Uroporphyrinogen I synthase induction in normal human bone marrow cultures: an early and quantitative response of erythroid differentiation.

Uroporphyrinogen I (URO) synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] activity increased when cultures of normal human bone marrow cells were incubated with erythropoietin. The increase of URO synthase activity was a linear function of erythropoietin concentration in the culture medium and was proportional to the extent of heme synthesis as determined by 55Fe incorporation into heme. The onset of the increase in URO synthase activity, which occurred on day 4 of incubation with erythropoietin, preceded by 3 days the appearance of hemoglobin in colonies derived from erythroid progenitor cells. These results indicate that induction of URO synthase in normal human bone marrow cells incubated with erythropoietin is an early event and that URO synthase activity is a useful quantitative index of erythroid differentiation.     

Endogenous BFU-E in peripheral blood in diagnosis of polycythemia vera

Erythroid progenitors (CFU-E and BFU-E) growth in vitro from bone marrow and peripheral blood of patients with polycythemia vera (PV) was studied using a methylcellulose culture technique. The aim of the study was to find out whether the in vitro colony formation of peripheral blood could be used in the differential diagnosis of PV. In all 25 patients studied, endogenous colonies were found in the bone marrow and peripheral blood. The parallel study of both bone marrow and peripheral blood erythroid progenitors indicates that the presence of endogenous BFU-E in peripheral blood is a dependable test for PV. The results presented here showed that the abnormalities in PV erythroid progenitors are expressed at the level of both CFU-E and BFU-E, suggesting multiple changes in the erythroid progenitors. Our finding indicate that peripheral blood BFU-E differ from bone marrow BFU-E with regard to their dependence for further differentiation on BPA, the activity present in PHA-LCM.     

Tissues of the Laron dwarf are sensitive to insulin-like growth factor I but not to growth hormone

Tissues from patients with Laron dwarfism are resistant to the actions of endogenous or exogenous GH. As a result, insulin-like growth factor I (IGF-I) levels are low, possibly contributing to the severe growth deficiency that occurs in patients with this syndrome. In this study, we found that erythroid progenitor cells and permanently transformed T-cell lines from two patients with Laron dwarfism responded in vitro to added IGF-I in concentrations ranging between 1-10 ng/mL despite no stimulatory response to added GH in concentrations of up to 500 ng/mL. Normal or near-normal responsiveness to insulin was also demonstrated. The persistence of GH resistance in the cultured T-cell lines confirms the primary genetic nature of the defect in Laron dwarfism. The preservation of in vitro growth responsiveness to IGF-I in hematopoietic tissue from the Laron dwarfs suggests that affected individuals are sensitive to this factor and may respond to it in vivo.     

Human P40 T-cell growth factor (interleukin-9) supports erythroid colony formation.

Because human P40 T-cell growth factor, tentatively designated interleukin-9 (IL-9), was isolated through its ability to stimulate a human IL-3-dependent leukemic cell line (M-O7E), we tested the ability of IL-9 to support the growth and differentiation of normal hematopoietic progenitor cells from peripheral blood and bone marrow. Although the M-O7E cell line was derived from a patient with megakaryoblastic leukemia, IL-9 has not proved to be a growth or maturation factor for megakaryocytes, but instead has proved to be effective in supporting the development of erythroid bursts (BFU-E) in cultures supplemented with erythropoietin. Using highly purified progenitors from peripheral blood, IL-3 showed a BFU-E plating efficiency of 46% compared with 20% for IL-9. Because of the purity of these cell preparations and the low cell density in culture, IL-9 is likely to interact directly with erythroid progenitors. Analysis of mixing experiments and of the morphology of the BFU-E in culture indicated that IL-9 interacts preferentially with a relatively early population of IL-3-responsive BFU-E. In cultures of human bone marrow or cord blood, IL-9 selectively supported erythroid colony formation, while IL-3 and granulocyte/macrophage colony-stimulating factor additionally yielded granulocyte/macrophage colonies. Therefore, IL-9 represents a new T cell-derived cytokine with the potential for selectively stimulating erythroid development in the hematopoietic system.     

Megaloblastic Change is a Feature of Colonies Derived from an Early Erythroid Progenitor (BFU-E) Stimulated by Monocytes in Culture

S ummary . The morphology of stained preparations of cells from human bone marrow and peripheral blood erythroid colonies cultured in methylcellulose, were examined by light microscopy. Although the morphology of 7 d erythroid colonies (CFU-E) was largely normoblastic, bone marrow and peripheral blood erythroid bursts (BFU-E) showed a variable degree of megaloblastic and dyserythropoietic change. This was not due to nutritional deficiencies of the culture system and the deoxyuridine suppression test demonstrated active thymidine synthesis.
Megaloblastic morphology was correlated with the growth induced by the addition of monocytes to erythroid progenitors. It was concluded that megalo-blastosis was a feature of the erythroblasts derived from an early BFU-E which required monocytes for their development.     

Enhancement of erythropoiesis in vitro by human growth hormone is mediated by insulin-like growth factor I

Insulin-like growth factor I (IGF-I) is the presumed paracrine or autocrine growth-promoting mediator of growth hormone in peripheral tissues. In order to evaluate the role of IGF-I as mediator of human growth hormone (hGH) in erythropoiesis, we compared the effects of both peptides upon in vitro colony formation by primitive (BFU-E) and relatively mature (CFU-E) human erythroid precursors. Biosynthetic IGF-I (2 ng/ml) and hGH (25 ng/ml) induced a significant increase in the growth of both BFU-E and CFU-E. BFU-E growth was maximally enhanced by 6 ng/ml IGF-I and by 50 ng/ml hGH, resulting in an increase in burst numbers of 62 ± 12% and 52 ± 12%, respectively. Maximal enhancement of CFU-E growth was detected at higher concentrations of IGF-I (20 ng/ml) and hGH (150 ng/ml), with respective increases of 121 ± 35% and 137 ± 18% in colony numbers. Enhancement of bone marrow and peripheral blood erythroid progenitor cell growth by hGH required the presence of monocytes and was abrogated by specific monoclonal antibodies directed against IGF-I membrane receptors. The in vitro growth-promoting effect of hGH upon human erythroid precursors thus appears to be mediated by paracrine IGF-I.     

Insulin and insulin-like growth factor I support the proliferation of erythroid progenitor cells in bone marrow through the sharing of receptors

The effects of insulin and insulin-like growth factor I (IGF-I) on the proliferation of erythroid progenitor cells in bone marrow were studied in serum-deprived culture. Primitive human bone marrow cells were purified by cell sorting on the basis of the expression of CD34 and the Kit receptor. Insulin and IGF-I with erythropoietin (EPO) dose dependently supported the formation of erythroid colonies of CD34+/Kit+ cells in bone marrow. The direct effect of insulin and IGF-I on the stimulation of primitive erythroid progenitor cells was confirmed by single-cell proliferation studies in serum-deprived liquid suspension culture. The addition of insulin and/or IGF-I to stem cell factor (SCF) resulted in an additive increase in the number of erythroid colonies. The erythroid colonies formed by insulin and IGF-I with EPO were different in size from those formed by SCF with EPO. These findings imply that erythroid progenitor cells responding to insulin and IGF-I might be at a different developmental stage of erythropoiesis from those responding to SCF in CD34+/Kit+ cells. Similarly, insulin and IGF-I with EPO supported the proliferation of the mature erythroid progenitor cells in light-density bone marrow mononuclear cells (LDBMCs). The addition of the anti-receptor antibody to IGF-I receptor or insulin receptor partially suppressed erythroid colony formation supported with insulin or IGF-I in both CD34+/Kit+ cells and LDBMCs. The simultaneous addition of both receptor antibodies completely abrogated the erythroid colony formation. These results suggest that insulin and IGF-I directly stimulate the proliferation of the late stage of primitive erythroid progenitor cells and mature erythroid progenitor cells through the sharing of receptors.     

Effect of murine mast cell growth factor (c-kit proto-oncogene ligand) on colony formation by human marrow hematopoietic progenitor cells.

Purified natural (n) and recombinant (r) murine (mu) mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with r human (hu) erythropoietin (Epo), rhu granulocyte-macrophage colony-stimulating factor (rhuGM-CSF), rhuG-CSF, and/or rhuM-CSF for effects in vitro on colony formation by multipotential (colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit erythroid [BFU-E]) and granulocyte-macrophage (CFU-GM) progenitor cells from normal human bone marrow. MGF was a potent enhancing cytokine for Epo-dependent CFU-GEMM and BFU-E colony formation, stimulating more colonies and of a larger size than either rhu interleukin-3 (rhuIL-3) or rhuGM-CSF. MGF, especially at lower concentrations, also acted with rhuIL-3 or rhuGM-CSF to enhance Epo-dependent CFU-GEMM and BFU-E colony formation. MGF had little stimulating activity for CFU-GM colonies by itself, but in combination with suboptimal to optimal amounts of rhuGM-CSF enhanced the numbers and the size of CFU-GM colonies in an additive to greater than additive manner. While we did not detect an effect of MGF on CFU-G colony numbers stimulated by maximal concentrations of rhuG-CSF, MGF did enhance the size of CFU-G-derived colonies. MGF did not enhance the activity of rhuM-CSF. In a comparative assay, maximal concentrations of rmu and rhuMGF were equally effective in the enhancement of human bone marrow colony formation, but rhuMGF, in contrast to rmuMGF, did not at the concentrations tested enhance colony formation by mouse bone marrow cells. MGF effects on BFU-E, CFU-GM, and CFU-GEMM may be direct acting ones as MGF-enhanced colony formation by these cells in highly enriched progenitor cell populations of CD34 HLA-DR+ and CD34 HLA-DR+CD33- sorted cells in which greater than or equal to 1 of 2 cells was a BFU-E plus CFU-GM plus CFU-GEMM. MGF appears to be an early acting cytokine that preferentially stimulates the growth of immature hematopoietic progenitor cells.     

Characterization of the potentiation effect of activin on human erythroid colony formation in vitro

Activin, also named FSH-releasing protein, was previously shown to induce hemoglobin accumulation in K562 cells and potentiate the proliferation and differentiation of CFU-E in human bone marrow cultures. Present studies indicate that the potentiation effect of activin is lineage specific. In addition to CFU-E, activin caused an increase in the colony formation of BFU-E from either bone marrow or peripheral blood. It had little effect on the colony formation of CFU- GM and the mixed colonies from CFU-GEMM. In serum-depleted culture, the effect of activin was shown to be dose-dependent with doses effective at picomolar concentrations. The potentiation effect of activin was exerted indirectly through mediation of both monocytes and T lymphocytes. Activin was also found to increase specifically the proportion of DNA-synthesizing erythroid progenitors from both bone marrow and peripheral blood. It had little effect on DNA synthesis in CFU-GM and in mitogen-stimulated lymphocytes. Addition of the monocytes or T lymphocytes to their respective depleted subpopulations of mononuclear cells reconstituted the enhancing effect of activin on the colony formation and DNA synthesis of erythroid progenitors. These results strongly suggest a specific role of activin in potentiating the proliferation and differentiation of erythroid progenitors in vitro.     

Production of colony-stimulating factor(s) for granulocyte-macrophage and multipotential (granulocyte/erythroid/megakaryocyte/macrophage) hematopoietic progenitor cells (CFU-GEMM) by clonal lines of human IL-2-dependent T-lymphocytes

Human T-lymphocyte lines that were selected for recognition of HLA-DR6 antigen and were dependent for growth in vitro on an added source of interleukin-2 (IL-2) were derived from the peripheral blood of normal individuals. Each was tested for production of a lymphokine(s) with properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) using as target cells nonadherent cells from human long-term bone marrow cultures (LTBMC) or fresh marrow. Each of eight T-lymphocyte lines that were OKT3, OKT4, and HLA-DR positive produced GM-CSF that stimulated colony formation by both LTBMC cells and fresh marrow. Individually examined single-cell-derived bone marrow colonies growing in T-cell GM-CSF contained peroxidase-positive neutrophils, and macrophage-monocytes (GM-CFUc). Supernatant from a single-cell-derived T-cell clonal line designated F1 stimulated formation of granulocyte-macrophage colonies, megakaryocyte colonies, macroscopic erythroid bursts, and multipotential colonies containing erythroid cells, megakaryocytes, neutrophilic and eosinophilic granulocytes, and monocyte-macrophages (CFU-GEMM) in the presence of added erythropoietin. These data indicate that human IL-2-responsive T-lymphocytes produce lymphokine(s) that stimulate proliferation of primitive as well as committed hematopoietic stem cells, and implicate human T-lymphocytes in regulation of human multipotential hematopoietic stem cells in vivo.     

Haem Biosynthesis in Refractory Sideroblastic Anaemia Associated with the Preleukaemic Syndrome

The activities of 5 enzymes of the haem biosynthetic pathway and the protoporphyrin concentrations have been measured in peripheral red blood cells of 23 patients having a preleukaemic syndrome with refractory sideroblastic anaemia. A decreased δ-aminolaevulinic acid synthase (ALA-S) activity, an increased uroporphyrinogen I synthase activity and an increased red cell protoporphyrin concentration were consistent findings. Patients with abnormal leucocyte and/or platelet counts in the peripheral blood as well as patients with an excess of blast cells in the bone marrow had the lowest ALA-S activities. A further decrease in ALA-S activity was observed in 3 patients after leukaemic change in the disease. Patients having cytogenetic abnormalities showed no unique enzyme abnormalities. These results indicate that enzymatic disturbances of haem synthesis cannot be used as prognostic indicator of leukaemic transformation in refractory sideroblastic anaemia, but a very low ALA-S activity appears to accompany the development of a leukaemia in such patients.     

Characterization of human erythroid burst-promoting activity derived from bone marrow conditioned media

Bone marrow conditioned media (BMCM) increases burst number and the incorporation of 59Fe into heme by bursts when peripheral blood or bone marrow cells are cultured at limiting serum concentrations. Burst- promoting activity (BPA) has now been purified approximately 300-fold from this source by ion-exchange chromatography on DEAE-Sephadex and absorption chromatography on hydroxyapatite agarose gel. Marrow BPA increased burst number and hemoglobin (Hb) synthesis in a dose- dependent manner. A larger increase in Hb synthesis than in burst number was consistently observed, which was probably a consequence of the increase in the number of cells per burst that occurs in the presence of BPA. The role of BPA in culture could be distinguished from erythropoietin (Ep), since no bursts grew in the absence of Ep, whether or not BPA was present, and since it had no effect on the growth of erythroid colonies scored at day 5 of culture. Our purified fraction did not support the growth of CFU-C in culture. Activity was stable at temperatures of 70 degrees C or lower for 10 min; exposure to 80 degrees C resulted in approximately 50% loss of activity. BPA was completely inactivated by treatment at 100 degrees C for 10 min. Thus, human bone marrow cells produce a heat-sensitive factor that specifically promotes the growth of early erythroid progenitors in culture.     

Growth of Human Erythroid and Erythroid-Granulocytic Colonies in Culture without Addition of Exogeneous Erythropoietin

S ummary . Growth of human erythroid and erythroid-granulocytic colonies was investigated in a fibrinogen clot culture system. CFU-E, BFU-E, CFU-C and the mixed colonies were grown in cultures in medium conditioned by phytohaemagglutinin-stimulated human mononuclear cells, with small amounts of aplastic anaemia serum, but no exogeneous erythropoietin (Epo). The level of Epo in the culture was 0·015 U. The mixed colonies constituted 8-22% of the total of colonies grown. The results indicate that high dose Epo is not required to grow BFU-E and the mixed colonies in cultures containing erythroid potentiating factor(s), and the frequency of bipotent haemopoietic stem cells in human bone marrow or peripheral blood may be higher than that reported previously.     

Long-acting peptidomimergic control of gigantism caused by pituitary acidophilic stem cell adenoma

Gigantism is caused by GH hypersecretion occurring before epiphyseal long bone closure and usually is associated with pituitary adenoma. A 15-yr-old female patient presented with accelerated growth due to a large pituitary tumor that was surgically resected to relieve pressure effects. Second surgery to remove residual tumor tissue was followed by administration of octreotide LAR, a long-acting depot somatostatin analog, together with long-acting cabergoline. Height was over the 95th percentile, with evidence of a recent growth spurt. Serum GH levels were more than 60 ng/mL (normal, <10 ng/mL) with no suppression to 75 g oral glucose, and serum PRL (>8,000 ng/mL; normal, <23 ng/mL) and insulin-like growth factor I levels (845 ng/mL; age-matched normal, 242-660 ng/mL) were elevated. Histology, immunostaining, and electron microscopy demonstrated a pituitary acidophil stem cell adenoma. Tumor tissue expressed both somatostatin receptor type 2 and dopamine receptor type 2. The Gs alpha subunit, GHRH receptor, and MEN1 genes were intact, and tumor tissue abundantly expressed pituitary tumor transforming gene (PTTG). Serum GH and PRL levels were controlled after two surgeries, and with continued cabergoline and octreotide LAR GH, PRL, and insulin-like growth factor I levels were normalized. In conclusion, administration of long-acting somatostatin analog every 4 weeks in combination with a long-acting dopamine agonist biweekly controlled biochemical parameters and accelerated growth in a patient with gigantism caused by a rare pituitary acidophil stem cell adenoma.     

The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro

Factors that regulate the growth and development of primitive bone marrow stromal cell precursors are not well defined. We have examined 25 purified recombinant growth factors for their ability to initiate and support clonogenic growth of fibroblast colony-forming cells (CFU- F) from adult human bone marrow. Assays were performed using bone marrow mononuclear cells (BMMNC) enriched in CFU-F by magnetic- activated cell sorting (MACS) using the monoclonal antibody (MoAb) STRO- 1. A serum-deprived assay was developed to avoid components of fetal calf serum (FCS) that may mask or otherwise modify the response of CFU- F to exogenously added factors. L-ascorbate and the glucocorticoid dexamethasone were found to be essential for CFU-F colony development under serum-deprived conditions. Importantly, clonogenic growth of CFU- F in this culture system was absolutely dependent on an exogenous source of growth factor. Platelet-derived growth factor-BB (PDGF) and epidermal growth factor (EGF) demonstrated the greatest ability to support colony growth. Colony formation was dose-dependent, with half- maximal colony numbers at approximately 0.2 ng/mL for either factor and plateau numbers at concentrations in excess of 1.0 ng/mL. Simultaneous addition of PDGF and EGF had no effect on the number of colonies initiated but resulted in dose-dependent increases in mean colony diameter that were significant (P < or = .05) when compared with the effect of either factor alone or with the size of colonies elicited in control cultures by 20% FCS. Fluorescence-activated cell sorting (FACS) of BMMNC using MoAbs to the alpha chain of the PDGF receptor and to the EGF receptor in combination with the Moab STRO-1 demonstrated constitutive expression of both receptors by greater than 90% on CFU-F. Receptors for insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) were also detected on STRO-1+ CFU-F, but in vitro both IGF- 1 and NGF did not support colony growth. This report demonstrates the development of a simple, reproducible, and stringent culture system for the growth and assay of stromal precursors under serum-deprived conditions and represents an important prerequisite for future studies of the role of growth factors in the regulation of stromal cell proliferation, differentiation, and development.     

Pure erythropoietic colony and burst formations in serum-free culture and their enhancement by insulin-like growth factor I

Recombinant human insulin-like growth factor I (IGF-I) increased human and murine erythropoietic colony formation in serum-free culture. In order to investigate the effects of purified factors such as IGF-I on hemopoietic progenitor cells, we have established a serum-free culture system which supports the clonal growth of CFU-E- and BFU-E-derived colonies. Exogenously supplied ingredients were bovine serum albumin (BSA), transferrin, lipid suspensions, 2-mercaptoethanol, and recombinant human erythropoietin (epo). Among these, BSA and cholesterol were found to be essential ingredients. The optimum concentration of BSA sufficient to grow BFU-E was 3%. Erythroid colony and burst formation of human and murine marrow cells was enhanced twofold (p less than 0.05) by a physiological concentration of recombinant human IGF-I. Potentiation was observed in a dose-dependent manner between 10(-9) and 10(-7) M. A few murine CFU-E colonies were formed in the absence of epo. These results suggest that IGF-I has a supportive effect on the proliferation and differentiation of erythroid precursor cells stimulated by epo and that its action is synergistic with that of epo.     

Differential effects of growth hormone and insulin-like growth factor I on colony formation of epiphyseal chondrocytes in suspension culture in rats of different ages

The effect of human GH (hGH) and insulin-like growth factor I (IGF-I) on the colony formation of rat epiphyseal chondrocytes was studied in suspension culture. Chondrocytes from epiphyseal growth plates of the proximal tibia of 7-, 20-, and 28-day-old normal male rats were enzymatically isolated and cultured in the presence of 10% newborn calf serum in suspension stabilized with 0.5% agarose. The cloning efficiency (number of formed colonies per 1000 seeded cells) was 13.7 +/- 1.9, 3.2 +/- 0.6, and 3.5 +/- 0.4 for chondrocytes isolated from 7-, 20- and 28-day-old rats and cultured for 14 days, respectively. The colonies were classified according to size (colony diameter), and the number of colonies was estimated as a function of colony size (distribution of cloning efficiency). IGF-I (25-200 ng/ml) increased the total colony number among chondrocytes isolated from the three different age groups and particularly increased the number of small colonies. However, IGF-I did not significantly change the distribution of cloning efficiency as compared to the control group. hGH potentiated colony formation at concentrations of 10-80 ng/ml, but no stimulatory effect of hGH was apparent at a concentration of 160 ng/ml. GH caused an assymetric distribution of cloning efficiency that was significantly different from the control group due to an increased number of large colonies (diameter exceeding 80 microns). The differential effects of GH and IGF-I were apparent after an extended period of culture (28 days) at various concentrations of the peptides. These results show that GH as well as IGF-I induced colony formation among epiphyseal chondrocytes in suspension culture, although the effects of GH and IGF-I are different in terms of distribution of cloning efficiency. The observation that GH particularly potentiated the formation of large size colonies suggests that GH promotes the differentiation of prechondrocytes, or young chondrocytes as suggested earlier. The finding that IGF-I stimulation resulted in a higher proportion of small size chondrocyte colonies, compared to the control group, suggests that IGF-I stimulates epiphyseal chondrocytes at a later stage of differentiation.     

3''-Azido-3''-deoxythymidine (AZT) inhibits proliferation in vitro of human haematopoietic progenitor cells

Peripheral blood cytopenias are a serious, dose-limiting toxicity of AZT therapy in patients infected by HIV. To evaluate the mechanism by which cytopenias develop, AZT effects of haematopoietic differentiation and growth were measured in serum-free, nucleoside-depleted cultures of normal human bone marrow. In contrast to native thymidine, AZT suppressed the proliferation of erythroid, granulocyte/macrophage and primitive haematopoietic stem cells in a dose-related and time-dependent fashion. Relative progenitor sensitivity varied, with half-maximal concentrations of 1-5 microM and 20-40 microM AZT for inhibition of erythroid and nonerythroid progenitor cell growth, respectively. Inhibition was observed over full ranges of concentrations of haematopoietic tissue-specific regulators (human erythropoietin, colony-stimulating activity, interleukin-3 and lymphocyte conditioned medium) and of platelet-derived growth factor (PDGF), an agent that enhances erythropoiesis in vitro via accessory marrow stromal elements. Furthermore, suppression was similar in cultures of marrow cells that were depleted of accessory populations, suggesting that its action is directed at progenitors. Finally, when deoxythymidine was added in increasing amounts to cultures with a half-maximal concentration of AZT, inhibition was abrogated. We conclude that AZT is a potent inhibitor of haematopoiesis in vitro, and that erythroid progenitors are particularly sensitive to its action. These results may explain the marrow hypoplasia that occurs during AZT administration in vivo.     

Increase of CD34 positive cells in polycythaemia vera

Abstract: The purpose of the present work was to evaluate the proliferative character of polycythaemia vera (PV). Therefore, in 15 patients with different stages of PV we assessed the level of CD34 positive (CD34+) cells in peripheral blood and bone marrow, erythroid colony growth of bone marrow cells and plasma erythropoietin (EPO). The mean concentration of CD34+ cells in blood was significantly increased in PV patients (9.0±11.2×10³/mL) compared to healthy controls (2.0±1.7×10³/mL). In aspirated bone marrow no such difference between PV and control subjects was present. Six patients with splenomegaly and/or requirement for chemotherapy had significantly higher mean blood levels of CD34+ cells compared to the remaining PV patients. All PV patients presented EPO independent erythroid colonies. Three PV patients with anaemia and long disease duration had high EPO levels.     

Decreased levels of myeloid progenitor cells associated with long-term administration of recombinant human granulocyte colony-stimulating factor in patients with autoimmune neutropenia

Summary. We studied the long-term in vivo effect of recombinant human granulocyte colony stimulating factor (rhG-CSF) on in vitro growth of granulocyte/macrophage colony forming cells (GM-CFC) in bone marrow and peripheral blood obtained from two patients with autoimmune neutropenia, who received rhG-CSF. Along with rhG-CSF treatment for more than 40 d, numbers of GM-CFC-derived colonies from both bone marrow and peripheral blood gradually decreased to a significant level though white blood cells in peripheral blood and nucleated cells in bone marow were increased in number. This observation suggests that long-term administration of rhG-CSF may preferentially activate a differentiation pathway for granulopoiesis while proliferation of GM-CFC is not induced as expected in response to rhG-CSF.