negative,cell lines,and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription,but cotreatment with both drugs resulted in a significantly greater induction,suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells. View Publication

The interleukin-3 receptor (IL-3R) subunits are overexpressed on acute myeloid leukemia (AML) blasts compared with normal hematopoietic cells and are thus potential targets for novel therapeutic agents. Both fluorescence-activated cell sorter (FACS) analysis and quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) were used to quantify expression of the IL-3Ralpha and beta(c) subunits on AML cells. QRT-PCR for both subunits was most predictive of killing of AML colony-forming cells (AML-CFCs) by diphtheria toxin-IL-3 fusion protein (DT(388)IL3). Among 19 patient samples,the relative level of the IL-3Ralpha was higher than the IL-3Rbeta(c) and highest in CD34(+)CD38(-)CD71(-) cells,enriched for candidate leukemia stem cells,compared with cell fractions depleted of such progenitors. Overall,the amount of IL-3Rbeta(c) subunit did not vary among sorted subpopulations. However,expression of both subunits varied by more than 10-fold among different AML samples for all subpopulations studied. The level of IL-3Rbeta(c) expression versus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (set at 1000) ranged from 0.14 to 13.56 in CD34(+)CD38(-)CD71(-) cells from different samples; this value was correlated (r = .76,P = .05) with the ability of DT(388)IL3 to kill AML progenitors that engraft in beta(2)-microglobin-deficient nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (n = 7). Thus,quantification of IL-3R subunit expression on AML blasts predicts the effectiveness IL-3R-targeted therapy in killing primitive leukemic progenitors. View Publication