技术资料

Murine hematopoietic stem and progenitor cells (HSPCs) home to bone marrow in part by rolling on P-selectin and E-selectin expressed on endothelial cells. Human adult CD34(+) cells,which are enriched in HSPCs,roll on endothelial selectins in bone marrow vessels of nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice. Many human umbilical cord blood (CB) CD34(+) cells do not roll in these vessels,in part because of an uncharacterized defect in binding to P-selectin. Selectin ligands must be alpha1-3 fucosylated to form glycan determinants such as sialyl Lewis x (sLe(x)). We found that inadequate alpha1-3 fucosylation of CB CD34(+) cells,particularly CD34(+)CD38(-/low) cells that are highly enriched in HSPCs,caused them to bind poorly to E-selectin as well as to P-selectin. Treatment of CB CD34(+) cells with guanosine diphosphate (GDP) fucose and exogenous alpha1-3 fucosyltransferase VI increased cell-surface sLe(x) determinants,augmented binding to fluid-phase P- and E-selectin,and improved cell rolling on P- and E-selectin under flow. Similar treatment of CB mononuclear cells enhanced engraftment of human hematopoietic cells in bone marrows of irradiated NOD/SCID mice. These observations suggest that alpha1-3 fucosylation of CB cells might be a simple and effective method to improve hematopoietic cell homing to and engraftment in bone marrows of patients receiving CB transplants. 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

Cell enrichment methods that deal with larger volumes of peripheral blood and BM are needed for increased sensitivity of detection,characterization and quantification of isolated tumor cells (ITC). This study was designed to evaluate a new procedure,the RosetteSep-Applied Imaging Rare Event (RARE) detection method,which depletes the majority of the erythrocytes and leucocytes in a peripheral blood (PB) sample,thereby negatively enriching tumor cells if present. This enrichment procedure allows for increased sensitivity,by analyzing a 5-10 fold larger volume of blood,compared with a direct immunocytochemical (ICC) technique,with minimal impact on laboratory workload. Model experiments showed comparable tumor cell recoveries between the two tested methods,both in PB and BM. Clinical samples were evaluated using paired PB and BM samples from 95 carcinoma patients. Analysis of PB results showed that 25.3% had textgreater or = 1 tumor cell detected by the RARE procedure,compared with 5.2% after direct ICC analysis,analyzing a 10-fold larger volume by the RARE procedure. The direct ICC analysis of BM from the same patients revealed 16.8% positive. The ITC detection differed both quantitatively and qualitatively between BM and PB,as samples with high numbers of ITC in BM were still negative in PB. The clinical significance of ITC in blood still needs to be established. However,the easy access of peripheral blood,and the increased sensitivity obtained by increasing the sample volume with the RARE procedure,suggests that the value of peripheral blood analysis should be tested in parallel in studies where ITC detection in BM is performed. View Publication

The colony-stimulating factors (CSFs) are a group of cytokines central to the hematopoiesis of blood cells,the modulation of their functional responses,as well as the maintenance of homeostasis and overall immune competence. This group consists of the macrophage-CSF (M-CSF),granulocyte-CSF (G-CSF),granulocyte/macrophage-CSF (GM-CSF),and multi-CSF (IL-3). M-CSF and G-CSF are relatively lineage-specific,having a role in the proliferation,differentiation,and survival of macrophages,neutrophils,and their precursors. In contrast,GM-CSF and multi-CSF function at earlier stages of lineage commitment regulating the expansion and maturation of primitive hematopoietic progenitors. Colony stimulating factor production and degradation are strictly controlled,thus allowing for effective modulation of their biological functions in steady-state conditions as well as under periods of stress. Moreover,the mechanisms behind their expression and that of their cognate receptors ensures that their actions are tightly coordinated,within the context of a network of complex but finely tuned regulatory pathways derived from a variety of local and endocrine hematopoietic regulators. In this review we present some of the most salient information on CSF biology collected over the last three decades. We examine the gene and protein structure of each of the four CSFs and their corresponding receptors,and consider the main determinants behind their biological activities. The components responsible for their functional redundancy as well as the mechanisms that mediate their specificity are also discussed. Although most of available knowledge about CSFs is on human and mouse CSFs,an attempt was made to integrate recent findings in other systems in order to highlight a more widespread role for CSFs throughout evolution. View Publication

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Granulomas are innate sequestration responses that can be modified by superimposed acquired immune mechanisms. The present study examined the innate stage of pulmonary granuloma responses to bead-immobilized Th1- and Th2-inducing pathogen antigens (Ags),Mycobacteria bovis purified protein derivative (PPD) and Schistosoma mansoni soluble egg Ags (SEA). Compared to a nonpathogen Ag,PPD and SEA bead elicited larger lesions with the former showing accelerated inflammation. Temporal analyses of cytokine and chemokine transcripts showed all Ag beads induced tumor necrosis factor-alpha mRNA but indicated biased interleukin (IL)-1,IL-6,and IL-12 expression with PPD challenge. All beads elicited comparable levels of CXCL9,CXL10,CCL2,CCL17,and CCL22 mRNA,but PPD beads caused biased CXCL2 CXCL5,CCL3,and CCL4 expression whereas both pathogen Ags induced CCL7. Immunohistochemical,electron microscopic,and flow cytometric analyses showed that Ag beads mobilized CD11c+ dendritic cells (DCs) of comparable maturation. Transfer of DCs from PPD Ag-challenged lungs conferred a Th1 anamnestic cytokine response in recipients. Surprisingly,transfer of DCs from the helminth SEA-challenged lungs did not confer the expected Th2 response,but instead rendered recipients incapable of Ag-elicited IL-4 production. These results provide in vivo evidence that lung DCs recruited under inflammatory conditions favor Th1 responses and alternative mechanisms are required for Th2 commitment. View Publication

The Aurora kinases are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumors,and lead to aneuploidy and tumorigenesis. Here we report the discovery of a highly potent and selective small-molecule inhibitor of Aurora kinases,VX-680,that blocks cell-cycle progression and induces apoptosis in a diverse range of human tumor types. This compound causes profound inhibition of tumor growth in a variety of in vivo xenograft models,leading to regression of leukemia,colon and pancreatic tumors at well-tolerated doses. Our data indicate that Aurora kinase inhibition provides a new approach for the treatment of multiple human malignancies. View Publication

High-titer,HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (textless 1%),reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier,we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo,3 rhesus macaques underwent transplantation with transduced,autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid,and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes,respectively,were observed 4 to 6 months after transplantation,consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models. View Publication

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

Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plastic-adherent mesenchymal stem cell able to generate tissues other than bone,increasing evidence suggests the existence of a more primitive cell that can differentiate to both hematopoietic and mesenchymal cells. We show here that the side population" (SP) of marrow stem cells� View Publication

Type 1 interferon-producing cells (IPCs),also known as plasmacytoid dendritic cell (DC) precursors,represent the key effectors in antiviral innate immunity and triggers for adaptive immune responses. IPCs play important roles in the pathogenesis of systemic lupus erythematosus (SLE) and in modulating immune responses after hematopoietic stem cell transplantation. Understanding IPC development from hematopoietic progenitor cells (HPCs) may provide critical information in controlling viral infection,autoimmune SLE,and graft-versus-host disease. FLT3-ligand (FLT3-L) represents a key IPC differentiation factor from HPCs. Although hematopoietic cytokines such as interleukin-3 (IL-3),IL-7,stem cell factor (SCF),macrophage-colony-stimulating factor (M-CSF),and granulocyte M-CSF (GM-CSF) promote the expansion of CD34+ HPCs in FLT3-L culture,they strongly inhibit HPC differentiation into IPCs. Here we show that thrombopoietin (TPO) cooperates with FLT3-L,inducing CD34+ HPCs to undergo a 400-fold expansion in cell numbers and to generate more than 6 x 10(6) IPCs per 10(6) CD34+ HPCs within 30 days in culture. IPCs derived from HPCs in FLT3-L/TPO cultures display blood IPC phenotype and have the capacity to produce large amounts of interferon-alpha (IFN-alpha) and to differentiate into mature DCs. This culture system,combined with the use of adult peripheral blood CD34+ HPCs purified from G-CSF-mobilized donors,permits the generation of more than 10(9) IPCs from a single blood donor. View Publication

The phosphatidylinositol-3 kinase (PI3K) pathway plays a central role in regulating numerous biologic processes,including survival,adhesion,migration,metabolic activity,proliferation,differentiation,and end cell activation through the generation of the potent second messenger PI-3,4,5-trisphosphate (PI-3,4,5-P(3)). To ensure that activation of this pathway is appropriately suppressed/terminated,the ubiquitously expressed 54-kDa tumor suppressor PTEN hydrolyzes PI-3,4,5-P(3) to PI-4,5-P(2),whereas the 145-kDa hematopoietic-restricted SH2-containing inositol 5'-phosphatase SHIP (also known as SHIP1),the 104-kDa stem cell-restricted SHIP sSHIP,and the more widely expressed 150-kDa SHIP2 break it down to PI-3,4-P(2). In this review,we focus on the properties of these phospholipid phosphatases and summarize recent data showing that the activities of these negative regulators often are modulated by simply altering their protein levels. We also highlight the critical role that SHIP plays in lipopolysaccharide-induced macrophage activation and in endotoxin tolerance. View Publication