concanavalin-a and Leukemia--Myeloid

Topics: Animals; Binding Sites; Carcinogens; Cell Adhesion; Cell Differentiation; Cell Membrane; Cell Transformation, Neoplastic; Chromosomes; Concanavalin A; Fibroblasts; Granulocytes; Hematopoietic Stem Cells; Lectins; Leukemia; Leukemia, Experimental; Leukemia, Myeloid; Macrophages; Models, Biological; Neoplasms; Neoplasms, Experimental; Phenotype; Polyomavirus; Simian virus 40

alpha-Melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide derived from pro-opiomelanocortin, has potent antiinflammatory activity in laboratory animals. alpha-MSH inhibits nitric oxide production by murine macrophages, an influence believed to reflect activation of an autocrine circuit in these cells, one that is based on production and release of alpha-MSH and subsequent stimulation of melanocortin receptors. We found that THP-1 cells, human monocytic cells, produced alpha-MSH; this production was increased by interleukin-6, tumor necrosis factor a, or concanavalin A. These cells also expressed the gene for the human alpha-MSH receptor MC1. Unlike murine macrophages, THP-1 cells produced little nitrite in response to interferon-gamma (IFN-gamma) and lipopolysaccharide, and a-MSH inhibited this production only slightly. However, production of neopterin, a presumed primate homologue of nitric oxide in lower animals, was increased in THP-1 cells stimulated with INF-gamma plus TNF-alpha and alpha-MSH significantly inhibited this production. The evidence indicates that an autocrine regulatory circuit based on alpha-MSH occurs in human monocyte/macrophages much as in murine macrophages. alpha-MSH-induced modulation of specific inflammatory mediators/cytotoxic agents appears to differ depending on the importance of the mediators in the myelomonocytic cells of different species.

Topics: alpha-MSH; Amino Acid Sequence; Animals; Base Sequence; Biopterins; Concanavalin A; Humans; Interferon-gamma; Leukemia, Myeloid; Lipopolysaccharides; Macrophages; Mice; Molecular Sequence Data; Monocytes; Neopterin; Nitrites; Polymerase Chain Reaction; Receptors, Corticotropin; Receptors, Melanocortin; Receptors, Pituitary Hormone; RNA, Messenger; Stimulation, Chemical; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Topics: Animals; Antineoplastic Agents; Blotting, Northern; Colony-Forming Units Assay; Concanavalin A; Cytokines; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunologic Factors; Interleukin-3; Leukemia, Myeloid; Lipopolysaccharides; Mice; Phosphorylcholine; Rats; Receptors, Granulocyte Colony-Stimulating Factor; Recombinant Proteins; RNA, Messenger; Tumor Necrosis Factor-alpha

Western blotting, indirect immunolocalization, flow cytometry, and a functional assay for drug-induced strand breakage were utilized to examine topoisomerase (topo) II levels during granulocytic maturation in HL-60 human progranulocytic leukemia cells and in samples of normal human marrow. Indirect immunofluorescence revealed that the intensity of the signal for topo II in unsynchronized log phase HL-60 cells varied widely. Indirect immunolabeling combined with propidium iodide staining and two-parameter flow cytometry revealed that topo II levels increased an average of 2-fold as cells progressed from G1 to G2/M. When HL-60 cells were induced to mature toward granulocytes, topo II levels progressively decreased and became undetectable by functional assays, by indirect immunoperoxidase staining, and by Western blotting with an antibody which identified Mr 170,000 and Mr 180,000 forms of topo II. Similar changes were detected during normal granulocytic maturation in human marrow in vivo. Western blotting revealed that levels of the Mr 170,000 (proliferation-associated) isoform of topo II were highest in marrow fractions enriched in progranulocytes and myelocytes, intermediate in unfractionated marrow from normal volunteers, and undetectable in mature granulocytes. The Mr 180,000 topo II polypeptide was also diminished or absent from mature granulocytes. In further experiments, marrow samples from normal volunteers were subjected to flow cytometry after labeling of topo II and various cell surface markers. Levels of the Mr 170,000 topo II polypeptide in CD34-positive cells (multipotent and committed progenitors from several hematopoietic lineages) were indistinguishable from levels observed in the HL-60 leukemia cell line. These results suggest that topo II levels in highly proliferative normal human myeloid cells in vivo approach levels found in corresponding neoplastic cell lines in vitro. Conversely, as the same cells mature into granulocytes in vivo or in vitro, levels of both molecular weight forms of topo II diminish. These results provide a framework for the further investigation of topo II levels and drug sensitivity in human leukemia.

Topics: Antigens, CD; Bone Marrow; Bone Marrow Cells; Cell Cycle; Cell Differentiation; Concanavalin A; DNA Topoisomerases, Type II; Granulocytes; Hematopoiesis; Humans; In Vitro Techniques; Lamins; Leukemia, Myeloid; Lymphocyte Activation; Lymphocytes; Molecular Weight; Nuclear Proteins; Tumor Cells, Cultured

A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.

Topics: Animals; Cell Division; Cell Survival; Colorimetry; Coloring Agents; Concanavalin A; Dose-Response Relationship, Drug; In Vitro Techniques; Interleukin-2; Interleukin-4; Leukemia, Myeloid; Mast Cells; Methylphenazonium Methosulfate; Mice; Spleen; T-Lymphocytes; Tetrazolium Salts; Thiazoles; Vitamin K

Using five different cell lines as reactive cells, the proliferative inhibitory effect of Con A induced suppressive cells (CISC) was observed. The percentage of inhibition of MLA144 was 93.1%, K562 77.5%, Raji 62.5%, Molt-4 46.4%, and P388 25.9%. It is possible that K562 and MLA144 can be used as reactive cells instead of the peripheral blood mononuclear cells. Supernatants of CISC were also shown to be inhibitory, although less so than CISC. The inhibitory activity of CISC did not seem to be related to prostaglandin. However, its activity could be diminished by anti-T11 monoclonal antibody and complement. These results indicate that CISC is a T11-positive lymphocyte population. The inhibitory activity of CISC from 48 normal donors was determined by this modified method and compared with that of 22 patients with non-Hodgkin's lymphoma. The CISC activity of the patients was significantly lower than that of the normal subjects. Addition of exogeneous IL-2 to the culture enhanced the inhibitory activity in both groups.

Topics: Animals; Antibodies, Monoclonal; Concanavalin A; Humans; Interleukin-2; Leukemia, Myeloid; Lymphoma; T-Lymphocytes, Regulatory; Tumor Cells, Cultured

The effect of TBG on the functional activity of different cell lines, spontaneous and Con A induced proliferation of PBL was studied. If concentration of TBG is higher than 50 mu kg/ml it suppresses the proliferation in many used cell lines, except choriocarcinoma and cancer of uterus. The reliable increasing of spontaneous proliferation of PBL, Jurkat and K-562 cells may be observed if concentration is more lower (0.5-15 mu kg/ml). However proliferation of other cell lines corresponds to control level, and Con A induced proliferation of PBL is inhibited. The effect was more marked at 48, as compared to 24 hours of cell incubation with TBG.

Topics: Biomarkers, Tumor; Cell Division; Cell Line; Choriocarcinoma; Concanavalin A; Female; HeLa Cells; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid; Leukemia, T-Cell; Lymphocytes; Lymphoma, B-Cell; Lymphoma, T-Cell; Pregnancy-Specific beta 1-Glycoproteins; Tumor Cells, Cultured; Uterine Neoplasms

A cytostatic factor (CF) with a molecular mass of 50 kDa was purified to more than 16,000-fold from the conditioned medium of LPS-treated mouse myelomonocytic leukemia (WEHI-3) cell cultures. The activity of CF was completely destroyed by heating at 70 degrees C for 10 min, 50 degrees C for 30 min, or by the treatment in pH 3 buffer for 2 h. CF showed a strong growth inhibitory effect on CHO cells, as well as several other unrelated cell lines, e.g., K562 and L1210, but not on L929 cells. It also inhibited Con A-induced mitogenesis in mouse and rat spleen cells. The growth inhibitory effect of CF, however, was highly reversible; CHO cells were able to regain the normal growth after removal of the factor from the culture medium. Our results suggest that CF is a protein secreted by WEHI-3 cells, which is distinct from other known macrophage- or tumor-derived cytotoxic proteins.

Topics: Animals; Antineoplastic Agents; Cell Division; Cell Line; Concanavalin A; Leukemia, Myeloid; Mice; Molecular Weight; Neoplasm Proteins; Spleen; Tumor Cells, Cultured

Isolated granulocytes from normal individuals and patients suffering from chronic myeloid leukemia (CML) displayed different fluorescent patterns on treatment with fluorescein isothiocyanate concanavalin A (Fl-Con A). The ligand was internalized by 86% of the normal granulocytes, while 80% of the leukemic granulocytes exhibited Fl-Con A localized on the cell periphery. In further experiments, pretreatment of the normal granulocytes with cytochalasin B, iodoacetamide, 2-deoxyglucose and sodium fluoride (but not with sodium azide or dinitrophenol) was found to drastically inhibit internalization of the ligand. However, pretreatment of granulocytes from CML patients with cytochalasin B and 2-deoxyglucose, caused only a little alteration in the pattern of Fl-Con A labelling relative to untreated cells. These results indicate that CML granulocytes are defective in their ability to endocytose Fl-Con A. We suggest that this differential interaction between Fl-Con A and normal and leukemic granulocytes is a convenient system to study the initial steps in receptor mediated endocytosis of Concanavalin A.

Topics: Azides; Cell Differentiation; Cell Membrane; Colchicine; Concanavalin A; Cytochalasin B; Endocytosis; Fluorescein-5-isothiocyanate; Fluoresceins; Granulocytes; Histocytochemistry; Humans; Iodoacetamide; Kinetics; Leukemia, Myeloid; Sodium Azide; Sodium Fluoride; Time Factors

The effects of BM 12 531, a 2-cyanaziridinyl derivative, on in vitro generation of Con A induced suppressor cells as well as generation of spontaneous suppressor cells in peripheral blood lymphocytes of 9 chronic myeloid leukemia (CML) patients in remission, 9 patients with active Hodgkin's disease (HD) and 12 normal healthy donors were studied. The suppressor cells generated spontaneously and with Con A, in the presence and absence of the drug, were tested for their modulating effect on mitogenic (PHA) response of autologous lymphocytes. The results indicate that the addition of the drug at the time of generation of suppressor cells decreased the spontaneous suppressor cell activity in 2/4 healthy donors, 3/7 CML patients and 4/6 HD patients. Con A induced suppressor cell activity generated in presence of the drug was significantly reduced in 7/12 healthy donors, 6/9 CML patients and 3/9 HD patients.

Topics: Aziridines; Azirines; Cells, Cultured; Concanavalin A; Hodgkin Disease; Humans; Leukemia, Myeloid; Lymphocyte Activation; T-Lymphocytes, Regulatory

Cell susceptibility to agglutination mediated by a plant lectin, concanavalin A (Con A), and the binding capacity of Con A to cells following gamma-irradiation have been examined in mouse myeloid leukaemia cells cultured in suspension. Irradiation caused an immediate decrease in the amount of Con A bound to the cell surface, whereas susceptibility of irradiated cells to agglutination by Con A was unchanged when compared to that of the unirradiated cells. Post-irradiation incubation of cells at 37 degrees C resulted in a temporary, more than 1.3-fold increase in cell susceptibility to agglutination 60 min after irradiation, whereas binding capacity of cells for Con A gradually recovered following irradiation, reaching a comparable level to that of unirradiated cells 3 h after irradiation. Cell susceptibility to agglutination by Con A does not depend strongly on its binding capacity.

Topics: Agglutination; Animals; Cell Line; Cell Membrane; Cobalt Radioisotopes; Concanavalin A; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Gamma Rays; In Vitro Techniques; Leukemia, Myeloid; Mice

Using the fluorescent activity staining procedure, transglutaminases in human monocytes, granulocytes and lymphocytes have been characterized with respect to agarose gel electrophoretic mobility and thrombin dependence. A thrombin dependent transglutaminase was found in concanavalin A stimulated peripheral monocytes. The electrophoretic mobility of this zymogen and of platelet and plasma factor XIII was similar. With stimulated peripheral lymphocytes a similar pattern was observed. However, the potential transglutaminase activity in the lymphocyte factor XIII was lower than that in monocytes. Similar results were obtained when lymphocytes from chronic myeloid and chronic lymphocytic leukaemia patients were examined. Quantitatively, the transglutaminase activity in the leukaemic cells was estimated to be lower than the activity in normal cells. With respect to agarose gel electrophoretic mobility, a similar transglutaminase was found in concanavalin A stimulated human peripheral granulocytes. Although electrophoretically clearly separated from tissues transglutaminase, the granulocyte enzyme did not seem to require thrombin for activation.

Topics: Acyltransferases; Blood Platelets; Cell Line; Concanavalin A; Electrophoresis, Agar Gel; Erythrocytes; Granulocytes; Humans; Leukemia, Lymphoid; Leukemia, Myeloid; Leukocytes; Lymphocyte Activation; Monocytes; Transglutaminases

In the present paper attempts have been made to investigate suppressor cell activity in CML patients in first and subsequent remissions in order to study the relationship between suppressor cell activity and progression of the disease. For this purpose, the ability of Con A activated suppressor cells from peripheral blood of CML patients in 1st, 2nd and 3rd remission to suppress PHA response of autologous lymphocytes is investigated and compared with that of normal healthy donors. The ability of Con A activated cell population to form rosettes with autologous RBCs (ARFC) is also investigated. The results indicate that lymphocytes from CML patients in 1st (61.8 +/- 6.1%), 2nd (62.6 +/- 3.0%) and 3rd (55.3 +/- 4.8%) remissions show significantly high suppressor cell activity than normal healthy donors (36.5 +/- 1.9%) when activated with Con A. Similarly, generation of spontaneous suppressor cell activity was also higher in 1st (23.3 +/- 4.7%) and 2nd (25.3 +/- 4.2%) remission lymphocytes than controls (10.1 +/- 2.5%). In the 3rd remission however, the spontaneous suppressor cell activity (14.5 +/- 3.2%) was comparable to controls. Thus it appears that a higher suppressor cell precursor population is present in CML patients in remission. However, this could not be correlated with the progression of the disease. CML patients in 1st remission also revealed an increased percentage of ARFC which correlated with the suppressor cell function. The ARFC activity tested in a few patients in subsequent remissions was comparable with controls although functional suppressor activity was increased.

Topics: Cells, Cultured; Concanavalin A; Humans; Leukemia, Myeloid; Lymphocyte Activation; Rosette Formation; T-Lymphocytes, Regulatory

A panel of lectins was used to analyse glycoproteins of normal granulocytes and leukaemic myeloid cells. The glycoproteins of detergent-solubilized whole cells were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their lectin-binding properties determined by incubation of the fixed gels with radioiodinated lectins. Normal granulocytes and leukaemic myeloid cells in different stages of maturation possess a cell-surface sialic acid-rich glycoprotein of apparent mol.wt. 115 000 (GP115), that can be labelled by both the lactoperoxidase and periodate/NaB3H4 cell-surface labelling techniques. The sialoglycoprotein of leukaemic myeloblasts has a slightly lower apparent mol.wt., 112000 (GP112). After neuraminidase treatment before cell solubilization, both GP115 and GP112 bind the lectins from Arachis hypogaea (peanut) and Helix pomatia (snail) and have an increased apparent molecular weight of 125000. Two concanavalin A-binding glycoproteins of apparent mol.wts. 98000 and 90000 are present in leukaemic myeloblasts. Concanavalin A binding to these glycoproteins is decreased in more mature leukaemic cells and absent in granulocytes. As concanavalin A binding decreases in the maturer forms, there is a concomitant increase in the binding of Ricinus communis (castor bean) and Maclura aurantiaca (osage orange) lectins to these glycoproteins. Whole granulocytes, but not leukaemic myeloblasts, contain a major cell-surface concanavalin A binding glycoprotein of apparent mol.wt. 130000, which is labelled by the periodate/NaB3H4 technique. Concanavalin A binding to this glycoprotein increases as the morphology of leukaemic cells approaches that of mature granulocytes.

Topics: Cell Membrane; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Granulocytes; Humans; Lectins; Leukemia, Myeloid; Membrane Proteins; Molecular Weight

Natural killer (NK) cell activity was studied in 23 patients with systemic lupus erythematosus (SLE). The overall NK activity was lower in patients with SLE than in normal female individuals. Patients with clinically active SLE disease had slightly lower NK activity than the patients with inactive disease. Other clinical parameters as well as treatment status did not correlate with NK activity. Interferon (IFN) enhanced the NK activity of normal individuals and of 11 SLE patients, while it did not enhance in the remaining 12 patients. The patients whose NK activity was enhanced by beta-IFN had significantly higher initial activity than those who did not respond to beta-IFN. Furthermore, peripheral mononuclear cells (MNC) from IFN responders produced gamma-IFN after stimulation with concanavalin A (Con A) in titres comparable to those of normals. In contrast, peripheral MNC from beta-IFN non-responders failed to produce significant titres of gamma-IFN after stimulation with Con A. These results indicate that certain patients with SLE have low NK activity, which is generally paralleled by an inability to respond to exogenous beta-IFN and by blunted production of gamma-IFN after stimulation with Con A.

Topics: Cells, Cultured; Concanavalin A; Female; Humans; Interferon Type I; Interferon-gamma; Killer Cells, Natural; Leukemia, Myeloid; Leukocytes; Lupus Erythematosus, Systemic

Conditioned media of mitogen- or antigen-stimulated spleen cells have been found to contain various lymphokines including macrophage activating factor (MAF), immune interferon (IFN-gamma) and a factor inducing differentiation of mouse myeloid leukemic M1 cells into macrophages and granulocytes (D-factor). We examined the properties and mutual relations of these lymphokines. Conditioned media of concanavalin A (Con A)-stimulated spleen cells and of purified protein derivative (PPD)-stimulated Bacillus Calmette-Guérin (BCG)-immune spleen cells contained the activities of D-factor, MAF and IFN. These lymphokines were similarly eluted on Sephadex G-100 in a peak corresponding to a molecular weight of 40,000 approximately 60,000. However, a rapidly eluted fraction contained MAF but not activities of D-factor and IFN. Treatment of conditioned medium of Con A-stimulated spleen cells at pH 2 abolished the activities of MAF and IFN but did not affect the activity of D-factor. Moreover, addition of cytochalasin B suppressed the productions of MAF and IFN but not that of D-factor by Con A-stimulated spleen cells. Antiserum against mouse IFN-gamma neutralized IFN activity but not MAF activity in the conditioned medium of Con A-stimulated spleen cells. These results indicate that D-factor, MAF and IFN-gamma are all distinct substances.

Topics: Animals; Cell Differentiation; Concanavalin A; Glycoproteins; Growth Inhibitors; Interferon-gamma; Interleukin-6; Leukemia Inhibitory Factor; Leukemia, Myeloid; Lymphokines; Macrophage-Activating Factors; Macrophages; Mice; Molecular Weight; Spleen

A monoclonal antibody with a broad anti-human leucocyte specificity, designated BK 19.45 and the plant lectin Concanavalin A have been labelled with the alpha-emitting cyclotron produced radiohalogen astatine-211. Both human and murine tumour cell lines and human leukemic bone marrow samples have been specifically labelled with these radioactive proteins. In all cases the amount of 211At bound to the cells is directly correlated with a decrease in cellular reproductive potential as shown by the ability of the cells to proliferate in a clonogenic assay. For the labelled monoclonal antibody experiments, the radiation dose needed to yield 37% cell survival, the D37 dose, may be achieved with an average of 12 211At atoms/cell.

Topics: Animals; Antibodies, Monoclonal; Astatine; Cell Line; Cell Survival; Colony-Forming Units Assay; Concanavalin A; Dose-Response Relationship, Radiation; Humans; Lectins; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Mice; Plasmacytoma

Topics: Animals; Cell Line; Clone Cells; Colony-Stimulating Factors; Concanavalin A; Culture Media; Growth Substances; Hematopoietic Stem Cells; Hydrogen-Ion Concentration; Leukemia, Myeloid; Macrophages; Mice; Molecular Weight; Temperature; Trypsin

Culture studies of peripheral blood mononuclear cells from seven typical cases of B-cell chronic lymphatic leukaemia (CLL) showed that, in the presence of PHA or Con A and greater than or equal to 20% autologous or allogeneic T-cells, CLL cells express true sheep erythrocyte (E) receptor after 2-5 d, but fail to stain with a panel of anti-T-cell sera. In contrast to similarly cultured hairy cells (HCs), the cultured CLL cells continue to express light-chain-restricted surface immunoglobulin (SIg) and show no change in their morphology. The continued SIg positivity and staining with a panel of monoclonal antibodies indicated that the high proportion of E+ cells could not be attributed to the outgrowth of normal T-cells or to mitogen-induced non-specific stickiness. Repeated addition of supernatants from cultured T-cells caused no phenotypic changes and therefore excluded the possibility of shedding and passive acquisition of E receptor. The points of similarity and difference between the responses of CLL cells and HCs cultured in a like manner are briefly considered in relation to the possible lineages of these two cell types.

Topics: B-Lymphocytes; Cells, Cultured; Concanavalin A; Erythrocytes; Humans; Leukemia, Myeloid; Leukocyte Count; Lymphocyte Activation; Phytohemagglutinins; Receptors, Antigen, B-Cell; Receptors, Immunologic; Rosette Formation; T-Lymphocytes

Procedures are described for the isolation of a mast cell growth factor (MCGF) from medium conditioned by mitogen-activated splenic leukocytes (CM). Although optimal conditions for the production of MCGF in CM are identical to those for the production of T cell growth factor (TCGF), MCGF can be dissociated from TCGF after the first stage of purification on a DEAE-cellulose column. MCGF elutes from the column in the breakthrough fraction, whereas TCGF binds avidly to DEAE and is eluted only at high salt concentration. MCGF also differs from TCGF with respect to m.w. (as estimated by Sephadex G-150 chromatography) and sensitivity to trypsin. In addition, MCGF is produced by the murine myelomonocytic leukemia WEHI-3 and the radiation induced thymic lymphoma LBRM-33 cells, whereas TCGF is produced only by the latter in the presence of a mitogen. Another hemopoietically active factor, granulocyte colony-stimulating factor (G-CSF) present in media conditioned by WEHI-3 and LBRM-33 cells, however, shares a number of properties with MCGF. Although studies with purified or partially purified MCGF have thus far failed to reveal a correlation between MCGF and G-CSF, further biochemical analyses are necessary to dissociate MCGF from G-CSF.

Topics: Animals; Cell Differentiation; Cells, Cultured; Chromatography, DEAE-Cellulose; Chromatography, Gel; Colony-Stimulating Factors; Concanavalin A; Female; Growth Substances; Interleukin-3; Leukemia, Myeloid; Lymphoma; Male; Mast Cells; Mice; Mice, Inbred DBA; Mice, Nude; Time Factors

T lymphocytic colony formation by peripheral lymphocytes separated by discontinuous albumin gradient centrifugation was evaluated in 8 patients with Philadelphia (Ph1)-positive chronic myeloid leukemia (CML). Colonies were obtained using a liquid-on-agar culture system recently introduced (PHA overlayer-leukocyte feeder layer assay) which has been shown to be simple and reliable. The pattern of colony growth in CML and in normal controls was similar, the peak ranging from the 4th to the 6th day. Also the morphological aspects of colonies did not differ in the two groups. Cells recovered from CML lymphocytic colonies were shown to belong to T cell lineage, as they are able to form spontaneous E-rosettes and to respond to mitogenic stimulation in vitro. In contrast, cells recovered from all other cultured fractions failed to display these properties. Cytogenetic analysis showed that T colony cells were Ph1-negative whereas the chromosome anomaly was found in nonlymphoid colonies of the same patients, thus suggesting a nonclonal origin of T lymphocytes in CML.

Topics: Agar; Cell Transformation, Neoplastic; Cells, Cultured; Centrifugation, Density Gradient; Chromosomes, Human, 21-22 and Y; Concanavalin A; Humans; Leukemia, Myeloid; Phytohemagglutinins; Pokeweed Mitogens; Rosette Formation; T-Lymphocytes

Topics: Animals; Cell Differentiation; Cell Line; Cell Membrane; Concanavalin A; Female; G(M1) Ganglioside; Glycosphingolipids; Guinea Pigs; Leukemia, Experimental; Leukemia, Myeloid; Lymphocytes; Lymphokines; Macrophages; Mice; Spleen; Tuberculin

Mouse myeloid leukemic Ml cells can be induced to differentiate into macrophages and granulocytes in vitro by a factor(s) stimulating differentiation of the cells (D-factor) or by various chemical compounds. Spleen lymphocytes and spleen macrophages have been shown to produce D-factor when treated with various mitogens including lipopolysaccharide or synthetic double-stranded polyribonucleotide, poly(I) . poly(C). Several immunostimulants from microorganisms were tested for ability to induce differentiation of Ml cells and to stimulate production of D-factor by spleen cells. Mycobacterium bovis BCG (500 micrograms/ml) and the cell wall skeleton of Nocardia rubra (50 micrograms/ml) induced differentiation of Ml cells; but Corynebacterium parvum CN6134 and the cell wall skeleton of Propionibacterium acnes C7 had no effect on Ml cells. On the other hand, all immunostimulants tested at concentrations of more than 10 micrograms/ml were found to stimulate production of D-factor by both spleen lymphocytes and spleen macrophages. Synthetic derivatives of N-acetylmuramyl dipeptide(MDP), the minimal adjuvant-active subunit of the bacterial cell wall, had no direct effect on the differentiation of Ml cells and only slightly stimulated the production of D-factor by spleen cells. Therefore, the structure of microorganisms required for induction of differentiation of Ml cells and for stimulation of production of D-factor are suggested to be different from that of MDP.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Antigens, Bacterial; BCG Vaccine; Cell Differentiation; Cell Wall; Cells, Cultured; Concanavalin A; Glycoproteins; Growth Inhibitors; Immunization; Interleukin-6; Leukemia Inhibitory Factor; Leukemia, Experimental; Leukemia, Myeloid; Lipopolysaccharides; Lymphokines; Mice; Nocardia; Poly I-C; Propionibacterium acnes; Spleen

Peripheral blood leucocytes from chronic myeloid leukaemia (CML) patients previously untreated or in relapse are agglutinated by concanavalin A (Con A) at concentrations of 25-400 microgram/ml. Leucocytes from normal subjects, from CML patients in remission and from patients with granulocytosis are not agglutinated by Con A. Normal bone marrow cells which contain immature and mature myeloid cells are not agglutinated either by Con A. These results suggest that an alteration occurs in the cell surface of myeloid cells in CML due to leukaemic change and results in the agglutination of these cells by Con A. If peripheral blood leucocytes of CML patients in relapse are separated into immature and mature fractions, both these fractions are agglutinated by Con A suggesting that this alteration occurs in the cell surface of both the immature and the mature myeloid cells.

Topics: Agglutination; Cell Membrane; Concanavalin A; Dose-Response Relationship, Drug; Granulocytes; Humans; Leukemia, Myeloid; Leukocyte Count

Topics: Animals; Concanavalin A; Cytotoxicity, Immunologic; Dose-Response Relationship, Immunologic; Fibroblasts; Humans; Immunoglobulin Fc Fragments; Interferons; Killer Cells, Natural; Kinetics; Leukemia, Experimental; Leukemia, Lymphoid; Leukemia, Myeloid; Lymphocytes

The mouse myeloid leukemia cell line (M1) is known to differentiate in vitro into macrophages and granulocytes upon treatment with various inducers including mouse ascitic fluid. Changes of cell surface proteins during differentiation of M1 cells were analyzed by the lactoperoxidase-catalyzed radioiodination method and SDS-polyacrylamide slab gel electrophoresis. Treatment of the cells with ascitic fluid changed the electrophoretic pattern of the iodinated proteins, the prominent change being the appearance of a new protein with a molecular weight of 180 000 (P180). Iodinated P180 was also detected in normal macrophages in granulocytes, which are similar to differentiated M1 cells. This protein was metabolically labeled with L-[14C]fucose, increasing with the period of the treatment. P180 was not expressed on ascitic fluid-treatment of a resistant clone of M1 cells that could not be induced to differentiate. These results indicate that P180 is a glycoprotein that is exposed on the outer surface of differentiated M1 cells, and that its expression is associated with differentiation of the cells. P180 was solubilized from 125I-labeled macrophages with detergents bound to concanavalin A-Sepharose. This suggests that P180 is one of the receptors for concanavalin A. Therefore, P180 may contribute partly to the increases in agglutinability by concanavalin A and in the number of concanavalin A binding sites on the surface of M1 cells, which are known to be associated with differentiation of M1 cells.

Topics: Agglutination; Animals; Cell Differentiation; Cell Membrane; Concanavalin A; Granulocytes; Kinetics; Leukemia, Myeloid; Macrophages; Membrane Proteins; Mice; Molecular Weight

Topics: Animals; Bone Marrow; Cell Separation; Cells, Cultured; Chromatography, Gel; Colony-Stimulating Factors; Concanavalin A; Dose-Response Relationship, Immunologic; Humans; Leukemia, Myeloid; Mice; Phagocytes; Spleen; Time Factors

Basophils possess membrane bound IgE molecules, and immunological activation leads to a secretory process with cell degranulation and histamine release. Heterologous anti IgE, concanavaline A, and phytohaemagglutinin are potent non-cytotoxic releasing agents. They operate by a mechanism similar to that of immunological activation. Heavy water is not a histamine releasing inducer but it increases histamine release of the cells. We studied the histamine release reaction of leukaemic basophils in 10 patients and found a physiological response such as that previously reported with normal human basophils.

Topics: Antibodies, Anti-Idiotypic; Basophils; Concanavalin A; Deuterium; Histamine Release; Humans; Immunoglobulin E; Leukemia, Myeloid; Phytohemagglutinins

Topics: Bone Marrow; Bone Marrow Cells; Child; Concanavalin A; Cytological Techniques; Horseradish Peroxidase; Humans; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute

The K562 cell line derived from a CML patient in blast crisis was examined for properties of B and T lymphocytes and cell lines. K562 lacks the B markers of immunoglobulins, Epstein-Barr virus (EBV) genome and associated nuclear antigen, and receptors for EBV. A low proportion of cells from rosettes with sheep erythrocytes, the frequency of which is considerably increased after neuraminidase treatment. Unlike B lines but like T lines, K562 cells are lysed rapidly by C'/Fc receptor-positive human blood leukocytes and do not stimulate MLC reactions. On the other hand, K562 lacks T antigen, high radiosensitivity and sensitivity to growth inhibition by thymidine. The cells do not contain N-APase, an enzyme found in all lines derived from lymphoid cells and in lymphoproliferative diseases. By scanning electron microscopy, K562 cells were seen to be rounded and relatively smooth, with small numbers of short microvilli resembling undifferentiated leukemic cells. A few cells had narrow ridge-like profiles and small ruffles similar to granulocytic leukemic cells. K562 is strongly positive for immunoglobuln Fc receptors and pinocytosis, but does not phagocytose or mediate antibody-dependent phagocytosis or cytolysis. Among histochemical stains, K562 is positive for esterase, lipid, and acid phosphatase. There seems to be no doubt that K562 is not a B cell line. While it has some T cell properties, these are not exclusive. Some of its characteristics indicate that it is probably not lymphoid. Due to its low level of differentiation, its nature cannot be stated with certainty. On the basis of the possible presence of the cellular marker of chronic myeloid leukemia, the Ph chromosome, it may be regarded as belonging to the granulocytic series of cells.

Topics: Alkaline Phosphatase; Antigens, Viral; B-Lymphocytes; beta 2-Microglobulin; Cell Line; Chromosomes; Concanavalin A; Humans; Immunoglobulin Fc Fragments; Immunoglobulins; Leukemia, Myeloid; Lymphocyte Activation; Lymphocytes; T-Lymphocytes

The ability of cells to bind to nylon fibers coated with lectin molecules interspaced with varying numbers of albumin molecules has been analyzed. The cells used were lymphoma cells, normal lymphocytes, myeloid leukemia cells, and normal and transformed fibroblasts, and the fibers were coated with different densities of concanavalin A or the lectins from soybean or wheat germ. Cells fixed with glutaraldehyde did not bind to lectin-coated fibers. The number of cells bound to fibers could be increased by increasing the density of lectin molecules on the fiber, the density of specific receptors on the cell, or the mobility of the receptors. It is suggested that binding of cells to fibers involves alignment and binding of specific cell surface receptors with lectin molecules immobilized on the fibers, and that this alignment requires short-range rapid lateral mobility (RLM) of the receptors. The titration of cell binding to fibers coated with different densities of lectin and albumin has been used to measure the relative RLM of unoccupied cell surface receptors for the lectin. The results indicate a relationship of RLM to lectin-induced cell-to-cell binding. The RLM or receptors for concanavalin A (Con A) was generally found to be higher than that of receptors for the lectins from wheat germ or soybean. Receptor RLM could be decreased by use of metabolic inhibitors or by lowering the temperature. Receptors for Con A had a lower RLM on normal fibroblasts than on SV40-transformed fibroblasts, and trypsinization of normal fibroblasts increased Con A receptor RLM. Normal lymphocytes, lymphoma cells, and lines of myeloid leukemia cells that can be induced to differentiate had a high receptor RLM, whereas lines of myeloid leukemia cells that could not be induced to differentiate had a low receptor RLM. These results suggest that the RLM of Con A receptors is related to the transformation of fibroblasts and the ability of myeloid leukemia cells to undergo differentiation

Topics: Animals; Binding Sites, Antibody; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Concanavalin A; Dinitrophenols; Fibroblasts; Glycine max; Lectins; Leukemia, Myeloid; Lymphocytes; Lymphoma; Mice; Models, Biological; Nylons; Plant Lectins; Rats; Serum Albumin, Bovine; Simian virus 40; Temperature; Triticum

The production of IgE-class antibody specific for polymyxin B is documented in an 18-year-old white female acute myelocytic leukemic patient in relapse. The patient was rendered T cell-deficient by total body X-irradiation and antihuman thymocyte globulin for the purpose of bone marrow transplatation. Thereafter, symptoms of nasal congestion, rhinorrhea, and perinasal urtication produced by topical application of a polymyxin solution were noted. Reaginic activity mediated by an IgE antibody against polymyxin is documented by Prausnbitz-Küstner-type passive transfer reactions and by an indirect hemagglutination technique developed for these studies. The occurrence of type I hypersensitivity to this topical antibiotic is rare. It is speculated that pharmaceuticals normally having a low sensitizing potential might demonstrate increased reaginic immunogenicity in a spontaneously or iatrogenically T cell-depleted patient.

Topics: Adolescent; Aerosols; Animals; Antibody Formation; Antibody Specificity; Antilymphocyte Serum; Bone Marrow Cells; Bone Marrow Transplantation; Concanavalin A; Female; Hemagglutination Tests; Hemocyanins; Humans; Immunization, Passive; Immunoglobulin E; Lectins; Leukemia, Myeloid; Lymphocyte Activation; Lymphocyte Depletion; Patient Isolators; Polymyxins; Rabbits; Radiation Effects; Remission, Spontaneous; Serum Albumin, Bovine; Skin Tests; T-Lymphocytes; Transplantation, Homologous

The cell-to-cell binding induced by concanavalin A (Con A) and the lectins from wheatgerm, soybean, and waxbean has been analyzed by measuring the ability of single cells to bind to lectin-coated cells immobilized on nylon fibers. The cells used were lymphoma, myeloid leukemia, and normal fibroblast cells. With all lectins, cell-to-cell binding was inhibited if both cells were prefixed with glutaraldehyde. However, in most cases cell-to-cell binding was enhanced when only the lectin-coated cell was prefixed. With normal fibroblasts, treatment of either one or both cells with trypsin enhanced the cell-to-cell binding induced by Con A and the wheatgerm lectin. Neuraminidase, which increases the number of receptors for soybean agglutinin, increased cell-to-cell binding only if both cells were treated. Although cell-to-cell binding induced by the lectins from soybean and wheatgerm could be partially reversed by the appropriate competitive saccharide inhibitor, binding induced by Con A could not be reversed. The experiments indicate that cell-to-cell binding induced by a lectin can be prevented by an insufficient density of receptors for the lectin, insufficient receptor mobility, or induced clustering of receptors. These effects can explain the differences in cell-to-cell binding and agglutination observed with different cell types and lectins. They also suggest that cell-to-cell binding induced by different lectins with a variety of cell types is initiated by a mechanism involving the alignment of complementary receptors on the colliding cells for the formation of multiple cell-to-lectin-to-cell bridges.

Topics: Agglutination; Animals; Binding Sites, Antibody; Cell Adhesion; Cell Line; Cells, Cultured; Clone Cells; Concanavalin A; Cricetinae; Fibroblasts; Glutaral; Glycine max; Lectins; Leukemia, Myeloid; Lymphoma; Mice; Models, Biological; Neuraminidase; Nylons; Plant Lectins; Triticum; Trypsin; Vegetables

Glucose utilization, energy metabolism and associated membrane changes, have been studied in D+ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D- myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D+ than in D- cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D- than in D+ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D+ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D+ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D- cells, were found before most of the D+ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D- cells to differentiate and the acquistion of the metabolic properties of normal granulocytes by differentiating D+ cells, were associated with differences in the organization of the cell surface membrane.

Topics: Adenosine Triphosphate; Agglutination; Cell Differentiation; Cell Line; Cell Membrane; Cholesterol; Concanavalin A; Culture Media; Cyanides; Deoxyglucose; Dinitrophenols; Fatty Acids; Fluorides; Glucose; Glycolysis; Hydrogen Peroxide; Iodoacetates; Lactates; Leukemia, Myeloid; Methylmannosides; Oligomycins; Sterols

Topics: Animals; Antigens, Neoplasm; Burkitt Lymphoma; Cell Line; Chromatography, Affinity; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Humans; Immune Sera; Iodine Radioisotopes; Leukemia; Leukemia, Myeloid; Mercaptoethanol; Molecular Weight; Papain; Rabbits; Sodium Dodecyl Sulfate; Urea

Topics: Animals; Cell Line; Concanavalin A; DNA, Neoplasm; Galactosamine; Galactose; Lectins; Leukemia, Myeloid; Lipopolysaccharides; Lymphoma; Mannose; Methylglycosides; Mice; Mitogens; Multiple Myeloma; Neoplasms, Experimental; Polysaccharides, Bacterial; Ricin; RNA, Neoplasm; Salmonella typhi; T-Lymphocytes

Topics: Cell Adhesion; Cell Line; Cells, Cultured; Concanavalin A; Cycloheximide; Dactinomycin; Edetic Acid; Galactose; Glass; Glycosides; Humans; Lectins; Leukemia, Myeloid; Lymphoma; Mannose; Mast-Cell Sarcoma; Microscopy, Phase-Contrast; Plant Lectins; Temperature; Trypsin; Vegetables