CSFs, from basic science to clinical trials.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
M Aglietta
Publications and source records attributed to M Aglietta.
Drug-mediated marrow purging: mafosfamide in adult acute leukemia in remission. The experience of the Italian study group.
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Adolescent↗
Interaction of transforming growth factor-beta 1 with hemopoietic growth factors in the regulation of human normal and leukemic myelopoiesis.
We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on the growth of normal and chronic myeloid leukemia (CML) granulo-monopoietic progenitors (CFU-GM) and erythroid progenitors (BFU-E) of different origins and degrees of maturation. In the presence of the supernatant of the 5637 cell line, used as a source of growth factors, TGF-beta 1 stimulates the growth of day-7 CFU-GM from Ficoll-isolated normal bone marrow cells. Maximum stimulation (172% of controls) is observed with 2.5 ng/ml TGF-beta. The results with a highly enriched progenitor cell population stimulated by recombinant granulocyte colony-stimulating factor (rG-CSF) and recombinant granulocyte-macrophage CSF (rGM-CSF) were similar, suggesting a direct effect of TGF-beta 1 on hemopoietic progenitors. In contrast to this stimulatory effect of TGF-beta 1 on normal day-7 bone marrow CFU-GM, TGF-beta 1 does not affect the growth of day-14 CFU-GM. The growth of normal bone marrow BFU-E is strongly inhibited. In the majority of cases (11/15) of CML, bone marrow day-7 CFU-GM growth is inhibited by TGF-beta 1. In few cases (4/15) leukemic progenitors respond to TGF-beta 1 as normal cells. TGF-beta 1 always inhibits the growth of day-14 bone marrow CFU-GM from CML patients.
Bone Marrow↗
Effect of interferon-gamma on HLA class II antigen expression and sensitivity to prostaglandin E1 by normal and leukemic myeloid progenitors.
Chronic myelogenous leukemia (CML) granulo-monocyte committed progenitors (CFU-GM) are markedly less sensitive than normal progenitors to the inhibitory action of prostaglandin E (PGE). This phenomenon has been ascribed to their abnormal expression of HLA class II (mainly DR) determinants. Since interferon gamma (IFN-gamma) is a potent inducer of the expression of HLA class II (DR and to a lesser extent DQ) antigens, we have sought to determine the extent to which this agent can modulate both the antigenic pattern of normal and leukemic progenitors and their sensitivity to PGE 1. 72-h preincubation of normal and CML bone marrow cells with or without IFN-gamma does not significantly change DR and DQ expression by CFU-GM. Pre-incubation for 72 h with and without IFN-gamma produces the following changes in PGE 1 sensitivity: (1) normal CFU-GM lose some sensitivity to PGE 1. This is only marginally counteracted by the presence of IFN-gamma. (2) CML CFU-GM, preincubated with IFN-gamma regain a significant sensitivity to high concentrations of PGE 1. Our data confirm the expression of DR molecules on normal and leukemic progenitors. They also show that, although incubation with IFN-gamma for 72 h in a liquid culture system does not significantly affect the expression of HLA class II molecules by progenitor cells, it may increase their sensitivity to PGE, particularly in the case of CML CFU-GM. Thus expression of HLA class II antigens and sensitivity to PGE may be dissociated.
Alprostadil↗
Human endothelial cells are target for platelet-activating factor. I. Platelet-activating factor induces changes in cytoskeleton structures.
Platelet-activating factor (PAF), but not its deacetylated and biologically inactive metabolite lyso-PAF, in a dose dependent-manner (0.1 to 10 nM), induces human endothelial cells (EC) in culture to change their shape. EC retract and tend to lose reciprocal contact, whereas the distribution of stress fibers is changed and the cells tend to assume a migratory phenotype. The normal localization of vinculin at streaks corresponding to adhesion plaques and located at stress fiber endings is mostly lost and replaced by diffuse distribution of this protein related to the cell-substratum adhesion complex. The effects of PAF are appreciable after 10 min, become maximal after 30 min, and are fully reversible. The disappearance of F-actin in stimulated EC was analyzed by measuring the fluorescence of the cells after staining with fluorosceinated phalloidin, PAF, but not lyso-PAF and the enantiomer of PAF ([S] form), decreases in a time- and concentration-dependent fashion the fluorescence of the cells stained with fluoresceinated phalloidin. EC grown on fibronectin-coated polycarbonate filters restrict the diffusion of 125I-albumin; PAF promotes 125I-albumin diffusion with a concentration dependency similar to that for the PAF-induced cytoskeleton changes. Four different PAF-receptor antagonists belonging to four different series, CV-3988 (PAF-related framework), BN52021 (a natural terpenoid), BN53013 (a natural lignan) and 48740 RP (a synthetic antagonist) prevent the alteration induced by PAF. These findings, coupled to the lack of effect of the enantiomer of PAF ([S] form), support the existence of a specific mechanism of PAF-mediated activation of EC, most probably mediated by a putative receptor. These results explain part of the PAF mechanism of action in inducing the increase of vascular permeability in vivo.
Actin Cytoskeleton↗
Studies on the mechanism of interleukin 1 stimulation of platelet activating factor synthesis in human endothelial cells in culture.
Interleukin 1 promotes the conversion of the biologically inactive lyso-platelet activating factor (lyso-PAF) to the bioactive platelet activating factor (PAF) by an acetylation reaction in cultured human endothelial cells. After 2 h stimulation with interleukin 1, 1-O-alkyl-2-lysoglycero-3-phosphocholine (GPC): acetyl CoA acetyltransferase is activated, reaching a plateau after 6 h and then declining to the basal value within 24 h. This time course is comparable to that of PAF production. These cells are able to incorporate [3H]acetate and [3H]lyso-PAF into PAF. Synthetized [3H]PAF is then catabolized in [3H]alkylacyl phosphoglycerides. 1-O-alkyl-2-acetylglycerol: CDP-choline cholinephosphotransferase and 1-O-alkyl-2-acetyl-GPC: acetylhydrolase activities are both present in endothelial cells, but are not activated under our conditions of stimuli. These findings indicate that interleukin 1 induces the PAF synthesis by a deacylation/reacetylation mechanism into human endothelial cells.
Acetates↗
Expression of HLA class II determinants by normal and chronic myeloid leukemia progenitors.
It has been suggested that the expression of some HLA class II antigens, derived from three loci (DR, DP, DQ) is important in the regulation of both the immune response and the response of haemopoietic progenitors to regulation factors, such as acidic isoferritins (AIF), as well as in the interaction between T lymphocytes and erythroid progenitors (BFU-E). Changes in the expression of class II antigens have been reported on the surface of granulo-monocyte progenitors in chronic myeloid leukemia (CML) and correlated to the abnormal proliferation of such cells. In this study, monoclonal antibodies against DR and DQ monomorphic determinants were used to investigate the expression of these antigens on the surface of normal and CML bone marrow and peripheral blood BFU-E by means of complement mediated cytotoxicity. It was found that most normal and leukemic BFU-E express DR antigens. Antigens density tends to be greater on marrow as opposed to peripheral precursors. In addition, leukemic BFU-E are more sensitive to cytolytic treatment than their normal counterparts. Normal BFU-E do not express detectable amounts of DQ antigens, whereas these are present on a proportion of leukemic BFU-E.
Antibodies, Monoclonal↗
Autologous bone marrow transplantation in acute leukemia.
We evaluated the therapeutic efficacy of autologous bone marrow transplantation (ABMT) in 49 patients with acute leukemia, using ex-vivo-treated marrow with mafosfamide. This report summarizes data collected from 8 italian centers, involving 27 cases with acute lymphoid leukemia (ALL) and 22 cases with acute nonlymphoid leukemia (ANLL). The analysis of the results shows that the disease-free survival of ALL and ANLL are similar (45 and 52%, respectively) and no significant difference can be demonstrated between patients transplanted in CR1 or CR2, although there is a trend in favor of ABMT in CR1.
Acute Disease↗
In vitro reappearance of myeloid progenitors killed by mafosfamide.
Cyclophosphamide derivatives active in vitro, such as mafosfamide, are potentially capable of reducing the number of leukemic cells remaining in marrow explanted for autografting. Although this treatment kills nearly all the committed hemopoietic progenitors, it does not prevent the reinstatement of hemopoiesis after chemoradiotherapy. This points to the persistence of more immature hemopoietic progenitors not detectable with current semi-solid culture techniques and resistant to cytotoxic treatment. Treatment of normal marrow with 80-140 micrograms/ml mafosfamide is followed in medium-term cultures by a gradual and dose-dependent reduction in total cellularity, whereas granulomonocyte progenitors (CFU-GM), virtually absent at the start of the culture, progressively reappear. The quantity of progenitors present after day 14 in liquid culture is, however, less in the treated marrows than in the controls, and the reappearance of CFU-GM is inversely related to the mafosfamide dose. In addition, the recovery of the more immature (day-14) CFU-GM is greater than that of the more mature (day 7) CFU-GM.
Bone Marrow↗
Interleukin 1 stimulates platelet activating factor production in cultured human endothelial cells.
Monocyte-derived interleukin 1 (IL 1) induced the platelet activating factor (PAF) production in cultured human endothelial cells (HEC). The product was identified as PAF by the chemical properties, the susceptibility to phospholipase A2 and C and to lipase A1, its behaviour in thin layer chromatography and in high pressure liquid chromatography and the recovery of biological activity tested on rabbit platelets. The action of IL 1 was concentration-dependent and took more than 2 hours to became apparent. Most of the PAF produced was cell-associated and only the 25% of the total was released.
Cells, Cultured↗
Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.
Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.
Amino Acids↗
Interleukin 1 stimulates platelet-activating factor production in cultured human endothelial cells.
Monocyte-derived interleukin 1 (IL-1) was found to be a potent inducer of platelet-activating factor (PAF) in cultured human vascular endothelial cells (HEC). The product was identified as PAF by its behavior in chromatographic systems, its recovery of biological activity, and its physico-chemical properties and susceptibility to lipases. The response of HEC to IL-1 was concentration-dependent, took more than 2 h to become apparent, and decreased after 18 h of incubation. Most of the PAF produced was cell-associated and only a small amount (about 25% of the total) was released in the culture medium. To study the mechanism of IL-1-induced HEC-PAF production we tested the activity of 1-O-alkyl-sn-glycero-3-phosphocholine:acetyl/coenzyme A acetyltransferase in HEC. Acetyltransferase activity measured in IL-1-stimulated HEC lysates showed a three to five times greater maximum velocity, but the same Michaelis constant, as untreated cells. The regulation of PAF generation in HEC by IL-1 may be an important aspect of the two-way interaction between immunocompetent cells and vascular tissue.
Acetyl Coenzyme A↗
Human gamma interferon enhances release from phytohemagglutinin-stimulated T4+ lymphocytes of activities that stimulate colony formation by granulocyte-macrophage, erythroid, and multipotential progenitor cells.
Human gamma interferon (HuIFN gamma) was evaluated for its effects on the release from human peripheral blood T lymphocytes (greater than 98% pure) stimulated by phytohemagglutinin (PHA) of activities that can stimulate granulocyte-macrophage (CFU-GM) colonies and clusters, erythroid (BFU-E) bursts, and mixed (CFU-GEMM) colonies. T lymphocytes did not release these activities in the absence of PHA with or without HuIFN gamma. In the presence of PHA, pure natural HuIFN gamma at concentrations of 0.1 to 100 U/mL significantly enhanced release of these colony-stimulating activities. Although enhanced release of granulocyte-macrophage colony-stimulating activities were noted when T lymphocytes were added to the conditioning medium in the presence of 0.1%, 0.5%, and 1% PHA, enhanced release of burst-promoting and mixed colony activities was seen only in the presence of 0.1% and 0.5% PHA. The enhanced release of colony-stimulating activities was not due to HuIFN gamma-suppression of the release from PHA-stimulated T lymphocytes of suppressor molecules. The enhancing effects of natural HuIFN gamma were neutralized with a monoclonal anti-natural HuIFN gamma, and recombinant HuIFN gamma mimicked the enhancing effects of the natural HuIFN gamma. This enhancing effect was noted only when HuIFN gamma was added with the T lymphocytes and PHA during the first 24 hours of incubation. T lymphocytes were separated into T4+, T8-, T8+, and T4- subsets (greater than 98% pure for the appropriate phenotypes) after incubation with OKT4- and OKT8- monoclonal antibodies and sorting on a fluorescence-activated cell sorter (FACS). All types of colony-stimulating activities were released from each population after stimulation with PHA, but enhanced release of these activities in the presence of HuIFN gamma was only detected with the T4+ or T8- subsets of lymphocytes. It cannot be concluded from these studies whether HuIFN gamma is enhancing the release of one or several types of colony-stimulating activities, but these studies suggest a role for HuIFN gamma and T4+ lymphocyte subsets in the regulation in vitro of the release of colony-stimulating activities.
Colony-Forming Units Assay↗
Expression of HLA class II (DR, DQ) determinants by normal and chronic myeloid leukemia granulocyte/monocyte progenitors.
It has been suggested that the expression of certain HLA class II antigens stemming from three distinct loci (DR, DP, and DQ) is important not only in the regulation of the immune response but also on the response of hemopoietic precursors to factors inhibiting myelopoiesis. Changes in the expression of DR antigens may be involved in the pathogenesis of altered cell proliferation in chronic myeloid leukemia, since they result in decreased sensitivity of the colony forming units, granulocyte-macrophage to prostaglandin E and acidic isoferritins. In studies using monoclonal antibodies against monomorphic DR or DQ determinants, in a complement-dependent cytotoxic assay, it was found that nearly all normal and chronic myeloid leukemia bone marrow colony forming units, granulocyte-macrophage express DR antigens. The dose response curve was similar for both normal and leukemic precursors. Leukemic peripheral blood precursors were more sensitive than were normal peripheral blood precursors. Normal colony forming units, granulocyte-macrophage did not express DQ antigens, whereas these were expressed in varying quantities by leukemic cells. This study shows that, in the patients we studied, leukemic cells express DR antigens in amounts comparable to normal. In addition, varying amounts of DQ antigens may be observed on leukemic but not on normal progenitors, perhaps as a consequence of an increase in the number of antigens also expressed by normal cells, though in an amount below the detection threshold of cytotoxicity techniques.
Animals↗
Effect of acidic and basic isoferritins on in vitro growth of human granulocyte-monocyte progenitors.
Acidic isoferritins have been previously found to be highly potent inhibitors of hematopoietic progenitors at concentrations of 10(-16) to 10(-18) mol/L, and it has been suggested that acidic isoferritin inhibitory activity plays a role in the regulation of normal hematopoiesis and also in the pathogenesis of leukemia. To characterize the ferritin species that affect the in vitro growth of human colony-forming unit-granulocyte-macrophage (CFU-GM), we tested different preparations of basic (L-subunit-rich) and acidic (H-subunit-rich) isoferritins. Three preparations of human liver (basic) ferritin did not show any effects on CFU-GM growth at concentrations up to 10(-9) mol/L, irrespective of the degree of glycosylation. Acidic isoferritins were purified both from HeLa cells and human heart. HeLa cell ferritin did not affect in vitro colony formation. One of two preparations of human heart ferritin, containing 5% glycosylated ferritin, showed a mean inhibition of 26% +/- 8% of the control at 10(-9) mol/L (P less than .02), whereas the other preparation, which contained no glycosylated ferritin, did not show any effect of CFU-GM growth. A preparation enriched for glycosylated acidic isoferritins from human heart was found to produce a mean inhibition of 32% +/- 11% of the control at 10(-9) mol/L (P less than .01), whereas another one was ineffective. A significant part of the inhibitory activity was removed by preincubation with the monoclonal antibody 2A4 directed against human heart ferritin. The present findings indicate that basic isoferritins, ie, the predominant ferritin type in human blood, have no effect on the growth of human CFU-GM, and this is in keeping with indirect clinical evidence. Inhibition of colony formation may be obtained by some preparations of acidic isoferritins that are rich in H subunits and bind to concanavalin A. The mechanism(s) responsible for this are not clear, but the effective concentrations are higher than those found in human blood both under normal conditions and in leukemia. At present, the physiologic significance of the observed inhibitory activity is uncertain.
Ferritins↗
H2 receptor antagonists and human granulopoiesis.
The effect of 2 H2 receptor antagonists (ranitidine and cimetidine) on the in vitro growth of human granulomonopoietic precursors (CFU-GM) was studied. Ranitidine, although having an anti H2 receptor activity much greater than that of cimetidine, displays the same toxicity for CFU-GM.
Cimetidine↗
In-vitro effect of retinoic acid on normal and chronic myeloid leukemia granulopoiesis.
The effect of increasing concentrations of retinoic acid (RA) on the in-vitro proliferation of normal and chronic myeloid leukemia (CML) granulo-monocyte precursors (CFU-GM) was studied. 10(-7)M RA added to semisolid cultures stimulated the growth of day 14 but not of day 7 normal CFU-GM, whereas in CML the growth of both populations was either unchanged or inhibited. Five-day and 10-day preincubation of normal bone marrow cells with RA augmented the number of day 14 CFU-GM (by up to 187% with 10(-6) M RA), whereas there was a marked decrease when CML cells were used. Total cellularity was not much affected, though a slight increase in liquid normal bone marrow cultures and a slight fall in CML cultures could be detected. These data point to a difference in the response to RA of normal and CML precursors. They may offer of preclinical basis for its employment to delay the blastic progression of CML.
Cell Differentiation↗
Alkyl-ether phosphoglycerides influence calcium fluxes into human endothelial cells.
Suspended or adherent human endothelial cells (HEC) treated with 5 to 100 nM 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, platelet-activating factor) showed a marked concentration and temperature-dependent increase in calcium uptake. This effect was also elicited by some AGEPC analogs. At 10 nM, the relative potencies were AGEPC = 100; 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid (AGEPA) = 52.9; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine (AGEPE) = 20; 1-O-octadecyl-2-deoxy-2-acetamido-sn-glycero-3-phosphocholine (2-acetamido-analog)-inactive at 100 nM = 25; 1-octadecyl-2-methoxy-sn-glycero-3-phosphocholine(2-methoxy analog)-inactive, and at 100 nM = 50. 1-O-octadecyl-2-lyso-sn-glycero-3-phosphocholine(lyso-GEPC) (100 nM) was inactive. The increase in calcium uptake was accompanied by a rise in membrane-associated calcium. The ratio between nonmembrane-bound intracellular calcium and membrane-associated calcium was constant for all agonists. CV-3988, a specific AGEPC antagonist, inhibited the effect of AGEPC. Preexposure of adherent HEC to AGEPC inhibited calcium uptake upon subsequent stimulation, suggesting a deactivation of the putative receptor. AGEPC (5 to 100 nM), but not lyso-GEPC, also stimulated calcium-efflux from calcium-preloaded, adherent HEC. AGEPC and 2-acetamido-analog, at concentrations able to induce calcium influx, did not elicit the production of 6-keto-PGF1 alpha.
Calcium↗