Chronic myelogenous leukemia: in vitro studies of hematopoietic regulation in a patient undergoing intensive chemotherapy.

A patient heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase and with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) was treated with combination chemotherapy and had a partial loss of Philadelphia chromosome accompanied by partial restoration of nonclonal hematopoiesis as determined by glucose-6-phosphate dehydrogenase. Studies of in vitro hematopoiesis were performed after chemotherapy to evaluate the influences of neoplastic stem cells on normal cells and to determine whether there were physical and cell kinetic differences between leukemic stem cells and their normal counterparts. The data revealed the following: (a) The frequencies of normal committed granulocytic stem cells (CFU-C) and erythroid stem cells (BFU-E) in blood did not differ from the frequencies in marrow. (b) Normal late erythroid progenitors (CFU-E) were found at a significantly lower frequency that the more primitive BFU-E. Calculations indicated that not only was there a decrease in CFU-E production by normal BFU-E, but there was also abnormal clonal expansion of CML BFU-E (CFU-E:BFU-E ratio for normal progenitors was 1.1, whereas for the CML clone it was 11.5). (c) No increase in frequency of normal CFU-C was found after marrow cells were exposed to high specific activity tritiated thymidine. (d) Normal CFU-C and those from the CML clone were not separable on the basis of density. (e) The frequency of normal BFU-E was consistently greater than that of CFU-C, suggesting that regulatory differences influence the commitment of normal progenitors to the two pathways.     

Identification of three accessory cell populations in human bone marrow with erythroid burst-promoting properties

Several laboratories have demonstrated a requirement for burst-promoting activity (BPA), a product of T cells, or T cell/monocyte collaboration in the induction of differentiation of peripheral blood erythroid burst-forming units (BFU-E) in vitro. The physiologic significance of this finding is brought into question by patients with severe mature T cell deficiency who have normal in vivo erythropoiesis. The studies described here were designed to determine whether the burst-promoting effects of marrow T cells and adherent cells are similar to those of peripheral blood, to define whether a third population of marrow cells is capable of production of BPA, and to describe the BPA requirements of immature and mature marrow erythroid progenitors. To that end we prepared adherence- and E-depleted low-density peripheral blood mononuclear cells as a source of BFU-E and demonstrated that their optimal erythropoietin-induced differentiation requires BPA. We then determined that both bone marrow and peripheral blood T cells and monocytes could provide the necessary BPA to induce their erythropoietin dependent differentiation. BPA production by T cells was sensitive to irradiation, but that of the whole bone marrow low-density population was considerably less sensitive. This in itself demonstrated that BPA production in marrow is not T cell dependent. We further demonstrated a potent, albeit infrequent, third population of BPA-producing marrow cells. These proved to be nonadherent, E receptor-negative, granulocyte antigen-negative, and gamma-Fc receptor-positive. Finally, we separated all of these BPA-producing cells from marrow erythroid progenitors and concentrated the latter into a population in which they comprised 6% of the cells. With this population we demonstrated that both immature (BFU-E) and mature (colony-forming units [CFU-E]) erythroid progenitors require BPA in addition to erythropoietin to induce them to form erythroid colonies in vitro. These results may explain the normal erythropoiesis found in patients with mature T cell deficiency. Though the differentiation of both BFU-E and CFU-E requires BPA, this need can be met by a special class of nonadherent, radioresistant, E receptor-negative, granulocyte antigen-negative, and gamma-Fc-positive cells.     

Retrovirus-induced feline pure red cell aplasia. Hematopoietic progenitors are infected with feline leukemia virus and erythroid burst-forming cells are uniquely sensitive to heterologous complement

Feline leukemia virus subgroup C/Sarma (FeLV-C) induces pure red cell aplasia (PRCA) in cats. Just before the onset of anemia, erythroid colony-forming cells (CFU-E) become undetectable in marrow culture, yet normal frequencies of erythroid burst-forming cells (BFU-E)- and granulocyte-macrophage colony-forming cells (CFU-GM) persist. To determine if erythroid progenitors were uniquely infected with retrovirus, marrow mononuclear cells from cats viremic with FeLV-C were labeled with monoclonal antibodies to gp70 and then analyzed with a fluorescence-activated cell sorter. Both erythroid and granulocyte-macrophage progenitors were among cells sorting positively, suggesting that infection of BFU-E alone did not result in PRCA. The results were confirmed by complement (C') lysis studies using baby rabbit or guinea pig sera as sources of C'. These studies also suggested that BFU-E from cats with PRCA were unusually sensitive to C' alone, without the addition of antibody. In further studies, we demonstrated that C' activation was via the classical pathway and that C' sensitivity was unique to BFU-E and not a property of CFU-E, CFU-GM, or progenitors that were capable of giving rise to BFU-E in suspension culture. As BFU-E from cats viremic with FeLV-A/Glasgow-1 or the Rickard strain of feline leukemia virus were not sensitive to C', this finding may relate to the pathogenesis of feline PRCA. We hypothesize that, in cats viremic with FeLV-C, the abnormal C' sensitivity of BFU-E leads to the absence of CFU-E and anemia.     

Erythroid Precursors in Congenital Hypoplastic (Diamond-Blackfan) Anemia

To explore the etiology of congenital hypoplastic anemia (CHA) or the Diamond-Blackfan anemia, erythropoietin responsive committed erythroid precursors were enumerated by the plasma clot method. These included blood and marrow erythroid burst-forming units (BFU-E) and marrow erythroid colony-forming units (CFU-E). The peripheral blood nucleated cells of 11 patients and the marrow cells of seven of these patients were examined. Studies were repeated in several patients during relapse and after induction of remission. BFU-E were undetectable in the marrow and blood of all but one relapsed patient, and the numbers of marrow CFU-E were depressed in all relapsed patients. Blood BFU-E remained low in all of the patients in remission. No evidence was obtained for suppression of normal CFU-E or BFU-E by CHA lymphocytes. Erythropoietin dose-response curves performed in two patients revealed a 10-fold increase in erythropoietin requirement for marrow CFU-E colony growth. This marked unresponsiveness to erythropoietin was strikingly improved by steroid therapy in one patient. We suggest that CHA is the result of a qualitative and/or quantitative deficiency of BFU-E. If BFU-E are produced, they must be relatively unresponsive to erythropoietin. The abnormal BFU-E give rise to erythropoietin unresponsive CFU-E and, thence, to proerythroblasts that are, in turn, trapped in that early stage of development because of their poor erythropoietic response. Hence, red cell production is deficient. Steroids appear to improve the erythropoietin response of CHA erythroid precursors.     

IL—6基因转染的骨髓基质细胞系对骨髓移植小鼠造血功能恢复？…

目的：探讨白细胞介素６（ＩＬ－６）基因转染的骨髓基质细胞系ＱＸＭＳＣ１ＩＬ－６对骨髓移植后造血功能的重建作用。方法：将骨髓造血细胞和骨髓基质细胞系一起经尾静脉注射给同系小鼠，建立骨髓移植（ＢＭＴ）模型。小鼠的造血功能用脾结节（ＣＦＵ－Ｓ）、粒－单系祖细胞（ＣＦＵ－ＧＭ）、红系祖细胞（ＣＦＵ－Ｅ、ＢＦＵ－Ｅ）测定及外周血各项血液学指标来确定。结果：ＷＸＭＳＣ１ＩＬ－６转基因骨髓基质细胞可明显增强ＢＭ     

Effect of haem arginate on colony formation by haematopoietic progenitors in vitro

The effect of haem arginate, a new stable haem compound, was studied on colony formation by erythroid (CFU-E, BFU-E) and granulocyte-macrophage (CFU-GM) progenitors from the bone marrow of 12 healthy persons. At the concentration of 10 mumol/l haem arginate significantly (p less than 0.05) increased and at the concentration of 200 mumol/l significantly decreased (p less than 0.005) the colony formation of CFU-E. A similar, though not significant, trend was seen in the BFU-E colony growth, while the CFU-GM colony formation was not influenced at any concentration between 1 and 200 mumol/l. We conclude that the stimulation of erythropoiesis in vitro is seen at lower concentrations with haem arginate than with former haemin preparations.     

IL-6基因转染的骨髓基质细胞系对骨髓移植小鼠造血功能恢复的促进作用

目的:探讨白细胞介素6(IL-6)基因转染的骨位基质细胞系QXMSC, IL6对骨髓移植后造血功能的重建作用。方法:将骨髓造血细胞和骨髓基质细胞系一起经尾静脉注射给同系小鼠,建立骨髓移植(BMT)模型。小鼠的造血功能用脾结节(CFU-S)、粒-单系祖细胞(CFU-GM)、红系祖细胞(CFU-E、BFU-E)测定及外周血各项血液学指标来确定。结果:QXMSC_1 IL-6转基因骨髓基质细胞可明显增强BMT小鼠形成的CFU-S、CFU-GM、CFU-E和BFU-E数,促进外周血象的恢复。结论:QXMSC_1 IL-6细胞可明显促进小鼠骨髓移植后早期造血功能重建。     

The inhibitory factors of hematopoiesis in chronic hemodialysis patients treated with recombinant human erythropoietin

In order to clarify possible factors responsible for varying responses in uremic patients treated with recombinant human erythropoietin (rHuEPO), we determined the inhibitory effects of ten uremic sera on the erythroid progenitors (CFU-E) and erythroid bursts (BFU-E). We also measured plasma EPO titers, Fe, UIBC, ferritin, PTH-C, beta 2-microglobulin, and aluminum in all ten patients. The inhibitor of CFU-E but not BFU-E, was present in the serum of the single anemic patient whose recovery took longer after the administration of rHuEPO. He did not have such conditions as iron deficiency, excess of aluminum, or chronic inflammation. The remaining patients, who had no CFU-E or BFU-E inhibitors, were good responders to rHuEPO. In none of ten patients were there inhibitors of granulocyte-macrophage progenitors (CFU-GM) or any differences in the other parameters. Although the inhibitory factor of CFU-E can be overcome with a larger dose, its prior determination may be useful to set out minimal effective dose of EPO treatment.     

Suppression of normal human erythropoiesis by gamma interferon in vitro. Role of monocytes and T lymphocytes.

Interferons (IFN) have been shown to suppress the proliferation of human erythroid progenitors (erythroid burst-forming units [BFU-E] and colony-forming units [CFU-E]) in vitro. To examine the mechanism(s) underlying this inhibitory activity, the effect of different doses (50-10,000 U) of a highly purified preparation of recombinant DNA produced human gamma-IFN on erythroid colony formation by normal human bone marrow BFU-E and CFU-E in the presence and absence of monocytes and/or T lymphocytes was studied. The addition of gamma-IFN to whole marrow caused suppression of BFU-E (6-65%) and CFU-E (31-79%) in a dose-dependent fashion. This inhibition occurred both with the direct addition of gamma-IFN to the culture plates as well as by the preincubation of marrow cells with gamma-IFN followed by the washing of the cells; at the highest concentration of gamma-IFN (10,000 U), near-maximal inhibition of colony formation occurred with as little as 15 min of preexposure (BFU-E, 50%; CFU-E, 81%). Removal of monocytes and/or T lymphocytes before the addition of gamma-IFN significantly reduced the inhibitory effects of this lymphokine (BFU-E, -1 to 38%; CFU-E, -8 to 67%). Co-culture of purified autologous monocytes or T cells preexposed to gamma-IFN with monocyte and T cell-depleted marrow cells resulted in highly significant inhibition of erythroid colony formation even when these treated cells comprised less than 1% of the total nucleated cell populations in culture. The inhibitory action of gamma-IFN was not prevented or reversed by erythropoietin. These results demonstrate that the inhibitory effects of gamma-IFN on erythropoiesis are mediated to a significant degree through accessory cell populations, and suggest that gamma-IFN may represent a useful tool in the study of the role of immunocompetent cells in the regulation of erythropoiesis in vitro.     

Effect of parathyroid hormone on erythropoiesis.

Inhibitors of erythropoiesis have been found in the blood of uremic patients but their nature has not been identified. These patients have excess blood levels of parathyroid hormone (PTH) and it is possible that PTH inhibits erythropoiesis. The present study was undertaken to examine the effect of intact PTH molecules and some of its fragments on human peripheral blood and mouse bone marrow burst-forming units-erythroid (BFU-E), on mouse bone marrow erythroid colony-forming unit (CFU-E), and granulocyte macrophage progenitors (CFU-GM), and evaluate the interaction between PTH and erythropoietin (Ep) on human BFU-E. Intact PTH (1-84 bPTH) in concentrations (7.5-30 U/ml;) comparable to those found in blood of uremic patients produced marked and significant (P less than 0.01) inhibition of BFU-E and mouse marrow GFU-GM, but not of mouse marrow CFU-E. Inactivation of 1-84 bPTH abolished its action on erythropoiesis. Increasing the concentration of Ep in the media from 0.67 to 1.9 U/ml overcame the inhibitory effect of 1-84 bPTH on BFU-E. The N-terminal fragment of PTH (1-34 bPTH) and 53-84 hPTH had no effect on BFU-E. The results demonstrate that (a) either the intact PTH molecule or a C-terminal fragment(s) bigger than 53-84 moiety exerts the inhibitory effect on erythropoiesis, and (b) adequate amounts of Ep can overcome this action of PTH. The data provide one possible pathway for the participation of excess PTH in the genesis of the anemia of uremia.     

Inhibition of bone marrow colony formation by human natural killer cells and by natural killer cell-derived colony-inhibiting activity

Incubation of human peripheral blood lymphocytes with bone marrow cells resulted in significant inhibition of colony formation by committed myeloid and erythroid cells. Using positively selected homogeneous natural killer (NK) cell preparations and lymphocyte subpopulations depleted of or enriched for NK cells, we definitively characterize as NK cells the cells in normal peripheral blood that are responsible for inhibition of bone marrow colony growth. The inhibitory effect of NK cells on hematopoiesis can be mediated by a soluble factor that is produced only by NK cells upon culture with HLA-DR+ hematopoietic cells and with NK-sensitive cell lines. Both NK cells and the NK-produced, colony-inhibiting activity (NK-CIA) are suppressive for allogeneic and autologous bone marrow CFU-GEMM (colony-forming units, granulocyte, erythroid, monocyte, megakaryocyte), CFU-E (CFU, erythroid), and early CFU-GM (CFU, granulocyte, monocyte), but not for either BFU-E (burst-forming units, erythroid) or late CFU-GM. [3H]Thymidine incorporation was inhibited by NK-CIA-containing supernatants in HLA-DR+ but not HLA-DR- bone marrow cell populations stimulated to proliferative by colony-stimulating factor (CSF). These data suggest that the NK cell-mediated inhibitory effect on proliferation and differentiation of hematopoietic precursor cells is mediated in part or completely by the secreted NK-CIA. The concentration of NK-CIA reached in the supernatant of the mixture of NK cell-containing lymphocyte populations with bone marrow cells is sufficient to account for the inhibitory effect mediated by NK cells. Our data support the hypothesis that human NK cells play a major role in the control of hematopoiesis, down-regulating it under conditions in which the NK cells are functionally activated.     

Polycythemia Vera: FURTHER IN VITRO STUDIES OF HEMATOPOIETIC REGULATION

Further in vitro studies of hematopoietic regulation were carried out in two patients with polycythemia vera who were also heterozygotes (Gd^B/Gd^A) for glucose-6-phosphate-dehydrogenase (G-6-PD). While only G-6-PD type A was detectable in circulating erythrocytes, granulocytes and platelets, cultures of peripheral blood and marrow from one patient revealed a substantial number of G-6-PD type B erythroid burst-forming units (BFU-E) and granulocyte/macrophage colony-forming units. Detailed analysis demonstrated: (a) where detectable, normal BFU-E and granulocyte/macrophage colony-forming units were found with similar frequencies; (b) the same frequencies for normal progenitors characterized cultures of peripheral blood and marrow; (c) inhibition of normal erythroid differentiation between BFU-E and the more mature erythroid colony-forming unit; (d) a decline in the prevalence of normal colony-forming units with time, suggesting that disease progression is associated with further suppression of normal hematopoiesis by products of the abnormal clone.     

In vitro hepatitis B virus infection of human bone marrow cells.

Infection of humans with hepatitis B virus (HBV) frequently results in suppression of hematopoiesis; in some cases this may lead to severe bone marrow failure. The mechanism whereby HBV infection affects hematopoiesis is unknown. In vitro exposure of human bone marrow to HBV results in a dose-dependent inhibition of erythroid (erythroid burst forming units, BFU-E; erythroid colony-forming units CFU-E), myeloid (colony-forming units-granulocyte macrophage CFU-GM), and lymphoid (CFU-[T-lymphocytic]-TL) hematopoietic stem cells. Inactivation or immunoabsorption of HBV from sera resulted in loss of HBV-induced inhibition of hematopoietic stem cells. De novo gamma interferon was not detectable in the supernatants of cultures of bone marrow cells with HBV. Antibodies to gamma interferon did not affect the suppression of hematopoietic stem cells by HBV. Hepatitis B surface antigen (HBsAg) was detected by immune electron microscopy in nuclei of greater than 70% of immature hematopoietic cells including myeloblasts, normoblasts, and lymphoblasts; granulocytes had mostly cytoplasmic HBsAg. Hepatitis B virus core antigen (HBcAg) was also detected in about 5% of HBV infected bone marrow cells by immunoperoxidase staining. These data indicate that HBV can infect hematopoietic cells and their progenitors, thus suggesting a wider range of tropism for HBV than previously reported. These results may provide a basis to study HBV infection in vitro, and the effects of HBV on hematopoiesis.     

Three-lineage hemopoietic precursor cells and effectiveness of recombinant human erythropoietin in patients with myelodysplastic syndromes.

Four-stem-cell assays, which evaluate megakaryocytic (CFU-Meg), immature and mature erythropoietic (BFU-E, CFU-E), and granulocyte-macrophage (CFU-GM) colony formation, were performed in nine patients with myelodysplastic syndromes (MDS). The CFU-Meg, BFU-E, and CFU-E colony growths were disturbed more often than the CFU-GM colony formation. A CFU-E increase was not recognized in most MDS patients, but a dose-dependent increase of bone marrow CFU-Es in response to erythropoietin (EPO) was recognized only in two refractory anemia (RA) patients whose CFU-Es were more than one tenth of normal controls. One patient with RA and the other with chronic myelomonocytic leukemia (CMML), both of whose bone marrow CFU-Es did not increase at the higher dose of EPO in vitro, were treated with recombinant human EPO (rHuEPO), resulting in no effects. The responsiveness of patients with MDS to various recombinant hemopoietic factors might be predicted by both the residual degree of bone marrow hematopoietic precursor cells and the response of stem cells to the higher doses of each hemopoietic factor.     

PNH患者骨髓红系祖细胞体外培养的研究

采用造血祖细胞体外培养技术研究了阵发性睡眠性血红蛋白尿症（ＰＮＨ）患者骨髓红系祖细胞（ＢＦＵ－Ｅ和ＣＦＵ－Ｅ）的增殖能力；骨髓细胞经新鲜酸化ＡＢ型血清处理后培养的ＢＦＵ－Ｅ和ＣＦＵ－Ｅ的增殖能力；以及ＢＦＵ－Ｅ和ＣＦＵ－Ｅ对红细胞生成素（Ｅｐｏ）的反应能力。发现ＰＮＨ患者骨髓ＢＦＵ－Ｅ和ＣＦＵ－Ｅ集落数明显低于正常；骨髓细胞经新鲜酸化ＡＢ型血清处理后培养的ＢＦＵ－Ｅ和ＣＦＵ－Ｅ集落数明显低于经热灭活酸化ＡＢ型血清处理后培养的集落数；ＢＦＵ－Ｅ和ＣＦＵ－Ｅ对Ｅｐｏ的反应回归直线呈低平状态。认为ＰＮＨ患者骨髓红系祖细胞膜缺陷可导致其在酸性条件下对补体的敏感性增高和导致其对Ｅｐｏ的敏感性降低。     

5-Azacytidine acts directly on both erythroid precursors and progenitors to increase production of fetal hemoglobin.

The effect of 5-azacytidine on erythroid precursors and progenitors was studied in nine patients with sickle cell anemia or severe thalassemia. Each patient received the drug intravenously for 5 or 7 d. 5-Azacytidine caused a four- to sixfold increase in gamma-messenger RNA concentration in bone marrow cells of eight of the nine patients and decreased the methylation frequency of a specific cytosine residue in the gamma-globin gene promoter in all nine patients. Within 2 d of the start of drug treatment there was a rise in the percentage of reticulocytes containing fetal hemoglobin (HbF; F-reticulocytes) without a significant change in the total number of reticulocytes, which suggested that there was a direct action of 5-azacytidine on erythroid precursors. Late erythroid progenitors (CFU-E), present in bone marrow after 2 d of drug administration, formed colonies containing an increased amount of HbF as compared with control colonies. Moreover, the number of CFU-E derived colonies was not decreased at these early times, which suggested that there was a direct action of 5-azacytidine on erythroid progenitors in the absence of cytotoxicity. Exposure of normal bone marrow cells in tissue culture to 5-azacytidine for 24 h reproduced both of these effects as judged during subsequent colony formation. The combined direct effects of 5-azacytidine on both the erythroid precursor and progenitor compartments resulted in an increase in HbF synthesis that was sustained for 2-3 wk. Toxicity to bone marrow as reflected by cytoreduction was evident after treatment in some patients but was not accompanied by an increase in HbF production. A correlation was found between the effects of 5-azacytidine on bone marrow, as assessed by in vitro measurements, and the hematological response of the individual patients to drug treatment.     

Determination of the hemoglobin F program in human progenitor-derived erythroid cells.

The absolute adult and fetal hemoglobin (HbF) contents of the erythroid cells derived from the differentiation of normal human and simian erythroid progenitors and of the peripheral blood erythroid burst-forming units (BFU-E) of patients with nondeletion hemoglobinopathies have been measured with a sensitive radioligand immunoassay. The HbF content varied between 0.13 and 2.96 pg/cell, representing between 0.7% and 19.6% of the total hemoglobin with a mean value of 7.0%. The absolute content of HbF was indistinguishable in the well-hemoglobinized progeny of marrow erythroid colony-forming units, marrow or blood BFU-E, or of mixed colony-forming units. The term HbF program refers to this inherent capacity to produce fetal hemoglobin (HbF) in the erythroid cells derived from these progenitors in vitro. The HbF content of marrow erythroblasts as determined by the same radioligand immunoassay was similar to that found in the peripheral blood, suggesting that the switch off of gamma-chain production occurs after the erythroid colony-forming unit stage of maturation. Increasing concentrations of a crude erythropoietin-containing preparation induced higher numbers of erythroid colonies, which were larger in size, but the HbF program was unaffected. In contrast to the hemoglobin accumulation in human progenitor-derived colonies, simian progenitor-derived colonies produced considerably more HbF, and the amount of HbF was strongly influenced by progenitor maturity. Assays of the HbF content of erythroblasts derived from culture of the peripheral blood BFU-E of patients with nondeletion hemoglobinopathies and their parents showed that the HbF program in the progenitors of such patients is highly variable. Some produce only a slight excess of HbF in progenitor-derived erythroblasts, whereas others have extraordinarily high HbF programs. The molecular basis of this variability is presently unknown.     

骨髓基质细胞系QXMSC_1体外促进造血及其作用机理的研究

本实验研究了骨髓基质细胞系QXMSC_1体外对CFU-F,CFU-GM,BFU-E及CFU-E集落形成的影响,并通过建立QXMSC_1基质细胞与骨髓造血细胞的长期共培养体系,研究了QXMSC-1在体外促进造血的机理。结果表明,QXMSC_1基质细胞条件培养上清液可促进小鼠CFU-F,CFU-GM,BFU-E及CFU-E集落的生长。在无GM-CSF和Epo存在的情况下,QXMSC_1细胞能维持CFU-GM,BFU-E和CFU-E集落生长。表明QXMSC_1细胞本身可分泌GM-CSF和Epo。QXMSC_1细胞与骨髓造血细胞共培养时,可明显提高CFU-GM集落数。共培养体系中,用微孔滤膜隔断QXMSC_1与造血细胞的直接接触,也能促进CFU-GM集落生成,但比混合培养时低,说明QXMSC_1通过直接接触和分泌细胞因子促进造血的过程。     

Value of in vitro models for the assessment of drug-induced haematotoxicity

BACKGROUND: A new antipsychotic compound induced unexpected red cell hypoplasia (reticulocytopenia, red marrow hypoplasia) in rats dosed orally for 7 days. MATERIALS AND METHODS: Since an erythropoietin-mediated pathogenesis was excluded, in vitro tests on rat and human bone marrow cells were performed with measurement of formation of late erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) colony-forming units after incubation with the drug. CFU-E together with growth factors were cultured for 2 days (rat) or 7 days (human) and CFU-GM was cultured for 7 days (rat) or 10 days (human). RESULTS: The drug induced inhibition of erythroid progenitors and myeloid progenitors for both species from 3 x 10(-5) mol/L, with the concentration inhibiting the growth of 50% (IC50) consistent with drug plasma levels measured in rats. CONCLUSION: These cloning assays on rat bone-marrow cells were shown to be adequate models for determining the haematotoxicity of the agent and to be predictive of human toxicity. With only a small amount of compound required, they can be used as screening tools to detect haematotoxic potential of candidate drugs.     

严重型再生障碍性贫血患者骨髓造血祖细胞集落形成与免疫抑…

OBJECTIVE: To explore the defect of hematopoietic stem cell and analyze the relationship between the colony formation capacity of bone marrow hematopoietic progenitor cells and the results of immunosuppressive therapy (IST) in severe aplastic anemia (SAA) patients. METHODS: Methylcellulose semisolid culture was used. RESULTS: Thirty patients with SAA at diagnosis were studied. In 90% of the patients, the CFU-E and CFU-GM yields were strikingly decreased and in 56.7% of the patients there was no colony formation at all. Nevertheless, there was still 10% of the patients having normal CFU-E, BFU-E or/ and CFU-GM yields. After IST, 59.1% of the patients showed colony formation improvement. The difference between the results obtained pre- and post-IST(for CFU-E and BFU-E, P < 0.01, for CFU-GM, P < 0.05) was significant. In the majority of the patients, the colony formation capacities were still under the normal post-IST. Moreover, 22.7% of the patients remained no colony formation post-IST. The IST response rates between the patients with or without colony formation had no statistically difference (80% versus 50%, P < 0.05). The increment of colony formation appeared later than the therapeutic effect did, but frequently concurred with the appearance of magkaryocytes in the bone marrow smear. CONCLUSION: In the majority of SAA patients, the hematopoietic stem cells or progenitors were defective perhaps caused by immune damage; IST can improve the hematopoiesis in vitro and in vivo of SAA patients.