搜索结果: 'gmp t cell activator'

Transcription factors are critical for instructing the development of B lymphocytes from multipotential progenitor cells in the bone marrow (BM). Here,we show that the absence of STAT3 impaired B-cell development. Mice selectively lacking STAT3 in BM progenitor cells displayed reduced numbers of mature B cells,both in the BM and in the periphery. The reduction in the B-cell compartment included reduced percentages and numbers of pro-B,pre-B,and immature B cells in the absence of STAT3,whereas the number of pre-pro-B cells was increased. We found that pro-B and pre-B-cell populations lacking STAT3 were hyporesponsive to IL-7 because of a decreased number of IL-7-responsive cells rather than decreased expression or signaling of IL-7Ralpha. Moreover,STAT3-deficient mice displayed enhanced apoptosis in the pro-B population when deprived of survival factors,suggesting that at least 2 mechanisms (impaired differentiation and enhanced apoptosis) are involved in the mutant phenotype. Last,BM transplantation confirmed that impaired B lymphopoiesis in the absence of STAT3 was caused by a cell autonomous defect. In sum,these studies defined a specific role for STAT3 in early B-cell development,probably acting at the pre-pro-B transition by contributing to the survival of IL-7-responsive progenitors. View Publication

While statin treatment may transiently mobilize endothelial progenitor cells (EPCs),the dose-dependent effects of a continuous statin therapy on EPCs in patients with chronic coronary artery disease (CAD) have not been analyzed. In 209 patients with angiographically documented CAD,144 of which received 10-40 mg/day of statins for textgreater8 weeks,the EPC number was determined by flow cytometry directly (CD34(+)/KDR(+),n=58) and after in vitro-culture (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled Ac-LDL (DiI-Ac-LDL(+))/lectin(+),n=209). EPC function was assessed by the formation of colony forming units (CFUs). Univariate analysis revealed that the dose of continuous statin therapy inversely correlated with the EPC number. Treatment with 40 mg/day significantly reduced EPC counts. Multivariate analysis unveiled the statin dose and extent of CAD as independent predictors of reduced EPC numbers. Conversely,obesity predicted increased counts,while CFU development was not detectable in all patients and augmented in females and smokers but not in statin-treated patients. Compared with matched controls,statin-treated patients showed significantly reduced absolute and relative EPC counts. In a prospective analysis,initiation of statin therapy significantly diminished the number of circulating and isolated EPCs after 3 but not after 1 month(s). Thus,the statin dose during chronic and continuous treatment independently predicts reduced numbers of circulating as well as isolated EPCs in patients with CAD. View Publication

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor,E.G7-OVA,was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice,although both routes primed OVA-specific immune responses,which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice,suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA,but few were detected in primary ocular tumors. Nevertheless,growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice,and CD8(+) T cell numbers were increased within eyes,suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However,CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus,CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye. View Publication

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells,we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells,whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover,long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage,strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether,these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors,and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis. View Publication

Differentiation of embryonic stem (ES) cells typically requires cell-cell aggregation in the form of embryoid bodies (EBs). This process is not very well controlled and final cell numbers can be limited by EB agglomeration and the inability to drive differentiation towards a desired cell type. This study compares three-dimensional (3D) fibrin culture to conventional two-dimensional (2D) suspension culture and to culture in a semisolid methylcellulose medium solution. Two types of fibrin culture were evaluated,including a PEGylated fibrin gel. PEGylation with a difunctional PEG derivative retarded fibrinogen migration during through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a result of crosslinking,similarly,degradation was slowed in the PEGylated gel. ES cell proliferation was higher in both the fibrin and PEGylated fibrin gels versus 2D and methylcellulose controls. FACS analysis and real-time-PCR revealed differences in patterns of differentiation for the various culture systems. Culture in PEGylated fibrin or methylcellulose culture demonstrated features characteristic of less extensive differentiation relative to fibrin and 2D culture as evidenced by the transcription factor Oct-4. Fibrin gels showed gene and protein expression similar to that in 2D culture. Both fibrin and 2D cultures demonstrated statistically greater cell numbers positive for the vascular mesoderm marker,VE-cadherin. View Publication

Nonmammalian glycan structures from helminths act as Th2 adjuvants. Some of these structures are also common on plant glycoproteins. We hypothesized that glycan structures present on peanut glycoallergens act as Th2 adjuvants. Peanut Ag (PNAg),but not deglycosylated PNAg,activated monocyte-derived dendritic cells (MDDCs) as measured by MHC/costimulatory molecule up-regulation,and by their ability to drive T cell proliferation. Furthermore,PNAg-activated MDDCs induced 2- to 3-fold more IL-4- and IL-13-secreting Th2 cells than immature or TNF/IL-1-activated MDDCs when cultured with naive CD4+ T cells. Human MDDCs rapidly internalized Ag in a calcium- and glycan-dependent manner consistent with recognition by C-type lectin. Dendritic cell (DC)-specific ICAM-grabbing nonintegrin (DC-SIGN) (CD209) was shown to recognize PNAg by enhanced uptake in transfected cell lines. To identify the DC-SIGN ligand from unfractionated PNAg,we expressed the extracellular portion of DC-SIGN as an Fc-fusion protein and used it to immunoprecipitate PNAg. A single glycoprotein was pulled down in a calcium-dependent manner,and its identity as Ara h 1 was proven by immunolabeling and mass spectrometry. Purified Ara h 1 was found to be sufficient for the induction of MDDCs that prime Th2-skewed T cell responses. Both PNAg and purified Ara h 1 induced Erk 1/2 phosphorylation of MDDCs,consistent with previous reports on the effect of Th2 adjuvants on DCs. View Publication

A cell-based screen of chemical libraries was carried out to identify small molecules that control the self-renewal of ES cells. A previously uncharacterized heterocycle,SC1,was discovered that allows one to propagate murine ES cells in an undifferentiated,pluripotent state under chemically defined conditions in the absence of feeder cells,serum,and leukemia inhibitory factor. Long-term SC1-expanded murine ES cells can be differentiated into cells of the three primary germ layers in vitro and also can generate chimeric mice and contribute to the germ line in vivo. Biochemical and cellular experiments suggest that SC1 works through dual inhibition of RasGAP and ERK1. Molecules of this kind may not only facilitate practical applications of stem cells in research and therapy,but also provide previously undescribed insights into the complex biology of stem cells. View Publication

The success rate is generally higher when cloning mice from embryonic stem (ES) cell nuclei than from somatic cell nuclei,suggesting that the embryonic nature or the undifferentiated state of the donor cell increases cloning efficiency. We assessed the developmental ability of cloned embryos derived from cultured neural stem cell (NSC) nuclei and compared the success rate with that of embryos cloned from other donor cells such as differentiated NSCs,cumulus cells,Sertoli cells and ES cells in the mouse. The transfer of two-cell cloned embryos derived from cultured NSC nuclei into surrogate mothers produced five live cloned mice. However,the success rate (0.5%) was higher in embryos cloned from cultured NSC nuclei than from differentiated NSCs (0%),but lower than that obtained by cloning mice from other cell nuclei (2.2-3.5%). Although the in vitro developmental potential to the two-cell stage of the cloned embryos derived from NSC nuclei (73%) was similar to that of the cloned embryos derived from other somatic cell nuclei (e.g.,85% in Sertoli cells and 75% in cumulus cells),the developmental rate to the morula-blastocyst stage was only 7%. This rate is remarkably lower than that produced from other somatic cells (e.g.,50% in Sertoli cells and 54% in cumulus cells). These results indicate that the undifferentiated state of neural cells does not enhance the cloning efficiency in mice and that the arrest point for in vitro development of cloned embryos depends on the donor cell type. View Publication

BACKGROUND: Endothelial progenitor cells (EPCs) are present in peripheral blood and can promote postnatal angiogenesis. The number and function of circulating EPCs are altered in diabetics. This study sought to investigate whether the number and functional properties of EPCs from patients with type II diabetes could be improved by pioglitazone. METHODS: For this randomized controlled study,we recruited 36 type II diabetic patients on metformin monotherapy with a glycohemoglobin A1c of textless7%. Patients were separated into pioglitazone (n = 24) and control (n = 12) groups. The number and functional activity of EPCs,and the brachial artery flow-mediated dilation were determined before and after pioglitazone treatment (8 weeks) as an add-on therapy to metformin. In addition,direct effects of pioglitazone on EPCs were also investigated. RESULTS: After pioglitazone treatment,the numbers of circulating EPCs significantly increased (from 0.44% +/- 0.14% to 0.89% +/- 0.29%,P = .01). The migratory response and the adhesive capacity to fibronectin and collagen were improved by 158%,34%,and 83%,respectively (all P textless .05). Treatment with pioglitazone significantly lowered triglyceride,very low density lipoprotein cholesterol,and high-sensitivity C-reactive protein (hsCRP) levels,and increased high-density lipoprotein levels and insulin sensitivity (all P textless .05). The increase in the number of circulating EPCs and the improvement in the migratory response after pioglitazone treatment were independently correlated to the decrease in hsCRP levels (P textless or = .01). The increase in the adhesive capacity was independently correlated to the decreases in very low density lipoprotein cholesterol (P = .01) and hsCRP levels (P = .03). In addition,pioglitazone was also demonstrated to have direct effects on increasing EPC proliferation and colony formation,and attenuating EPC apoptosis (all P textless .05,versus the controls). There were no significant changes in flow-mediated dilation in either group. CONCLUSIONS: Pioglitazone significantly increased the number and improved the functional properties of EPCs in type II diabetic patients through direct effects and/or anti-inflammation and lipid modification effects. View Publication

Human embryonic stem cells (hESCs) provide great opportunities for regenerative medicine,pharmacological and toxicological investigation,and the study of human embryonic development. These applications require proper derivation,maintenance,and extensive characterization of undifferentiated cells before being used for differentiation into cells of interest. Undifferentiated hESCs possess several unique features,including their extensive proliferation capacity in the undifferentiated state,ability to maintain a normal karyotype after long-term culture,expression of markers characteristic of stem cells,high constitutive telomerase activity,and capacity to differentiate into essentially all somatic cell types. This chapter will summarize the current development in culture conditions and provide technical details for the evaluation and characterization of hESCs. View Publication

The transcription factor PAX5 is a critical regulator of B-cell commitment and development. Although normally not expressed in myeloid progenitors,PAX5 has recently been shown to be frequently expressed in myeloid malignancies and to suppress expression of myeloid differentiation genes,compatible with an effect on the differentiation or maintenance of myeloid progenitors. However,previous studies in which PAX5 was ectopically expressed in normal myeloid progenitors in vivo and in vitro provided conflicting results as to the effect of PAX5 on myeloid development. Herein,we demonstrate that on ectopic expression of PAX5 in bone marrow multipotent stem/progenitor cells,cells with a biphenotypic B220(+)GR-1/MAC-1(+) phenotype are produced. These remain cytokine-dependent,but unlike control-transduced cells they sustain long-term generation of myeloid progenitors in vitro and remain capable of myeloid differentiation. Notably,PAX5(+)B220(+)GR-1/MAC-1(+) myeloid progenitors coexpress,at the single-cell level,myeloid genes and otherwise B-cell-specific PAX5 target genes. These findings establish that ectopic expression of PAX5 introduces extensive self-renewal properties in otherwise short-lived myeloid progenitors. Along with the established ectopic expression of PAX5 in acute myeloid leukemia,this motivates a careful investigation of the potential involvement of ectopic PAX5 expression in myeloid and biphenotypic leukemias. View Publication

Hepatocellular carcinoma (HCC) is a common malignancy in Asia and Africa. We previously reported that overexpression of extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) and ERK1/2 was detected in HCC,and that their activation was required for liver cancer cell proliferation and survival. In the present study,we determined the efficacy of a specific MEK1/2 inhibitor AZD6244 (ARRAY-142886) in treatment of HCC. Treatment of primary HCC cells with AZD6244 led to growth inhibition,elevation of the cleavage of caspase-3 and caspase-7,and cleaved poly(ADP)ribose polymerase,but inhibition of ERK1/2 and p90RSK phosphorylation. Studying the protein expression profile of seven HCC xenografts revealed that their growth rate was positively correlated with the levels of phosphorylated MEK. AZD6244,when given p.o. to mice bearing these xenografts,resulted in a dose-dependent inhibition of tumor growth. AZD6244-induced growth suppression was associated with inactivation of ERK1/2 and p90RSK,and up-regulation of activated caspase-3 and caspase-7,and cleaved poly(ADP)ribose polymerase. Our data suggest that the MEK-ERK pathway plays an important role in the growth and survival of liver cancer cells and that the HCC xenograft models are excellent tools for screening preclinical drugs. Targeted inhibition of the MEK-ERK pathway with AZD6244 may represent an alternative approach for the treatment of this disease. View Publication