搜索结果: 'gmp t cell activator'

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB),used in the Aldeflour assay,has been considered a specific inhibitor for ALDH1A1 isoform. In this study,we explore the effects of human ALDH isoenzymes,ALDH1A2 and ALDH2,on drug resistance and proliferation,and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay,Western blot,RT-PCR,and Aldefluor assay. Both cell lines,with either ALDH1A2 or ALDH2,exhibited higher cell proliferation rates,higher clonal efficiency,and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors,DEAB and disulfiram,we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore,our TLDA results revealed that ALDH1,ALDH7,ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition,other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells. View Publication

Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified,but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved,we performed Northern analyses on RNA extracted from human marrow LTC adherent layers,or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL-1 beta),IL-6,and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF,GM-CSF,and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast,transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC,and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures,respectively. Together,these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC,with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF,GM-CSF,and IL-6. Given the similarities between the LTC system and the marrow microenvironment,it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells. View Publication

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted,self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani,K. Hobson,and M. S. Rao,1997,Dev. Biol. 186,202-223; M. S. Rao and M. Mayer-Proschel,1997,Dev. Biol. 188,48-63; M. Mayer-Proschel,A. J. Kalyani,T. Mujtaba,and M. S. Rao,1997,Neuron 19,773-785). We now show that cells identical to rat NEPs,NRPs,and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs,respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive,undergo self-renewal in defined medium,and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus,lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells. View Publication

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells,up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking,however,decreased to 0.1% or lower within 2 weeks. In contrast,EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks,the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis,and EGFP expression was observed in CD4(+),CD8(+),CD20(+),and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452) View Publication

Green tea has shown remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays,cell culture systems,and epidemiological studies. Many of these biological effects of green tea are mediated by epigallocatechin 3-gallate (EGCG),the major polyphenol present therein. We have earlier shown that EGCG treatment results in apoptosis of several cancer cells,but not of normal cells (J. Natl. Cancer Inst. 89,1881-1886 (1997)). The mechanism of this differential response of EGCG is not known. In this study,we investigated the involvement of NF-kappaB during these differential responses of EGCG. EGCG treatment resulted in a dose-dependent (i) inhibition of cell growth,(ii) G0/G1-phase arrest of the cell cycle,and (iii) induction of apoptosis in human epidermoid carcinoma (A431) cells,but not in normal human epidermal keratinocytes (NHEK). Electromobility shift assay revealed that EGCG (10-80 microM) treatment results in lowering of NF-kappaB levels in both the cytoplasm and nucleus in a dose-dependent manner in both A431 cells and NHEK,albeit at different concentrations. EGCG treatment was found to result in a dose-based differential inhibition of TNF-alpha- and LPS-mediated activation of NF-kappaB in these cells. The inhibition of NF-kappaB constitutive expression and activation in NHEK was observed only at high concentrations. The immunoblot analysis also demonstrated a similar pattern of inhibition of the constitutive expression as well as activation of NF-kappaB/p65 nuclear protein. This inhibition of TNF-alpha-caused NF-kappaB activation was mediated via the phosphorylative degradation of its inhibitory protein IkappaBalpha. Taken together,EGCG was found to impart differential dose-based NF-kappaB inhibitory response in cancer cells vs normal cells; i.e.,EGCG-mediated inhibition of NF-kappaB constitutive expression and activation was found to occur at much higher dose of EGCG in NHEK as compared to A431 cells. This study suggests that EGCG-caused cell cycle deregulation and apoptosis of cancer cells may be mediated through NF-kappaB inhibition. View Publication

AC133 antigen is a novel marker for human hematopoietic stem/progenitor cells. In this study,we examined the expression and proliferation potential of AC133(+) cells obtained from steady-state peripheral blood (PB). The proportion of AC133(+) cells in the CD34(+) subpopulation of steady-state PB was significantly lower than that of cord blood (CB),although that of cytokine-mobilized PB was higher than that of CB. The proliferation potential of AC133(+)CD34(+) and AC133(-)CD34(+) cells was examined by colony-forming analysis and analysis of long-term culture-initiating cells (LTC-IC). Although the total number of colony-forming cells was essentially the same in the AC133(+)CD34(+) fraction as in the AC133(-)CD34(+) fraction,the proportion of LTC-IC was much higher in the AC133(+)CD34(+) fraction. Virtually no LTC-IC were detected in the AC133(-)CD34(+) fraction. In addition,the features of the colonies grown from these two fractions were quite different. Approximately 70% of the colonies derived from the AC133(+)CD34(+) fraction were granulocyte-macrophage colonies,whereas more than 90% of the colonies derived from the AC133(-)CD34(+) fraction were erythroid colonies. Furthermore,an ex vivo expansion study observed expansion of colony-forming cells only in the AC133(+)CD34(+) population,and not in the AC133(-)CD34(+) population. These findings suggest that to isolate primitive hematopoietic cells from steady-state PB,selection by AC133 expression is better than selection by CD34 expression. View Publication

The application of multi-parameter flow cytometry for the assessment of T-cell and cytokine functioning has been used by several groups for studying human and mouse samples,although little has been reported for the rat. Here we report the optimisation of immunofluorescent staining for cell surface and intracellular antigens using three-colour flow cytometric analysis to measure the frequency of rat CD3(+)4(+) T-cells that produce IFN-gamma,IL-4 and IL-10. In vitro stimulation of IFN-gamma production required incubation of splenocytes with PMA and ionomycin in the presence of the protein transport inhibitor brefeldin A for 6 h. Three stimulation protocols for IL-4 and IL-10 production were evaluated. In vitro priming of splenic T-cells with antibodies against CD3 and CD28 and recombinant cytokines (IL-2 and IL-4) for 5 days followed by restimulation with PMA and ionomycin was required to stimulate cells to produce either IL-4 or IL-10. Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems. View Publication

DYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1,an inhibitor of cell cycle progression in yeast. At present,mDYRK-3 and mDYRK-2 have been cloned,and mDYRK-3 has been characterized with respect to kinase activity,expression among tissues and hematopoietic cells,and possible function during erythropoiesis. In sequence,mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a,but is 91.3% identical overall to hDYRK-3. Catalytically,mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro. Expression of mDYRK-1a,mDYRK-2,and mDYRK-3 was high in testes,but unlike mDYRK1a and mDYRK 2,mDYRK-3 was not expressed at appreciable levels in other tissues examined. Among hematopoietic cells,however,mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells. In developmentally synchronized erythroid progenitor cells,expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone),and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver. Interestingly,antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit-erythroid colony formation. Thus,it is proposed that mDYRK-3 kinase functions as a lineage-restricted,stage-specific suppressor of red cell development. (Blood. 2001;97:901-910) View Publication

High expression of the Breast Cancer Resistance Protein (BCRP) gene has been shown to be involved in resistance to chemotherapeutic drugs. Knowledge of the localization of BCRP protein in normal tissues may help unravel the normal function of this protein. Therefore,we characterized the tissue distribution and cellular localization of BCRP in frozen sections of normal human tissues. For this purpose,we used the recently described monoclonal antibody BXP-34 and another independently developed monoclonal antibody directed against BCRP,BXP-21. Both monoclonal antibodies show specific BCRP plasma membrane staining on cytospins obtained from topotecan- or mitoxantrone-selected cell lines,as well as from BCRP-transfected cell lines. Immunoprecipitation experiments using either BXP-21 or BXP-34 yielded a clear M(r) 72,000 BCRP band from BCRP-overexpressing tumor cells. In the topotecan-selected T8 and mitoxantrone-selected MX3 tumor cell lines,BCRP turned out to be differentially glycosylated. In contrast to BXP-34,BXP-21 is able to detect the M(r) 72,000 BCRP protein on immunoblots and is reactive with BCRP in formalin-fixed,paraffin-embedded tissues. Using BXP-21 and BXP-34,prominent staining of BCRP was observed in placental syncytiotrophoblasts,in the epithelium of the small intestine and colon,in the liver canalicular membrane,and in ducts and lobules of the breast. Furthermore,BCRP was present in veinous and capillary endothelium,but not in arterial endothelium in all of the tissues investigated. In the tissues studied,the mRNA levels of BCRP were assessed using reverse transcription-PCR,and these corresponded with the levels of BCRP protein estimated from immunohistochemical staining. The presence of BCRP at the placental syncytiotrophoblasts is consistent with the hypothesis of a protective role of BCRP for the fetus. The apical localization in the epithelium of the small intestine and colon indicates a possible role of BCRP in the regulation of the uptake of p.o. administered BCRP substrates by back-transport of substrate drugs entering from the gut lumen. Therefore,it may be useful to attempt to modulate the uptake of p.o. delivered BCRP substrates,e.g.,topotecan or irinotecan,by using a BCRP inhibitor. Clinical trials testing this hypothesis have been initiated in our institute. View Publication

Although the source of embryonic stem (ES) cells presents ethical concerns,their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas,insulin is produced and secreted by specialized structures,islets of Langerhans. Diabetes,which affects 16 million people in the United States,results from abnormal function of pancreatic islets. We have generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells. The cells self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons. Glucose triggers insulin release from these cell clusters by mechanisms similar to those employed in vivo. When injected into diabetic mice,the insulin-producing cells undergo rapid vascularization and maintain a clustered,islet-like organization. View Publication