scholarly journals CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 682-688 ◽  
Author(s):  
RB Jenkins ◽  
WL Nichols ◽  
KG Mann ◽  
LA Jr Solberg
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Protein Synthesis ◽  
Bone Marrow Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Glycoprotein ◽  
Factor X ◽  
Membrane Glycoproteins ◽  
Molecular Weight Range

Abstract Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with greater than or equal to 95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1–1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1–1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1–1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.

scholarly journals CFU-M-derived human megakaryocytes synthesize glycoproteins IIb and IIIa

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 682-688 ◽  
Author(s):  
RB Jenkins ◽  
WL Nichols ◽  
KG Mann ◽  
LA Jr Solberg
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Protein Synthesis ◽  
Bone Marrow Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Glycoprotein ◽  
Factor X ◽  
Membrane Glycoproteins ◽  
Molecular Weight Range

Human megakaryocytes have been shown by immunofluorescent techniques to express platelet glycoprotein IIb/IIIa antigen. We report evidence that megakaryocytes derived from human committed megakaryocytic progenitor cells in vitro (CFU-M) synthesize glycoproteins IIb and IIIa. Nonadherent light-density human bone marrow cells were cultured in human plasma and methylcellulose using conditions that promote large megakaryocytic colonies. On day 13 the megakaryocytic colonies were picked, pooled, and pulsed with 35S-methionine in methionine-free media. Populations of approximately 100,000 cells with greater than or equal to 95% viability and containing 70% to 90% megakaryocytes were obtained reliably for study. After the radioactive pulse, the cell suspension was solubilized with nonionic detergent. To reduce nonspecific binding of 35S-labeled proteins to agarose, the lysate was chromatographed sequentially on glycine-quenched Affi-gel and antihuman factor X-Sepharose. The unbound material from these resins was then chromatographed on an antiglycoprotein IIb/IIIa monoclonal antibody resin (HP1–1D-Sepharose) or on a control monoclonal antibody resin. Bound fractions were eluted and analyzed by polyacrylamide gel electrophoresis and autoradiography. Autoradiograms of diethylamine eluates from HP1–1D-Sepharose revealed two labeled proteins with electrophoretic mobilities identical with those of human platelet membrane glycoproteins IIb and IIIa, isolated using similar conditions. Autoradiograms of material synthesized by control macrophages from the same donors revealed no significant labeling of proteins in the glycoprotein IIb/IIIa molecular weight range, nor were such proteins bound by HP1–1D-Sepharose. Our observations show that protein synthesis by CFU-M-derived human megakaryocytes can be readily studied using a small amount of bone marrow aspirate as starting material. This approach will allow the study of protein synthesis by megakaryocytes from normal subjects or from subjects with clinical disorders, and it will circumvent the need to obtain large amounts of bone marrow to prepare enriched populations of megakaryocytes.

scholarly journals A monoclonal antibody that recognizes B cells and B cell precursors in mice.

1981 ◽  
Vol 153 (2) ◽  
pp. 269-279 ◽  
Author(s):  
R L Coffman ◽  
I L Weissman
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Cell Lineage ◽  
Hematopoietic Stem ◽  
B Cell Precursors

The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

scholarly journals A glycoprotein inhibitor of in vitro granulopoiesis associated with AIDS

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1267-1272 ◽  
Author(s):  
IZ Leiderman ◽  
ML Greenberg ◽  
BR Adelsberg ◽  
FP Siegal
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Conditioned Media ◽  
Normal Individuals ◽  
Immunodeficiency Virus ◽  
Aids Related Complex ◽  
Significant Deficit ◽  
Normal Controls

Patients infected with the human immunodeficiency virus (HIV) often present with neutropenia. To elucidate the mechanism(s) of this HIV- related neutropenia, we assessed the proliferative capacity of the granulocyte-macrophage progenitor cell (CFU-GM) from the bone marrow (BM) of 78 patients within the AIDS spectrum manifesting symptoms or signs related to HIV infection. Of these, 70 had a significant deficit in the growth of this committed progenitor when compared with normal controls (P less than .01). Further analysis revealed that the nucleated bone marrow cells from AIDS and AIDS-related complex (ARC) patients inhibited the growth of CFU-GMs from normal individuals when cocultured in agar (P less than .001). Control CFU-GMs were also inhibited when they were cultured over feeder layers containing patients' BM cells (P less than .001). Conditioned media obtained from the liquid culture of patients' BM cells did not inhibit normal control CFU-GM growth to a degree different from that of the cells themselves (P greater than .4). Analysis of these conditioned media by polyacrylamide gel electrophoresis (PAGE) revealed a unique glycoprotein (gp) with a mol wt of 84 kd. Further studies revealed that this gp possessed the inhibitory activity. These data suggest that this gp may be an important factor in HIV-related neutropenia. The presence of gp84 was independent of drugs administered to the patients.

scholarly journals Immunofluorescent identification of human megakaryocyte colonies using an antiplatelet glycoprotein antiserum

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 277-286 ◽  
Author(s):  
EM Mazur ◽  
R Hoffman ◽  
J Chasis ◽  
S Marchesi ◽  
E Bruno
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Human Peripheral Blood ◽  
Immunofluorescent Staining ◽  
Platelet Glycoprotein ◽  
Plasma Clot ◽  
Phenotypic Marker ◽  
Vitro Assay

The development of a satisfactory in vitro assay system for human megakaryocyte colony forming progenitor cells has been delayed by the lack of a suitable marker for cells of human megakaryocyte lineage. For this purpose we raised an antiserum directed against a purified human platelet glycoprotein preparation. In conjunction with indirect immunofluorescent staining of human bone marrow, this antiserum labeled only platelets, megakaryocytes, and an infrequent population of small mononuclear cells. These small mononuclear cells, not otherwise identifiable as members of the megakaryocyte series, constituted 22.9% of the total fluorescein positive nucleated bone marrow cells. This antiserum was also used to label colonies cultured from human peripheral blood mononuclear cells using a modified plasma clot technique. A mean of 123 fluorescein-labeled colonies were cloned per 10(6) mononuclear cells cultured. Granulocyte-macrophage and erythroid burst colonies did not label using this method. No augmentation of colony numbers was found with varying concentrations of erythropoietin, human embryonic kidney cell conditioned media (a source of thrombopoietin), or media conditioned by a human T lymphoblast cell line (a source of both colony stimulating and burst promoting activities). Immunofluorescent labeling for platelet glycoproteins is a convenient phenotypic marker for cells of human megakaryocyte lineage useful in the study of in vitro human megakaryocytopoiesis.

scholarly journals Monoclonal antibody BA-1 does not bind to hematopoietic precursor cells

Blood ◽  
1982 ◽  
Vol 59 (5) ◽  
pp. 1029-1035 ◽  
Author(s):  
J Jansen ◽  
RC Ash ◽  
ED Zanjani ◽  
TW LeBien ◽  
JH Kersey
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Bone Marrow ◽  
Hematopoietic Precursor ◽  
Proliferation In Vitro

Abstract Monoclonal antibody BA-1 binds to B lymphocytes, to cells from most cases of non-T acute lymphoblastic leukemia (ALL), and weakly to neutrophils. To determine whether BA-1 also reacts with hematopoietic progenitor cells (HPC), we studied the effect of removal of BA-1+ cells from human bone marrow on the proliferation in vitro of the trilineage precursor cell CFU-GEMM, and on the committed progenitor cells of granulopoiesis (CFU-C) and erythropoiesis (BFU-E/CFU-E). Complement- mediated cytotoxicity using BA-1 at concentrations far beyond those required to lyse BA-1+ bone marrow cells and ALL cells did not result in inhibition of colony formation in any of the assays. A rosette separation method, using ox red blood cells coated with BA-1, resulted in enrichment of HPC in the BA-1-depleted interface, whereas very few HPC were found in the BA-1-enriched pellet. Both methods indicate that BA-1 does not bind to HPC, although binding of the antibody to the lymphohematopoietic stem cell cannot be excluded yet. The high cytotoxic capacity of the IgM antibody BA-1, and the lack of reactivity with HPC, make the antibody particularly suitable for use in autologous bone marrow transplantation for patients with ALL.

scholarly journals Monoclonal antibody BA-1 does not bind to hematopoietic precursor cells

Blood ◽  
1982 ◽  
Vol 59 (5) ◽  
pp. 1029-1035
Author(s):  
J Jansen ◽  
RC Ash ◽  
ED Zanjani ◽  
TW LeBien ◽  
JH Kersey
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Bone Marrow ◽  
Hematopoietic Precursor ◽  
Proliferation In Vitro

Monoclonal antibody BA-1 binds to B lymphocytes, to cells from most cases of non-T acute lymphoblastic leukemia (ALL), and weakly to neutrophils. To determine whether BA-1 also reacts with hematopoietic progenitor cells (HPC), we studied the effect of removal of BA-1+ cells from human bone marrow on the proliferation in vitro of the trilineage precursor cell CFU-GEMM, and on the committed progenitor cells of granulopoiesis (CFU-C) and erythropoiesis (BFU-E/CFU-E). Complement- mediated cytotoxicity using BA-1 at concentrations far beyond those required to lyse BA-1+ bone marrow cells and ALL cells did not result in inhibition of colony formation in any of the assays. A rosette separation method, using ox red blood cells coated with BA-1, resulted in enrichment of HPC in the BA-1-depleted interface, whereas very few HPC were found in the BA-1-enriched pellet. Both methods indicate that BA-1 does not bind to HPC, although binding of the antibody to the lymphohematopoietic stem cell cannot be excluded yet. The high cytotoxic capacity of the IgM antibody BA-1, and the lack of reactivity with HPC, make the antibody particularly suitable for use in autologous bone marrow transplantation for patients with ALL.

scholarly journals Acquired amegakaryocytic thrombocytopenic purpura: a syndrome of diverse etiologies

Blood ◽  
1982 ◽  
Vol 60 (5) ◽  
pp. 1173-1178 ◽  
Author(s):  
R Hoffman ◽  
E Bruno ◽  
J Elwell ◽  
E Mazur ◽  
AM Gewirtz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Additional Patient ◽  
Platelet Glycoprotein ◽  
Intrinsic Defect ◽  
Thrombocytopenic Purpura ◽  
Marrow Cells

Abstract The possible pathogenetic mechanisms responsible for the production of acquired amegakaryocytic thrombocytopenic purpura (AATP) were investigated in a group of patients with this disorder. Absence of megakaryocytes and small platelet glycoprotein-bearing mononuclear cells, as determined by immunochemical staining of patient marrows with an antisera to platelet glycoproteins, suggested that the defect in AATP occurs in an early progenitor cell of the megakaryocytic lineage. Using an in vitro clonal assay system for negakaryocytic progenitor cells or megakaryocyte colony-forming units (CFU-M), the proliferative capacity of AATP marrow cells was then assessed. Bone marrow cells from three of four patients formed virtually no megakaryocyte colonies, suggesting that in these individuals the AATP was due to an intrinsic defect in the CFU-M. Bone marrow cells from an additional patient, however, formed 12% of the normal numbers of colonies, providing evidence for at least partial integrity of the CFU-M compartment in this patient. Serum specimens from all six patients were screened for their capacity to alter in vitro megakaryocyte colony formation. Five of six sera enhanced colony formation in a stepwise fashion, demonstrating appropriately elevated levels of megakaryocyte colony- stimulating activity. The serum of the patient with partial integrity of the CFU-M compartment, however, stimulated colony formation only at low concentrations. At higher concentrations, this patient's serum actually inhibited the number of colonies cloned, suggesting the presence of a humoral inhibitor to CFU-M. Serum samples from all patients were further screened for such humoral inhibitors of megakaryocyte colony formation using a cytotoxicity assay. The patient whose serum was inhibitory to CFU-M at high concentrations was indeed found to have a complement-dependent serum IgG inhibitor that was cytotoxic to allogeneic and autologous marrow CFU-M but did not alter erythroid colony formation. These-studies suggest that AATP can be due to at least two mechanisms: either an intrinsic effect at the level of the CFU-M or a circulating cytotoxic autoantibody directed against the CFU-M.

scholarly journals A glycoprotein inhibitor of in vitro granulopoiesis associated with AIDS

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1267-1272 ◽  
Author(s):  
IZ Leiderman ◽  
ML Greenberg ◽  
BR Adelsberg ◽  
FP Siegal
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Conditioned Media ◽  
Normal Individuals ◽  
Immunodeficiency Virus ◽  
Aids Related Complex ◽  
Significant Deficit ◽  
Normal Controls

Abstract Patients infected with the human immunodeficiency virus (HIV) often present with neutropenia. To elucidate the mechanism(s) of this HIV- related neutropenia, we assessed the proliferative capacity of the granulocyte-macrophage progenitor cell (CFU-GM) from the bone marrow (BM) of 78 patients within the AIDS spectrum manifesting symptoms or signs related to HIV infection. Of these, 70 had a significant deficit in the growth of this committed progenitor when compared with normal controls (P less than .01). Further analysis revealed that the nucleated bone marrow cells from AIDS and AIDS-related complex (ARC) patients inhibited the growth of CFU-GMs from normal individuals when cocultured in agar (P less than .001). Control CFU-GMs were also inhibited when they were cultured over feeder layers containing patients' BM cells (P less than .001). Conditioned media obtained from the liquid culture of patients' BM cells did not inhibit normal control CFU-GM growth to a degree different from that of the cells themselves (P greater than .4). Analysis of these conditioned media by polyacrylamide gel electrophoresis (PAGE) revealed a unique glycoprotein (gp) with a mol wt of 84 kd. Further studies revealed that this gp possessed the inhibitory activity. These data suggest that this gp may be an important factor in HIV-related neutropenia. The presence of gp84 was independent of drugs administered to the patients.

scholarly journals The 175 antigen expressed on myeloid and erythroid cells during differentiation is associated with serine protease activity

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1215-1219 ◽  
Author(s):  
D Deane ◽  
L Inglis ◽  
D Haig
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Surface ◽  
Serine Protease ◽  
Protease Activity ◽  
Bone Marrow Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Erythroid Cells ◽  
Serine Protease Activity

Monoclonal antibody 175 recognizes a cell-surface antigen on more than 80% of nucleated ovine bone marrow cells (BMC). The distribution is unusual, as the majority of differentiated myeloid and erythroid cells express the antigen (175 antigen), whereas mast cells, basophils, and the majority of lymphocytes do not. The level of 175 antigen expression has been shown to increase as BMC differentiate during hematopoiesis. Previous attempts to identify the 175 antigen have been unsuccessful. In this study, the 175 antigen was affinity-purified and shown to contain serine protease activity. Immunoblot analysis following sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of bone marrow cell lysates run under reducing or nonreducing conditions showed two closely adjacent protein bands (a doublet) of 28 to 30 kD molecular weight. N-linked deglycosylation showed that the 30-kD band was a glycosylated form of the 28-kD protein. Both protein bands shared the same N-terminal amino acid sequence over 20 residues, with high homology with serine proteases. Affinity-purified 175 antigen was proteolytic in substrate gels, the activity being inhibited by the 175 monoclonal antibody (Mab) and the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), but not by metallo, thiol, or acid protease-specific inhibitors. The 175 antigen is therefore part of a growing family of cell-surface proteases associated with hematopoietic cell differentiation.

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