A case of drug-induced hematotoxicity: from in vivo to in vitro assessment

As part of the early preclinical development of a new antipsychotic compound, Wistar rats of both sexes were dosed orally for upto 7 days. At high doses, expected changes in appearance and behavior, decreases in bodyweight gain and feed intake but also a fluid and pale bone marrow (BM) were observed. Blood cell counts were normal as were clinical chemical values. BM sections showed a red cell hypoplasia. Circulating reticulocytes and erythroblasts on BM smears were decreased suggesting that the compound might have a selective toxicity for the erythroid lineage. In a mechanistic experiment, rats were dosed for 9 days and phlebotomized after 7 days of exposure to stimulate erythroid regeneration. Red-blood cell mass, reticulocytes and erythropoietin (EPO) levels were monitored before and upto 48 h after bleeding. Results showed that an EPO-mediated pathogenesis could be excluded. The effect of the drug on the formation of Colony-forming units (CFU)-E and CFU-GM was then quantitatively measured in vitro after direct exposure to the compound. In two successive assays, rat or human BM cells were incubated with the drug dissolved in the collection medium at final concentrations of 0.3×10^–7 –3×10^–5 M. In the presence of adequate growth factors, CFU-E and CFU-GM were cultured and cell proliferation was compared between treated and control groups. Our results showed an expected inhibition by the drug of the growth of erythroid progenitors associated to a similar effect on myeloid progenitors. The CFU-E and CFU-GM of both human and rat sources were totally inhibited from the concentration of 3×10^–5 M. The IC₅₀ values were consistent with rat peak plasma levels reached in vivo by the drug. Therefore, the short-term cloning assays performed on rat BM cells were sensitive indicators of the hematotoxicity of the compound and were considered as predictive for human toxicity.     

急性非淋巴细胞白血病过程细胞克隆的变化

18例未治或复发ANLL,2例慢粒急变及10例完全缓解(CR)的ANLL患者作L-CFU,CFU-E及CFU-GM骨髓体外造血祖细胞培养。ANLL未治和复发组的L-CFU、CFU-E及CFU-GM集落数与正常对照有非常显著差异。ANLL的CR组,CFU-E和CFU-GM恢复正常,但L-CFU仍较正常对照组明显多,提示CR后,骨髓中仍残留有白血病细胞。初步认为PHA-MNCCM作为L-CFU的生长因子,有助于CR后ANLL残留白血病细胞检测意义。     

几种造血刺激剂拮抗干扰素对粒-单系祖细胞的抑制效应

本实验用C_(57)BL/6j小鼠CFU-GM集落形成的方法观察了三种CSF和氢化可的松、锂盐及硒盐对CFU-GM的影响及其对IFN的CFU-GM抑制作用的阻断作用。结果表明,三种CSF能部分拮抗IFN的CFU-GM抑制效应;微量的硒酸钠,碳酸锂均可刺激CFU-GM增殖,并使IFN200IU抑制的CFU-GM产率提高200％以上;微量的氢化可的松也能部分阻断IFN的CFU-GM抑制。     

骨髓增生异常综合征及慢性原因不明性粒细胞减少症患者骨髓CFU—GM和CFU—F的初步观察

利用人类骨髓粒一单核系祖细胞集落(CFU—GM)和成纤维细胞集落(CFU—F)体外检测技术,比较观察32例骨髓增生异常综合征(MDS)和11例慢性原因不明性粒细胞减少症(CIN)患者的造血改变。结果表明,MDS 和 CIN 的造血状态存在显著区别,69%的 MDS 患者 CFU—GM集落显著受抑,集簇、簇/落比值明显升高;其 CFU—F 集落亦存在明显缺陷。而 CIN 患者的上述指标均类似于正常。这提示体外集落检测技术有助于 MDS 和 CIN 的鉴别。此外,CFU—GM 系列培养对 MDS 患者具有一定预后价直。     

胎肌条件培养液抗损伤效应的观察

胎肌条件培养液（ＦＭＣＭ）可在ＣＦＵ－ＧＭ体外培养中提供集落刺激活性。研究发现它还能在体外培养中保护ＣＦＵ－ＧＭ，减轻理化损伤因素对其生长的抑制效应。所用化学损伤因素为５×１０－７Ｍ的阿糖胞苷与９×１０－９Ｍ的三尖杉酯碱，物理损伤因素为放射（３．５Ｇｙ），热（４４℃，６０分钟）与冷（—２０℃，１６分钟）。在损伤因素作用下，ＣＦＵ－ＧＭ的集落产率受到明显抑制，用２０％ＦＭＣＭ处理后，抑制现象可以减轻。ＦＭＣＭ可以对抗多种因素对ＣＦＵ－ＧＭ的抑制作用，没有特异性。用胰蛋白酶或热（８５℃，３０分钟）处理后的ＦＭＣＭ不具上述保护效应，用酸（ＰＨ＝２），碱（ＰＨ＝１０），ＤＮＡ酶与透析处理则无明显影响。提示ＦＭＣＭ中起保护作用的活性物质可能为分子量大于１００００的蛋白质。     

4℃保存外周血造血干细胞动态观察

本文对4℃条件下不同时间保存后的外周血造血干细胞(peripheral bloodhematopoietic stem cells,PBHSC)数进行了动态观察。在本实验中以定向造血祖细胞(CFU-GM)作为观察PBHSC的指标。结果表明:随着保存时间的延长,PBHSC集落数逐渐下降;0小时与24小时保存后的干细胞数无显著差异;24小时与48、72、96、120小时保存后的干细胞数有高度显著差异;48小时与72、96、120小时保存后的干细胞数无显著差异。同时发现,红细胞的存在有利于PBHSC的4℃保存。     

白血病相关抑制物对CFU－GM增殖的抑制作用

用半固体双层琼脂培养法，将白血病细胞植入底层，正常ＣＦＵ－ＧＭ植入上层，作白血病细胞与正常ＣＦＵ－ＧＭ的共同培养。结果发现，底层琼脂中加入０．５×１０５／ｍｌ的白血病细胞后，上层琼脂中的ＣＦＵ－ＧＭ从对照组的１９３．５下降至１２３．１／１×１０５个骨髓有核细胞（ＢＭＮＣ），加入底层的白血病细胞增加时，ＣＦＵ－ＧＭ生成进行性受抑制，当植入的白血病细胞数达１５×１０５／ｍｌ时，几乎无ＣＦＵ－ＧＭ生成。此抑制作用亦见于白血病细胞的培养上清液中。量－效关系测定发现，正常ＣＦＵ－ＧＭ培养体系中加入１０％的白血病细胞培养上清液时，ＣＦＵ－ＧＭ生成率从对照纪的１１２．２下降到８８．６／１×１０５ＢＭＮＣ，以后抑制效应不再增加。以上结果揭示，白血病细胞对ＣＦＵ－ＧＭ的抑制是经某种体液因子实现的。     

Monoclonal antibodies against human granulocytes and myeloid differentiation antigens

Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315–43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1. 80H.3. and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (lgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (lgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence. i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited BFU-GM and CFU-E. Antigens recognized by 80H.3. 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.     

The time course of leucocyte colony-formation in cultures of bone-marrow progenitor cells from rats and dogs

The colony-forming units granulocyte and macrophage (CFU-GM) assay, using either rat or dog haematopoietic progenitor cells, assesses the toxicity of new compounds. To identify the characteristics of colony formation in this system, a time-course study of CFU-GM assays using rat and dog bone marrow cells was tested. Neutrophil colonies, macrophage colonies and mixed colonies of neutrophils and macrophages were formed in soft agar medium. Neutrophil colonies reached their maximum number on days 3–4 and decreased markedly thereafter. Macrophage colonies reached their maximum number on days 7–8 and remained steady thereafter. Only a small number of mixed colonies of neutrophils and macrophages were formed beginning around day 4. There were no significant differences between rat and dog bone marrow cells in the occurrence of these maxima, or in any other growth phenomenon. This result suggests that to evaluate the influence of compounds on neutrophil colonies and macrophage colonies, observations should be made on days 4 and 8, respectively.     

植物血凝素(PHA)对小鼠骨髓粒—单祖细胞增殖的影响

用体外琼脂培养法研究了PHA对小鼠骨髓CFU-GM的影响,结果表明,小鼠经腹腔注射PHA后,对小鼠CFU-GM产率和自杀率的影响随PHA的剂量不同而异,20～40mg/kg的PHA能明显提高CFU-GM的产率,其作用时间维持3天,剂量大于50mg/kg,可抑制CFU-GM产率。PHA组自杀率较对照组明显提高,提示适量的PHA在体内可促进小鼠CFU-GM的增殖,使更多的CFU-GM由Go期进入S期。将PHA直接加入无CSF的培养体系中,未见集落生长。此项结果提示PHA可能通过间接机制而非直接作用于CFU-GM。