PURE red-cell aplasia is a syndrome characterized by severe anemia, reticulocytopenia, and an absence of erythroid precursor cells in bone marrow that is otherwise normal. This failure of erythropoiesis has been shown to be antibody-mediated in almost half the patients with the adult form of red-cell aplasia.1 2 3 4 Immunosuppressive agents such as corticosteroids, cyclophosphamide, and antithymocyte globulin have been used to treat immunologically mediated red-cell aplasia.2 , 5 6 7 8 Results of in vitro assays for hematopoietic progenitor cells may assist in predicting which patients will respond to immunosuppressive therapy.9 10 11 Red-cell aplasia in children usually differs from the disorder in adults. There is a constitutional . . .
Nonadherent low density T‐lymphocyte depleted (NALT ‐ ) marrow cells from normal donors were sorted on a Coulter Epics 753 Dye Laser System using Texas Red labelled My 10 and phycoerythrin conjugated anti HLA‐DR monoclonal antibodies in order to obtain enriched populations of colony forming unit‐megakaryocyte (CFU‐MK). The CFU‐MK cloning efficiency (CE) was 1.1 ± 0.5% for cells expressing both high densities of My 10 and low densities of HLA‐DR (My 10 +++ DR + ). This procedure resulted in an 18‐fold increase in CE over NALT ‐ cells. The effect of purified or recombinant human haematopoietic growth factors including erythropoietin (Epo), thrombocytopoiesis stimulating factor (TSF), interleukin 1α (IL‐1α), granulocyte colony stimulating factor (G‐CSF), granulocyte‐macrophage colony stimulating factor (GM‐CSF), macrophage colony stimulating factor (M‐CSF or CSF‐1) and interleukin‐3 (IL‐3) on MK colony formation by My10 +++ DR + cells was determined utilizing a serum depleted assay system. Neither Epo, TSF, CSF‐1, IL‐lα nor G‐CSF alone augmented MK colony formation above baseline (2.5 ± 0.8/5 × 10 3 My 10 +++ DR + cells plated). In contrast, the addition of GM‐CSF and IL‐3 each increased both CFU‐MK colony formation and the size of colonies with maximal stimulation occurring following the addition of 200 units/ml of IL‐3 and 25 units/ml of GM‐CSF. At maximal concentration, IL‐3 had a greater ability to promote megakaryocyte colony formation than GM‐CSF. The stimulatory effects of GM‐CSF and IL‐3 were also additive in that the effects of a combination of the two factors approximated the sum of colony formation in the presence of each factor alone. The CFU‐MK appears, therefore, to express HPCA‐1 and HLA‐DR antigens. These studies also indicate that GM‐CSF and IL‐3 are important in vitro regulators of megakaryocytopoiesis, and that these growth factors are not dependent on the presence of large numbers of macrophages or T cells for their activity since the My 10 +++ DR + cells are largely devoid of these accessory cells.
Abstract Polyethylene glycol (PEG) conjugation to proteins has emerged as an important technology to produce drug molecules with sustained duration in the body. However, the implications of PEG conjugation to protein aggregation have not been well understood. In this study, conducted under physiological pH and temperature, N‐terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to (1) prevent protein precipitation by rendering the aggregates soluble, and (2) slow the rate of aggregation relative to GCSF. Our data suggest that PEG‐GCSF solubility was mediated by favorable solvation of water molecules around the PEG group. PEG‐GCSF appeared to aggregate on the same pathway as that of GCSF, as evidenced by (a) almost identical secondary structural transitions accompanying aggregation, (b) almost identical covalent character in the aggregates, and (c) the ability of PEG‐GCSF to rescue GCSF precipitation. To understand the role of PEG length, the aggregation properties of free GCSF were compared to 5kPEG‐GCSF and 20kPEG‐GCSF. It was observed that even 5kPEG‐GCSF avoided precipitation by forming soluble aggregates, and the stability toward aggregation was vastly improved compared to GCSF, but only marginally less stable than the 20kPEG‐GCSF. Biological activity measurements demonstrated that both 5kPEG‐GCSF and 20kPEG‐GCSF retained greater activity after incubation at physiological conditions than free GCSF, consistent with the stability measurements. The data is most compatible with a model where PEG conjugation preserves the mechanism underlying protein aggregation in GCSF, steric hindrance by PEG influences aggregation rate, while aqueous solubility is mediated by polar PEG groups on the aggregate surface.
In order to study the effects of recombinant and purified hematopoietic growth factors on megakaryocyte (MK) progenitor cells (CFU-MK), enriched populations of human CFU-MK were isolated utilizing fluorescence activated cell sorting after labelling of cells with monoclonal antibodies exhibiting specificity to the My10 (HPCA-1) antigen and the major histocompatibility (MHC) class II (HLA-DR) locus. The CFU-MK cloning efficiency (CE) was 1.1 +/- 0.5% for cells expressing both high densities of My10 and low densities of HLA-DR (My10 DR+). This procedure resulted in an 18 fold increase in CE over NALT- cells. The effects of natural or recombinant human hematopoietic growth factors including erythropoietin (Epo), thrombocytopoiesis stimulating factor (TSF), interleukin 1 alpha (IL-1 alpha), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (CSF-1), and interleukin 3 (IL-3) on MK colony formation by My10 DR+ cells were determined utilizing a defined medium assay system. Neither Epo, TSF, CSF-1, IL-1 alpha nor G-CSF alone augmented MK colony formation above baseline (2.5 +/- 0.8 per 5 x 10(3) My10 DR+ cells plated). By contrast, the addition of GM-CSF and IL-3 each increased CFU-MK colony formation with maximal stimulation occurring following the addition of 200 units/ml of IL-3 and 100 units/ml of GM-CSF. At maximal concentration, IL-3 had a greater ability to promote megakaryocyte colony formation than GM-CSF.
INCREASING evidence over the past decade has indicated that certain patients with aplastic anemia and single-lineage deficiencies of hematopoietic cells have disorders of immunologic origin.1 2 3 Recently, a family of specific glycoproteins collectively known as colony-stimulating factors, which interact to control the differentiation of hematopoietic cells, have been identified, purified, and cloned.4 5 6 Granulocyte–macrophage colony-stimulating factor (GM-CSF) stimulates the development of megakaryocyte colony-forming units (CFU-MK) as well as granulocytes and macrophages. We report evidence that cyclic amegakaryocytic thrombocytopenia may be due to an antibody that selectively blocks the action of GM-CSF on megakaryocyte progenitor cells.Case ReportA 21-year-old woman first experienced . . .
Stem cell factor (SCF) is a hematopoietic growth factor which acts on both primitive and mature progenitors cells. In animals, high doses of SCF alone stimulate increases in cells of multiple lineages and mobilize peripheral blood progenitor cells (PBPC). Phase I studies of rhSCF have demonstrated dose related side effects which are consistent with mast cell activation. Based upon in vitro synergy between SCF and G-CSF we have demonstrated the potential of low doses of SCF to synergize with G-CSF to give enhanced mobilization of PBPC. These PBPC have increased potential for both short and long term engraftment in lethally irradiated mice and lead to more rapid recovery of platelets. On going Phase I/II studies with rhSCF plus rhG-CSF for mobilization of PBPC, demonstrated similar increases in PBPC compared to rhG-CSF alone. These data suggest a clinical role of rhSCF in combination with rhG-CSF for optimal mobilization of PBPC.
