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Query: UNIPROT:P04141 (granulocyte-macrophage colony-stimulating factor)
 6,790 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously demonstrated that murine MS-5 and SI/SI4 cell lines induce the proliferation of human factor-dependent UT-7 cells in the absence of normally required human cytokines and also stimulate the differentiation of CD34+/CD38-LTC-ICs. We report in this study that the effect of MS-5 cells on UT-7 cells can be completely explained by the synergistic action of nerve growth factor (NGF) and stem cell factor (SCF) produced by these murine stromal cells. Purified murine NGF was able to support short-term clone formation and long-term growth of UT-7 cells in suspension cultures as efficiently as rhu-granulocyte-macrophage colony-stimulating factor. NGF action was mediated through the TrkA receptor, in which messenger RNA (mRNA) was easily detected in UT-7 cells by Northern blot. MS-5 cells strongly expressed NGF mRNA in Northern blot, and direct implication of MS-5-derived NGF in the induction of UT-7 cells proliferation was demonstrated in inhibition assays with an anti-NGF monoclonal antibody (MoAb) that neutralized by 84% +/- 4.1% (n = 5) UT-7 clone formation. However, NGF did not act alone, and several arguments demonstrated the synergistic action of MS-5-derived SCF: (1) an anti-c-kit partially inhibited UT-7 cells clone formation in coculture assays, (2) SCF and NGF synergized in an H3-TdR incorporation assay, and (3) the stimulatory effect of 10x-concentrated MS-5 supernatant was completely inhibited by an anti-c-kit but not by an anti-NGF, and levels of soluble NGF (1.2 ng/mL) detected by enzyme-linked immunosorbent assay in 10x supernatant of MS-5 cells cultures were below the biologically active concentrations. In contrast, although MS-5 cells also promoted the differentiation of very primitive CD34+/CD38- human stem cells both in colony assays and long-term cultures, we could not incriminate MS-5-derived NGF in the observed effect: an anti-NGF MoAb did not inhibit the synergistic effect of MS-5 cells in colony assays or long-term cultures nor did soluble muNGF duplicate MS-5 effect and survival of CD34+/CD38- clonogenic progenitor cells promoted by MS-5 was unaffected by an anti-NGF and was not induced by soluble NGF alone or combined with SCF. In contrast, NGF in synergy with SCF supported the short-term maintenance of high numbers of CD34+/CD38+ mature erythroid progenitors probably through an indirect mechanism implying macrophages. These results suggest that NGF, in which the primary target cells are outside the hematopoietic system, is present in the marrow environment and might act at some steps of hematopoietic stem cell development. These results also underline that the response of cell lines and normal stem cells to stromal cells is mediated by different pathways.
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PMID:Nerve growth factor is involved in the supportive effect by bone marrow--derived stromal cells of the factor-dependent human cell line UT-7. 878 16

Based on initial observations of human CD34+ Thy-1+ cells and long-term culture-initiating cells (LTC-IC) in the bone marrow of some sublethally irradiated severe combined immunodeficient (SCID) mice transplanted intravenously with normal human marrow cells, and the subsequent finding that the NOD/LtSz-scid/scid (NOD/SCID) mouse supports higher levels of human cell engraftment, we undertook a series of time course experiments to examine posttransplant changes in the number, tissue distribution, cycling activity, and in vivo differentiation pattern of various human hematopoietic progenitor cell populations in this latter mouse model. These studies showed typical rapid posttransplant recovery curves for human CD34- CD19+ (B-lineage) cells, CD34+ granulopoietic, erythroid, and multilineage colony-forming cells (CFC), LTC-IC, and CD34+ Thy-1+ cells from a small initial population representing <0.1% of the original transplant. The most primitive human cell populations reached maximum values at 5 weeks posttransplant, after which they declined. More mature cell types peaked after another 5 weeks and then declined. A 2-week course of thrice weekly injections of human Steel factor, interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (administered just before the mice were killed for analysis) did not alter the pace of regeneration of either primitive or mature human hematopoietic cells, or their predominantly granulopoietic and B-lymphoid pattern of differentiation, although a significant enhancing effect on the level of human cell engraftment sustained after 3 months was noted. Cycling studies showed the human CFC present at 4 to 5 weeks posttransplant to be rapidly proliferating even in mice not given human growth factors. However, by 10 weeks and thereafter, only quiescent human CFC were detected; interestingly, even in mice that were given the 2-week course of growth factor injections. These studies indicate the use of this model for future analysis of the properties and in vivo regulation of primitive human hematopoietic cells that possess in vivo repopulating ability.
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PMID:Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice. 919 53

Time course studies of sublethally irradiated non-obese mice with severe combined immunodeficiency (NOD/ SCID mice) transplanted intravenously with 10(7) human cord blood cells showed a rapid and parallel regeneration of human erythroid, granulopoietic, megakaryopoietic and B-lymphoid progenitors, as well as more primitive subpopulations of CD34+ cells (defined by their multi-lineage in vitro colony-forming ability, coexpression of Thy-1, or functional activity in long-term culture-initiating cell [LTC-IC] assays), in the marrow, spleen and blood. Maximum numbers of human cells were reached within 6 weeks and were then sustained for another 18-20 weeks. 3H-thymidine suicide studies showed all types of in vitro clonogenic human progenitors tested and the human LTC-IC to be proliferating in vitro throughout this period. A 2-week course of injections of human Steel factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin given just prior to assessment of the mice had no effect on any of these human engraftment parameters. 4-6 weeks post-transplant, the marrow of primary NOD/SCID recipients contained human cells that were able to regenerate lymphopoiesis and/or myelopoiesis in secondary irradiated NOD/SCID mice. These findings establish a baseline for the kinetics of engraftment, multi-lineage differentiation and self-renewal of human cord blood stem cells in this xenogeneic transplant model and thus set the stage for future studies of their regulation in vivo.
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PMID:Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haemopoietic cells in NOD/SCID mice transplanted with human cord blood. 932 7

The application of ex vivo expansion to cell products pharmacologically purged in vitro may provide sufficient numbers of cells for rapid engraftment in a product with reduced tumor burden. To pursue this possibility we evaluated the effect of 4-hydroperoxycyclophosphamide (4-HC) treatment on granulocyte colony-stimulating factor-mobilized peripheral blood stem cells (G-PBSC) and their subsequent expansion potential. A small number of G-PBSC CD34+ cells are resistant to 4-HC and are phenotypically and functionally immature. 4-HC-resistant G-PBSC cells are CD34+ bright, CD38+/-, DR(lo), CD13(lo), CD33-, CD71-, and rhodamine dull. In six experiments, treating G-PBSC with 60 microg/mL of 4-HC at 37 degrees C for 30 minutes reduced the number of colony-forming units (CFUs) per 5000 CD34+ cells by 96.3% (from 1333 +/- 137 to 46.5 +/- 11). This purging also reduced the frequency of 5-week long-term culture initiating cells (LTC-ICs) from 1/39 (range 1/27 to 1/62) to <1/1680 (range 1/1180 to 1/2420). Ex vivo expansion cultures were used to compare the proliferative potential of treated and untreated CD34+ cells. These cells were cultured with either the HS-5 stromal cell line serum-deprived conditioned media supplemented with 10 ng/mL kit ligand (HS-5CM/KL) or a recombinant growth factor mix (GFmix) containing 10 ng/mL each of interleukin (IL)-1, IL-3, IL-6, KL, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and 3 U/mL of erythropoietin. Culturing untreated CD34+ G-PBSC with 10% HS-5CM/KL increased total nucleated cells by 460-fold after 15 days. Progenitors, which were measured as CFUs, also increased by 47-fold over the same period. More significantly, culturing the 4-HC-treated CD34+ cells with HS-5/KL increased CFUs 98-fold and the nucleated cells increased 4573-fold. The absolute number of CFUs present after expansion of the 4-HC-resistant cells with HS-5CM/KL was threefold higher than that detected before purging and significantly higher than that obtained with GFmix. These data indicate that G-PBSC contain a very immature pool of cells not detectable using the 5-week LTC-IC assay, but have extremely high proliferative potential. Additionally, pharmacological purging of G-PBSC greatly reduces mature cells while retaining an immature population. Also significant is the finding that supernatant from the HS-5 bone marrow stromal cell line plus KL can fully regenerate progenitors from the 4-HC-resistant CD34+ G-PBSC.
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PMID:Ex vivo expansion of immature 4-hydroperoxycyclophosphamide-resistant progenitor cells from G-CSF-mobilized peripheral blood. 976 8

Ex vivo stroma-free static liquid cultures of granulocyte colony-stimulating factor (G-CSF)/chemotherapy-mobilized CD34+ cells were established from patients with epithelial solid tumors. Different culture conditions were generated by adding G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt3 ligand (Flt3), megakaryocyte growth and development factor (Peg-rHuMGDF), GM-CSF/erythropoietin (EPO) hybrid protein (MEN11303), and interleukin-15 (IL-15) to the basic stem cell factor (SCF) + interleukin-3 (IL-3) + EPO combination. This study showed that, among the nine different combinations tested in our 5% autologous plasma-containing cultures, only those containing IL-3/SCF/Flt3/MEN11303 and IL-3/SCF/Flt3/MEN11303/IL-15 significantly expanded colony-forming unit granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), long-term culture-initiating cells (LTC-IC), CD34+, and CD34+/CD38- cells after 14 days of culture. Particularly, the addition of IL-15 to IL-3/SCF/Flt3/MEN11303 combination produced a significant increase of LTC-IC, with an average 26-fold amplification as compared to input cells, without any detrimental effect on CFU-GM and BFU-E expansion. This combination also produced a statistically significant 3.6-fold expansion of primitive CD34+/CD38- cells. Moreover, this study confirms the previously described erythropoietic effect of MEN11303, which, in our experience, was the only factor capable of expanding BFU-E. Compared to equimolar concentrations of GM-CSF and EPO, MEN11303 hybrid protein showed a significantly higher capacity of expanding CFU-GM, BFU-E, LTC-IC, CD34+, and CD34+/CD38- cells when these cytokines were tested in combination with IL-3/SCF/Flt3. These cultures indicated that Peg-rHuMGDF addition to IL-3/SCF/EPO/Flt3 does not affect CFU-GM and BFU-E expansion but, unlike G-CSF or GM-CSF, it does not decrease the ability of Flt3 to expand primitive LTC-IC. These studies indicate that, starting from G-CSF/chemotherapy-mobilized CD34+ cells, concomitant expansion of primitive LTC-IC, CFU-GM, BFU-E, CD34+, and CD34+/CD38- cells is feasible in simple stroma-free static liquid cultures, provided IL-3/SCF/Flt3/MEN11303/IL-15 combination is used as expanding cocktail in the presence of 5% autologous plasma.
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PMID:Expansion of granulocyte colony-stimulating factor/chemotherapy-mobilized CD34+ hematopoietic progenitors: role of granulocyte-macrophage colony-stimulating factor/erythropoietin hybrid protein (MEN11303) and interleukin-15. 1008 3

Evidence has been provided recently that shows that high concentrations of cytokines can fulfill functions previously attributed to stromal cells, such as promote the survival of, and led to a net increase in human primitive progenitors initiating long-term cultures in vitro (LTC-IC) or engrafting NOD-SCID (nonobese diabetic severe-combined immunodeficient) recipients in vivo. These data prompted us to re-evaluate whether stromal cells will further alter the properties of primitive progenitor cells exposed to cytokines. Single CD34(+)CD38(low) and CD38(neg) cells were incubated 10 days in serum-containing or serum-free medium in the presence or in the absence of murine marrow-derived stromal cells (MS-5). Recombinant human cytokines stem cell factor (SCF), pegylated-megakaryocyte growth and differentiation factor (PEG-MGDF), FLT3-L, Interleukin (IL)-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were systematically added at various concentrations (10 to 300 ng/mL). Cell proliferation and LTC-IC potential were evaluated in each clone after 10 days. A striking and consistent observation was the retention of a high LTC-IC potential in clones exposed to cytokines in the presence of stromal feeders, whereas clones exposed to cytokines alone in the absence of stromal feeders rapidly lost their LTC-IC potential as they proliferated. This was reflected both by the higher proportion of wells containing LTC-IC and by the high numbers of CFC produced after 5 weeks in clones grown with MS-5 during the first 10 days. We further showed by analyzing multiple replicates of a single clone at day 10 that MS-5 cells promoted a net increase in the LTC-IC compartment through self-renewal divisions. Interestingly, these primitive LTC-IC were equally distributed among small and large clones, as counted at day 10, indicating that active proliferation and loss of LTC-IC potential could be dissociated. These observations show that, in primitive cells, stromal cells counteract differentiation events triggered by cytokines and promoted self-renewal divisions. Furthermore, the almost identical distribution of the size of the clones with or without MS-5 suggests that proliferation and function of human primitive cells may be independently regulated by external signals, and that the former is primarily under the control of cytokines.
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PMID:Murine stromal cells counteract the loss of long-term culture-initiating cell potential induced by cytokines in CD34(+)CD38(low/neg) human bone marrow cells. 1039 20

We have previously shown that when human umbilical cord blood (UCB) cells are cultured in standard Dexter-type long-term cultures (D-LTC), adherent cells develop forming a discrete net on the bottom of the culture flask. The identity of such cells, however, has not been defined. Accordingly, the major goal of the present study was to characterize the adherent cells developed in standard UCB D-LTC. Cultures were established from 14 UCB samples and from nine bone marrow (BM) samples, as controls. Both UCB and BM cultures were initiated with the same number of mononuclear cells (MNC) (2.5 x 10(6) MNC/ml). After three weeks in culture, adherent cell numbers in UCB D-LTC were 24%-30% of the numbers found in BM cultures. More than 90% of the adherent cells in UCB D-LTC expressed the acid phosphatase enzyme, whereas no alkaline phosphatase-positive cells were observed. This was in contrast to BM D-LTC, in which alkaline and acid phosphatase were expressed by 60%-75% and 20%-45% of the adherent cells, respectively. Immunochemical analysis showed that CD61 (osteoclast marker) and Factor VIII (endothelial cell marker) were not expressed by the adherent cells developed in UCB cultures. Interestingly, the majority of such cells expressed CD1a (dendritic cell marker), CD14, CD68 and CD115 (antigens mainly expressed by macrophagic cells). When the cultures were supplemented with the recombinant cytokines epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor or granulocyte-macrophage colony-stimulating factor (GM-CSF), only GM-CSF had a significant positive effect on adherent cell number. In order to test for some functional properties of the adherent cells developed in culture, production of stem cell factor (SCF), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) was assessed. IL-6 and TNF-alpha showed elevated levels in UCB D-LTC, whereas SCF levels were always below detection. Finally, analysis of fibroblast progenitors (fibroblast colony-forming units [CFU-F]) showed that these cells were present in BM samples (6 CFU-F/10(5) MNC) and were totally absent in UCB samples. Taken together, the results of the present study indicate that the vast majority of the adherent cells developed in standard UCB D-LTC belong to the macrophage lineage and that fibroblasts seem to be absent. Interestingly, the high proportion of CD1a+ cells suggests that dendritic cells are also present in these cultures.
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PMID:Characterization of the adherent cells developed in Dexter-type long-term cultures from human umbilical cord blood. 1066 71

We assessed the effect of anti-CD3-stimulated secretion of cultured human Th1- and Th2-like cells on leukotriene C(4) (LTC(4)) secretion in isolated human eosinophils. T helper (Th) cell subsets were generated from human naive CD4(+) T cells cocultured with irradiated human transformed B cells and either recombinant human interleukin (rhIL)-1beta plus rhIL-6 plus rhIL-12 for Th1-like cells or rhIL-1beta plus rhIL-6 plus rhIL-4 for Th2-like cells. Coincubation of eosinophils with 1:5 dilution of Th2-supernatant (Sup) caused an increase in LTC(4) secretion caused by 0.1 microM formyl-Met-Leu-Phe and 5 microg/ml cytochalasin B from 921 +/- 238 to 3,067 +/- 1,462 pg/10(6) eosinophils (P < 0.01). Th1-Sup at the same dilution had no augmenting effect on stimulated secretion of LTC(4) in eosinophils despite substantial concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant. Dilution of Th1-Sup caused increased LTC(4) that returned to baseline after immunoabsorption of GM-CSF, suggesting the presence of a possible inhibitory factor. We demonstrate that pretreatment of eosinophils with 1:5 dilution of Th2-Sup but not of Th1-Sup causes substantial augmentation of LTC(4) secretion in vitro and establishes that human Th2 cells cause direct augmentation of LTC(4) secretion within 15-30 min of exposure.
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PMID:Augmentation of LTC(4) synthesis in human eosinophils caused by CD3-stimulated Th2-like cells in vitro. 1083 22

Eosinophilia is a feature of airway inflammation associated with asthma. Leukotriene antagonists provide therapeutic benefit in asthma, but their potential antiinflammatory actions have not been fully explored. We have examined the role of eosinophil-derived cysteinyl leukotrienes in the maintenance of eosinophil survival, and the involvement of leukotrienes in the paracrine stimulation of eosinophil survival by mast cells and lymphocytes. We obtained eosinophils and autologous lymphocytes from peripheral blood of asthmatic subjects. Leukotriene (LT)-B(4), LTC(4) and LTD(4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and fibronectin promoted eosinophil survival. LTD(4) (10(-)(6) M) was as effective as GM-CSF (5 ng/ml) and fibronectin (400 ng/ml) in promoting survival. Lymphocytes and conditioned medium from a human mast cell line (HMC-1) induced eosinophil survival. Blockade of cysteinyl leukotriene receptors with SKF 104353 (pobilukast, 3 nM), and inhibition of 5-lipoxygenase (5-LO) with BW A4C (1 microM) and of 5-LO activating protein with MK 886 (1 microM), all increased basal rates of eosinophil apoptosis and reversed GM-CSF-induced eosinophil survival. Fifty percent reversal of GM-CSF- induced survival was achieved with SKF 104353 at 0.3 nM. The potency of SKF 104353 was two orders of magnitude greater than that of the LTB(4) receptor antagonist SB 201146. Mast cell- and lymphocyte-induced eosinophil survival were completely reversed by SB 201146, SKF 104353, BW A4C, and MK 886. These findings provide evidence for the involvement of an autocrine cysteinyl leukotriene pathway that supports eosinophil survival in response to a range of survival stimuli. They also suggest that LTB(4) could act as a paracrine stimulus of eosinophil survival.
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PMID:Leukotriene receptor antagonists and synthesis inhibitors reverse survival in eosinophils of asthmatic individuals. 1085 61

Long-term bone marrow culture (LTBMC) supports both differentiation and conservation of hematopoietic progenitor cells. During the culture period, the frequency and proliferative potential of early repopulating stem cells that give rise to progenitors detectable after 5 weeks in LTBMC--so-called long-term culture-initiating cells (LTC-IC)--can be investigated. The adherent stroma cell layer was modified by interleukin-3 (IL-3) + granulocyte-macrophage colony-stimulating factor (GM-CSF)+ stem-cell factor (SCF) with an increased cellularity and higher percentage of differentiated myeloid cells and a reduced percentage of stroma cells. We have studied the effects of stimulative cytokines, such as GM-CSF, SCF, and IL-3, on proliferation of committed colony-forming unit cells (CFU-C), LTC-IC, and stroma cell formation in LTBMC of unseparated bone-marrow cells. IL-3, GM-CSF, and SCF significantly stimulated the cumulative proliferation of nucleated cells and committed CFU-C. Weekly stimulation of LTBMC during the culture period did not exhaust the proliferative capacity of stem cells as seen in maintenance of LTC-IC after 5 weeks in LTBMC. In contrast, no LTC-IC were seen in limiting dilution analyses from LTBMC cultured 5 weeks without cytokine supplementation. Our data indicate that an increased proliferation of committed stem cells can be achieved by the addition of stimulatory growth factors, and maintenance of LTC-IC is possible over a period of 5 weeks. A net expansion of LTC-IC if compared with the starting bone-marrow suspension could not be obtained after a 5-week LTBMC period.
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PMID:Effect of interleukin-3, stem cell factor and granulocyte-macrophage colony-stimulating factor on committed stem cells, long-term culture initiating cells and bone marrow stroma in a one-step long-term bone marrow culture. 1087 Apr 78


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