技术资料

Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF-dependent block in B cell development occurs near the onset of B-lineage commitment,which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted,multipotential progenitor cells. To test this hypothesis,we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF-expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were textgreater95 and 90% GFP+EBF+ mature B cells,respectively. Both NK and lymphoid-derived dendritic cells were also significantly reduced compared with control-transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid-derived cell pathways. View Publication

Raloxifene reduces bone loss and prevents vertebral fractures in postmenopausal women. Its skeletal effects are mediated by estrogen receptors (ER) and their modulation of paracrine osteoblastic factors. Receptor activator of nuclear factor-kappa B ligand is essential for osteoclasts and enhances bone resorption,whereas osteoprotegerin (OPG) neutralizes receptor activator of nuclear factor-kappa B ligand. Here,we assessed the effects of raloxifene on OPG production in human osteoblasts (hOB). Raloxifene enhanced gene expression of ER-alpha and progesterone receptor. Moreover,raloxifene increased OPG mRNA levels and protein secretion by hOB in a dose- and time-dependent fashion by 2- to 4-fold with a maximum effect at 10(-7) M and after 72 h (P textless 0.001). Treatment with the ER antagonist ICI 182,780 abrogated the effects of raloxifene on OPG production. Moreover,raloxifene enhanced osteoblastic differentiation markers,type 1 collagen secretion,and alkaline phosphatase activity by 3- and 2-fold,respectively (P textless 0.001). In addition,raloxifene inhibited expression of the bone-resorbing cytokine IL-6 by 25-45% (P textless 0.001). In conclusion,our data suggest that raloxifene stimulates OPG production and inhibits IL-6 production by hOB. Because OPG production increases with osteoblastic maturation,enhancement of OPG production by raloxifene could be related to its stimulatory effects on osteoblastic differentiation. View Publication

1. The mechanism of Ca2+ release from intracellular stores was studied in defolliculated Xenopus laevis oocytes by measuring whole-cell currents using the two-electrode voltage-clamp method. 2. The extracellular application of ionomycin,a selective Ca2+ ionophore,evoked an inward current consisting of a spike-like fast component followed by a long-lasting slow component with few superimposed current oscillations (fluctuations). The ionomycin response occurred in a dose-dependent manner and was dependent on Cl-. 3. No apparent refractory period was observed for repetitively evoked small ionomycin responses when the concentration of ionomycin was low (0.1 microM). In contrast,a larger ionomycin response (1 microM),consisting of fast and slow components,was followed by refractory period. Washing for 50-90 min was necessary for full recovery of the ionomycin response. 4. The response to ionomycin was suppressed by the extracellular application of acetoxymethyl ester of bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA AM,1-10 microM),a membrane-permeable intracellular Ca2+ chelator. 5. The ionomycin response was not affected by pertussis toxin (PTX,0.3-2.0 microgram/ml),a blocker of guanine nucleotide-binding regulatory proteins (G proteins). In contrast,the response to acetylcholine (ACh),which is known to occur via a G protein,was suppressed by PTX. 6. The fast component was not affected by removing Ca2+ from the bathing medium or by replacing extracellular Ca2+ with Ba2+ or Mn2+ (all of these solutions were supplemented with 2 mM EGTA),whereas the slow component was suppressed. 7. Injection of inositol 1,4,5-trisphosphate (IP3) following a response to extra-cellularly applied ionomycin did not evoke an appreciable membrane current. In contrast,ionomycin evoked a small inward current when it was applied after an inward-current response evoked by IP3 injection,whereas a second injection of IP3 did not evoke any appreciable current. 8. The results indicate that (a) ionomycin releases Ca2+ from its intracellular stores without the involvement of G proteins,resulting in activation of Ca(2+)-activated Cl- channels,(b) ionomycin mainly acts on the same intracellular Ca2+ stores as IP3,and (c) entry of Ca2+ from outside the cell considerably contributes to the slow component of the ionomycin response,whereas its fast component is predominantly dependent on the release of Ca2+ from the intracellular stores. View Publication

Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist,RAR alpha,RAR beta,and RAR gamma. The receptors are differentially expressed in different cell types and stages of development,suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation. Beyond its potential practical relevance,this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions. View Publication

The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR alpha,beta and gamma. Since RAR gamma is predominantly expressed in adult skin,specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RAR gamma in skin and (ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay,we have identified three compounds with high RAR gamma selectivity. View Publication

Optimal induction of clonal expansion by normal CD4 T cells requires a ligand that can engage the T cell receptor as well as functionally defined costimulatory activity on the same antigen-presenting cell surface. While the presence of effective costimulation induces proliferation,T cell receptor ligation in its absence renders T cells inactive or anergic. The molecular basis of this costimulatory activity remains to be defined. Here we describe a monoclonal antibody that can block the costimulatory activity of splenic accessory cells. Treatment with this antibody not only blocks the proliferation of CD4 T cells to a T cell receptor ligand,but also induces T cell nonresponsiveness to subsequent stimulation. Sequence analysis of the antigen recognized by this antibody indicates that it recognizes a protein that is identical to heat-stable antigen. Gene transfer experiments directly demonstrate that this protein has costimulatory activity. Thus,heat-stable antigen meets the criteria for a costimulator of T cell clonal expansion. View Publication

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Undifferentiated pluripotent stem cells with flexible developmental potentials are not normally found in peripheral blood. However,such cells have recently been reported to reside in the bone marrow. Herein are reported methods of inducing pluripotency in cells derived from unmobilised adult human peripheral blood. In response to the inclusion of purified CR3/43 monoclonal antibody (mAb) to well-established culture conditions,mononuclear cells (MNC) obtained from a single blood donor are converted into pluripotent haematopoietic,neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells. The haematopoietic stem cells are CD34+,clonogenic and have been shown to repopulate non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The neuronal precursors transcribe the primitive stem cell markers OCT-4 and nestin,and on maturation,differentially stain positive for neuronal,glial or oligodendrocyte-specific antigens. The cardiomyogenic progenitor stem cells form large bodies of asynchronously beating cells and differentiate into mature cardiomyocytes which transcribe GATA-4. The undifferentiated stem cells do not express haematopoietic-associated markers,are negative for major histocompatibility complex (MHC) class I and II antigens,transcribe high levels of OCT-4 and form embryoid body (EB)-like structures. This induction of stem cell-like plasticity in MNC may have proceeded by a process of retrodifferentiation but,in any case,could have profound clinical and pharmacological implications. Finally,the flexibility and the speed by which a variety of stem cell classes can be generated ex vivo from donor blood could potentially transfer this novel process into a less invasive automated clinical procedure. View Publication

Deamination of the nucleoside analogues ARA-C and 5-AZA-CdR by CR deaminase results in a loss of antileukemic activity. To prevent the inactivation of these analogues,inhibitors of CR deaminase may prove to be useful agents. In the present study we investigated the effects of the deaminase inhibitors Zebularine,5-F-Zebularine,and diazepinone riboside on the deamination of CR,ARA-C,and 5-AZA-CdR using highly purified human CR deaminase (EC 3.5.4.5). These inhibitors produced a competitive type of inhibition with each substrate,the potency of which followed the patterns diazepinone riboside greater than 5-F-Zebularine and THU greater than Zebularine. 5-AZA-CdR was more sensitive than ARA-C to the inhibition produced by these deaminase inhibitors. The inhibition constants for diazepinone riboside lay in the range of 5-15 nM,suggesting that this inhibitor could be an excellent candidate for use in combination chemotherapy with either ARA-C or 5-AZA-CdR in patients with leukemia. View Publication

To directly study the biological properties of purified hematopoietic colony-forming cell precursors,cells with a CD34+ CD45RAlo CD71lo phenotype were purified from human bone marrow using density separation and fluorescence-activated cell sorting,and were cultured in serum-free culture medium supplemented with various cytokines. In the presence of interleukin 3 (IL-3),IL-6,erythropoietin,and mast cell growth factor (a c-kit ligand),cell numbers increased approximately 10(6)-fold over a period of 4 wk,and the percentage of cells that expressed transferrin receptors (CD71) increased from less than 0.1% at day 0 to greater than 99% at day 14. Interestingly,the absolute number of CD34+ CD71lo cells did not change during culture. When CD34+ CD71lo cells were sorted from expanded cultures and recultured,extensive cell production was repeated,again without significant changes in the absolute number of cells with the CD34+ CD71lo phenotype that were used to initiate the (sub)cultures. These results document that primitive hematopoietic cells can generate progeny without an apparent decrease in the size of a precursor cell pool. View Publication

CD34+ cells devoid of detectable mature and immature T and B lymphocytes,expressing the CD2,CD10,and CD20 antigens,were isolated from marrows of three pairs of sex-mismatched,mixed lymphocyte culture (MLC) nonreactive,sibling baboons. Reciprocal transplants were performed between members of each pair,using the sex chromosomes,identified by standard cytogenetic techniques,as markers of the transplanted cells. Five animals from these three pairs were transplanted with 0.6 to 2.1 x 10(6)/kg of isolated cryopreserved and/or fresh isolated cells that were greater than 95% to 97% CD34+. Before transplantation,animals were treated with either single (920 or 1,020 cGy) or split (700 cGy x 2) dose total body irradiation. All animals engrafted with donor cells,as demonstrated by cytogenetic analysis of bone marrow metaphase cells 4 weeks after transplantation,with days to white blood cell count (WBC) greater than 500 being 19 +/- 2,to WBC greater than 1,000 23 +/- 2,to absolute neutrophil count greater than 500 24 +/- 3,and to platelets greater than 20,000 30 +/- 7. Three animals died of infectious-related complications at 34,42,and 109 days after transplantation with evidence of host and donor cells (mixed chimerism) in marrow. Two animals remain alive and healthy more than 545 and 455 days after transplantation with stable mixed chimerism in marrow and blood. For these two animals,cytogenetic analysis of granulocyte/macrophage and erythroid colonies derived from marrow precursors between weeks 25 and 42 posttransplant showed evidence of mixed chimerism. Cytogenetic studies of CD2+ T cells and CD20+ B cells isolated from blood of these two animals between weeks 21 and 51 posttransplant showed the presence of mixed chimerism in both lymphocyte populations. Thus,isolated allogeneic CD34+ marrow cells devoid of detectable mature and immature T and B lymphocytes can engraft and reconstitute stable long-term myelopoiesis and lymphopoiesis in lethally irradiated baboons. These results are consistent with the hypothesis that CD34+ marrow cells contain pluripotent hematopoietic stem cells capable of fully reconstituting lymphohematopoiesis in the transplanted host. View Publication

Embryonic stem cells can be induced in vitro,by coculture with the stromal line RP.0.10 and a mixture of interleukins 3,6,and 7,to differentiate into T (Joro75+) and B (B-220+) lymphocyte progenitors and other (Thy-1+,PgP-1+,c-kit+,Joro75-,B-220-,F4/80-,Mac-1-) hemopoietic precursors. The progeny of in vitro-induced embryonic stem cells can reconstitute the lymphoid compartments of T- and B-lymphocyte-deficient scid mice and generate mature T and B lymphocytes in sublethally irradiated normal mice. Exogenous cytokines can dramatically alter the developmental fate of embryonic stem cells in culture. The in vitro system described here should facilitate the study of molecular events leading to cell-lineage commitment and to the formation of hemopoietic stem cells and their immediate lymphoid progeny. View Publication