Hemopoietic Cell Lines Research Articles

Investigation of immunosuppressive properties of inactivated human immunodeficiency virus and possible neutralization of this effect by some patient sera

Retroviral infections are accompanied by immunosuppression in a variety of species. For feline leukemia virus, the immunosuppression has been ascribed to the transmembrane envelope protein, p15E, which suppresses the proliferative responses of cat, mouse, and human lymphocytes. A similar suppressive effect has been shown for a lysate of human immunodeficiency virus (HIV), strain HTLV-IIIB. Here we determined that detergent-disrupted HTLV-IIIB lysate exerted a strong suppressive effect on PHA-stimulated lymphocytes. Preparations of whole virions, a lysate of a local HIV isolate grown on MP-6 cells, and a commercially obtained UV and psoralene-inactivated lysate were examined and demonstrated to have a similar suppressive effect. The HIV lysate was not directly cytotoxic to lymphocytes and did not contain tumor necrosis factor or lymphotoxin. The HIV lysate specifically suppressed the proliferation of a range of hemopoietic cell lines from man and mouse including three EBV transformed CD4and IL-2 receptor-negative B-cell lines. The lysate also suppressed the formation of human bone marrow colonies, whereas the lysate had only a slight or no effect on fibroblasts. The suppression of lymphocyte proliferation was not abrogated by addition of IL-2 or IL-1 and the HIV lysate inhibited the expression of IL-2 receptors on suboptimal PHA-stimulated mononuclear cells. The suppressive factor(s) has not been characterized in molecular terms, but suppressive activity was recovered in fractions with a molecular weight of about 67,000 and in both the glycoprotein fraction and in the glycoprotein-depleted fraction of the HIV lysate. Sera from one-third of a small series (N = 13) of individuals with antibodies to HIV seem to be able to neutralize the suppressive properties of HIV lysate in cultures.

Biological activity of recombinant murine interleukin-6 in interleukin-1 T cell assays

Interleukin-6 (also called B cell stimulatory factor 2, hepatocyte activating factor, interferon-β 2) has been shown to have effects on various lineages of hemopoietic cells. Some of its activities appear to overlap those of interleukin-1. In particular, recombinant murine IL-6 induced proliferation of phytohemagglutinin-activated thymocytes, an assay widely used to detect IL-1. In this report, we compared several features of IL-1 and IL-6 dependent thymocyte proliferation. The results indicate that IL-2 is the major second mediator of both IL-1 and IL-6 dependent proliferation. Finally, we tested whether IL-6 would also have activity in other T cell-based IL-1 assays using the T cell lymphoma LBRM33 1A5 and the T cell clone D10-G4.1. IL-6 had no activity in the latter two assays. These results indicate that IL-1 assays using LBRM33 1A5 and D10-G4.1 selectively detect Il-1, and are more specific assays for the detection of IL-1 in samples that may also contain IL-6.

Metabolic properties of a homogeneous proteoglycan of a haemopoietic stem cell line, FDCP-mix

A biochemical analysis has been carried out of metabolically labelled proteoglycans and glycosaminoglycans synthesized by a haemopoietic multipotential stem cell line, FDCP-mix. The only proteoglycan identified in these multipotential cells was a homogeneous component that contained chondroitin 4-sulphate chains (Mr approximately 10,000) arranged in close proximity in a proteinase-resistant domain of the protein core. Small quantities of free chondroitin 4-sulphate were also detected. Following a 48 h incubation with Na2 35SO4 the majority of the 35S-radiolabelled proteoglycans (approximately 80%) were associated with the cells, mainly in an intracellular compartment, and the remaining 20% were in the culture medium. Pulse-chase studies demonstrated two turnover pathways for the newly synthesized cellular proteoglycans. In the minor pathway, the proteoglycans were secreted rapidly into the medium without any discernable structural modification. In the major pathway the proteoglycans seemed to be transferred into a storage compartment from which the intact macromolecules were not secreted. Eventually, these proteoglycans were degraded to yield free polysaccharide chains and these chains were then released into the medium, but only at a relatively slow rate. There was very little intracellular degradation of chondroitin sulphate chains. The pathway to polysaccharide secretion was a slow stepwise process with a time-lag of about 5 h between proteoglycan synthesis and the appearance of free chondroitin sulphate and a second time-lag, also of about 5 h, before these chains began to be secreted. The existence of separate secretory pathways for proteoglycans and chondroitin sulphate chains is an interesting characteristic that seems to distinguish proteoglycan metabolism in primitive multipotent stem cells from related metabolic processes in mature haemopoietic cells.

Interaction of serum and colony‐stimulating factor for survival of a factor‐dependent hemopoietic progenitor cell line

The growth in vitro of the murine myeloid cell line FDC-P1 depends on the presence of serum and a murine hemopoietic growth factor (either granulocyte/macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (multi-CSF, IL3]. To determine the differential roles of serum and colony-stimulating factor (CSF) during the growth of FDC-P1 cultures, we investigated the kinetics of proliferation and death after withdrawal of serum or CSF, using flow cytometry to quantitate the numbers of vital and dead cells. After withdrawal of CSF, the cells died without entering a quiescent state. The life span of cultures lacking CSF increased with increasing concentrations of serum (greater than 50 h at 30% serum), and the cells kept dividing until they died. During the period of population death caused by the absence of CSF, the re-addition of CSF immediately prevented further cells from dying. After the withdrawal of serum in the presence of CSF, the cells continued to live and proliferate for weeks, but required high cell densities (much greater than 10(5)/ml), which suggests that the cells produced an active substance that can substitute for serum. Serum as well as serum-free conditioned medium from dense cultures made the survival and growth of FDC-P1 cultures independent of cell density. Without sufficient quantities of this activity, all cells of the population died within an interval that was much shorter than one cell cycle, which indicates that the factor acts throughout most of the cell cycle. The results suggest that both the CSF and the serum factor act together to permit cell survival, rather than to drive proliferation.

Multidrug resistance in haemopoietic cell lines, myelodysplastic syndromes and acute myeloblastic leukaemia

Resistance to cytotoxic agents is a common clinical problem encountered in the treatment of human myelodysplastic syndromes (MDS) and acute myeloblastic leukaemia (AML). Cellular acquisition of the multidrug resistance (MDR) phenotype confers loss of sensitivity to a wide range of structurally dissimilar anti-neoplastic agents. This state can arise through increased expression of the mdrl (P-glycoprotein) gene. We have used the mdrl gene probe to investigate adriamycin resistant (HL60/AR) and vinblastine resistant (CEM/VLB100) human leukaemic cell lines. In addition, peripheral blood or bone marrow cells from 66 patients with MDS and AML have been screened for gene amplification and 40 cases for increased mRNA expression. P-glycoprotein gene amplification was observed only in the (CEM/VLB100) and not in the HL60/AR on any other leukaemic cell line. Gene amplification was not found in any patient's cells. Eighteen out of 40 patients showed an increase (2----20) of mdrl mRNA expression. These results are not only of significance in understanding the biology of human drug resistance but have practical importance in the design of anti-leukaemic therapy.

Molecular Cloning of 114/A10, a Cell Surface Antigen Containing Highly Conserved Repeated Elements, Which Is Expressed by Murine Hemopoietic Progenitor Cells and Interleukin-3-dependent Cell Lines

Monoclonal antibody 114/A10, raised against the murine multipotential hemopoietic progenitor cell line B6SUtA, identifies an antigen highly expressed by primary myeloid progenitor cells, the myelomonocytic leukemia cell line WEHI-3, and various interleukin-3 (IL-3)-dependent cell lines. Western blotting studies indicate that the 114/A10 antigen has a mean relative molecular mass of 150,000 but varies greatly in size range between different cell types. cDNA clones encoding this protein were isolated from a plasmid-based expression vector library prepared from B6SUtA RNA. Three clones corresponding in size to the two major mRNA species detected in IL-3-dependent cell lines (3.0 and 2.2 kilobase pairs) and differing in their utilization of alternative polyadenylation signals were obtained. These clones contain a single long open reading frame of 573 amino acids possessing the typical characteristics of an integral membrane protein. A particularly striking feature of this sequence is the presence at the extracellular amino terminus of a series of eight highly conserved 27-amino acid, serine/threonine-rich (55%) tandem repeats that may serve as sites of extensive glycosylation. The extracellular domain also contains three epidermal growth factor-like cysteine-rich repeats. The distribution and structural characteristics of the 114/A10 antigen suggest a possible regulatory role in the cellular response to IL-3.

Detection of Granulocyte Colony‐stimulating Factor Produced by a Newly Established Human Hepatoma Cell Line Using a Simple Bioassay System

A colony‐stimulating factor(CSF)‐producing tumor cell line (KX‐87) was established from a patient with hepatocellular carcinoma and marked granuloeytosis. This cell line formed tumors on nude mice in high frequency and the mice revealed marked granuloeytosis. In clonogenic assays of human bone marrow cells, KX‐87 conditioned medium (CM) supported the formation of colonies mainly consisting of neutrophilic granulocytes but had no burst‐promoting activity. The molecular weight of the colony‐stimulating activity (CSA) in KX‐87CM was estimated about 25,000 daltons by gel filtration and a new bioassay system. In principle, a subline of murine hemopoietic cell line NFS‐60 was cloned which was dependent on KX‐87CM. Then the growth of this subline was examined by a rapid and sensitive colorimetric tetrazolium assay. From these results, it was concluded that the CSA which KX‐87 cell line produced was G‐CSF.

Characterization of membrane homing receptors in two cloned murine hemopoietic progenitor cell lines.

To characterize homing receptors that are responsible for the recognition and specific binding of hemopoietic progenitor cells to the stroma, we synthesized and 125I-labeled a number of neoglycoproteins. We used these neoglycoproteins as ligand to detect receptors on the membrane of two cloned murine hemopoietic progenitor cell lines, B6SUT and FDCP-1. Both cell lines demonstrated membrane receptors with galactosyl and mannosyl, but not fucosyl, specificities. B6SUT galactosyl receptors showed a single receptor population with a Kd of about 2.3 X 10(-7) M and 10(6) receptors per cell. Mannosyl receptors demonstrated two components with high and low affinities respectively with Kd of about 2.5 X 10(-8) M and 1.0 X 10(-7) M, and respectively about 7.4 X 10(5) and 3.7 X 10(5) receptors per cell. Comparable data were also obtained for FDCP-1. Displacement experiments indicated that radioactive ligands bound to receptors could be increasingly displaced by homologous cold ligand giving typical sigmoid-shaped curves. Cold mannosyl probe could also displace radioactive galactosyl probe in a similar manner, but cold galactosyl probe displaced radioactive mannosyl ligand with a curve demonstrating two phases, further suggesting two receptor components for the mannosyl ligand. Mature murine neutrophils and red cells as well as human neutrophils, monocytes, and red cells showed no receptors. The functional significance of these receptors in binding to stromal cells was demonstrated by quantitation of the binding of 51Cr-labeled progenitor cells to the cloned stromal cell line, D2X, before and after enzymatic removal of various carbohydrate residues of membrane glycoconjugates. Enzymatic removal of galactosyl and mannosyl, but not fucosyl, residues almost totally eliminated the binding. The findings strongly suggest that these homing receptors are present on the surface of early hemopoietic progenitor cells. With maturation the cells lose their receptors, so that mature cells can be released into the blood stream.

Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction.

The pivotal role that protein-tyrosine kinases (PTKs) play in the growth regulation of eukaryotic cells is manifest in the frequent appearance of members of the PTK family as growth factor receptors or as the transforming agents of acutely transforming retroviruses. A feature common to all members of the PTK family is a highly conserved catalytic domain which is characteristic of the group as a whole and whose activity appears to be tightly regulated within the cell by other domains of the PTK. Degenerate oligonucleotide probes corresponding to two invariant amino acid sequence motifs within the catalytic domains of all PTK family members were synthesized and employed in the polymerase chain reaction (PCR) to amplify cDNA sequences between them. An M13 PCR library was produced in this way from cDNA prepared against mRNA from the murine hemopoietic cell line FDC-P1. The PCR library was then screened by DNA sequencing for PTK-related sequences. Two sequences were identified that, on the basis of sequence comparison with known PTKs, may encode representatives of a new class of PTK.

Structure and biological activity of the transcriptional initiation sequences of the murinec-myboncogene

To study the control mechanism(s) that govern the transcription of c-myb, genomic clones corresponding to the 5' region of the murine c-myb gene have been isolated and characterized structurally and functionally. Primer extension and nuclease protection analysis have revealed the presence of multiple transcriptional initiation sites, that are utilized in several hemopoietic cell lines (WEHI3B(D+). FDC-P1 and RB22.2). Some of the sites are used in all cell lines but others are unique; all are located in a region of the c-myb gene that is G-C rich, contains a number of potential Sp1 binding sites and lacks classical promoter consensus sequences. Experiments in which well characterized promoters controlling expression of a reporter gene have been replaced by fragments of c-myb DNA (including the observed cap sites) were performed in an attempt to demonstrate promoter activity in various cell types. It was shown that a region of the c-myb gene (approximately 1.0 kbp upstream from the splice donor site of the first exon) contains a weak promoter that has a low level of transcriptional activity in hemopoietic as well as in fibroblastic cells. These results support the suggestion that c-myb expression is not regulated primarily at the level of initiation of transcription.

Treatment of myelodysplastic syndromes with recombinant human granulocyte colony-stimulating factor: A preliminary report

purpose: The expansion of an abnormal hemopoietic stem cell line is responsible for the myelodysplastic syndromes, which are characterized by pancytopenias, often resulting in lethal infections. Cloned granulocyte colony-stimulating factor (G-CSF) was recently shown to enhance the growth and differentiation of normal granulocyte progenitor cells in vitro. The aim of our study was to examine the effects of recombinant human G-CSF in patients with myelodysplastic syndromes. patients and methods: Four patients with myelodysplastic syndromes and one patient with smoldering acute myelogenous leukemia following the occurrence of a myelodysplastic syndromes received recombinant human G-CSF by intravenous infusion for six days. Patients received different dosage levels (50 to 1,600 μg/m 2). results: A response was seen in all patients with an increase in both immature myeloid cells in the bone marrow and mature granulocytes in the peripheral blood. The dose levels that could stimulate granulocyttopoiesis differed among patients. conclusion: These results suggest that, at least in some cases of myelodysplastic syndromes, granulocytopenia can be improved by G-CSF, although it still remains to be determined whether the increase in the number of granulocytes is due to the differentiation and maturation of the myelodysplastic clone or restoration of a residual normal clone.

An autocrine activity capable of substituting for serum in cell cultures.

Provided that high cell densities (above 10(6)/ml) are maintained, a factor-dependent murine hemopoietic progenitor cell line (FDC-P1) will proliferate in serum-free medium. The conditioned medium (CM) from high-density FDC-P1 cells permits the serum-free survival of FDC-P1 cells even at low density, indicating the existence of a diffusible autocrine factor. The requirement of FDC-P1 for a colony-stimulating factor (either IL-3 or GM-CSF) is not abrogated by culturing the cells at high cell density or in the conditioned medium. Furthermore, the CM from FDC-P1 enhances the mitogenic stimulation of normal human skin fibroblasts (HSF) by epidermal growth factor (EGF): i.e., the lag period before entry into the cell cycle is shortened by up to 6 hr. The fibroblasts themselves secrete an activity into serum-free medium that appears to be required during mitogenic stimulation by EGF. The HSF-CM also allows FDC-P1 cells to survive and proliferate serum-free at low cell densities. Low concentrations of fetal calf serum or human plasma (0.2-2%) have the same effect as FDC-P1-CM and HSF-CM. We have tested many of the known growth factors, and none of them mimicked the autocrine serum replacing activity (ASRA). The activity in human plasma elutes from a gel-filtration column with an apparent molecular weight of 60,000. It appears as if cultured normal cells and cell lines produce molecules capable of complementing the growth factors required for the survival and proliferation of a range of cells in serum-free cultures.

Haemopoietic growth factors: their role in acute myeloblastic leukaemia.

The chromosome alterations specifically associated with leukaemia are found largely in the regions where the genes for the haemopoietic growth factors (as well as other regulatory molecules or their receptors) are located, indicating a crucial role of the growth factors in leukaemogenesis. However, growth factor genes per se do not generally induce leukaemia when inserted into normal haemopoietic cells, although they will do so if they are inserted into immortalized haemopoietic stem cell lines. The response of AML cells to these growth factors is extremely heterogeneous, and the tilting of the balance between self-reproduction (leading to perpetuation of the leukaemic process) and differentiation ('death' of the malignant cells) depends on several parameters, on the type and combination of factors to which the cells are exposed, with IL-3 and GM-CSF tending to favour self-renewal, and G-CSF and M-CSF tending to favour differentiation. These findings open the possibility to consider the use of growth factors to control the leukaemic process, although such treatment should be approached with considerable caution, and on an individual patient basis.

Gene transfer into hemopoietic stem cells using retroviral vectors.

Gene transfer to hemopoietic cells offers a variety of new approaches to the experimental hematologist as well as potentially providing a means for correcting a number of genetic disorders of humans. From the experimental viewpoint, gene transfer utilizing retroviral vectors introduces new methods for analyzing hemopoietic cell lineages, and the effects of over-expression of genes affecting hemopoietic cell proliferation and differentiation. The unique properties of retroviral vectors and the optimized methods currently in use to infect hemopoietic cells are represented as a brief review of a rapidly expanding new field of experimental hematology.

Direct demonstration of an autocrine mechanism in EMS-induced, tumorigenic mutants of the growth factor-dependent hemopoietic cell line, FDC-P1.

A growth factor-dependent hemopoietic cell-line, FDC-P1, was treated with a chemical mutagen, EMS, and a number of growth factor-independent variants isolated. Six lines have been extensively analyzed with respect to their growth kinetics, morphology, karyotype, tumorigenicity, and hemopoietic growth factor production. Four lines produced at least one growth factor, subsequently demonstrated to be GM-CSF, while two lines showed no evidence of hemopoietic growth factor production. The observation that the autonomous proliferation of those EMS-derived cell lines that produced GM-CSF can be inhibited by incubation in growth media containing 10-50 microM anti-sense GM-CSF oligonucleotides demonstrated directly that the autonomous behavior of these cells is based on an autocrine mechanism. The induction of the expression of the GM-CSF gene represents a rare class of EMS-induced mutants, and is strongly suggestive of repressor inactivation rather than promoter activation.

Interleukin 3 mediates interleukin 6 production in murine interleukin 3-dependent hemopoietic cells.

A series of murine interleukin 3 (IL-3)-dependent hemopoietic cell lines was studied for the capacity to produce interleukin 6 (IL-6) in vitro. These included a bone marrow-derived mast cell line (L138.8A) and several early myeloid cell lines described in the literature (DA-1, DA-3, NFS-60, NFS-78, FDC-P1, FDC-P2, FDC-PmixA4, and 32Dcl.23). All of these cell lines produced growth factor activity for IL-6-dependent hybridoma cells (7TD1), which was completely neutralized by the monoclonal anti-IL-6-antibody 6B4. IL-6 expression was also evident at the mRNA level using a murine IL-6-specific cDNA probe. In 32Dcl.23 cells (2 x 10(5)/ml) stimulated for 24 hr with serial dilutions of purified murine IL-3, a positive correlation was found between the IL-3 dose and the amount of IL-6 measured in the conditioned media. At 24 hr this correlation was not evident at the mRNA level. However, prolonged exposure of 32Dcl.23 cells (up to 72 hr) to either a high (60 U/ml) or a low IL-3 concentration (1 U/ml) revealed a time-dependent increase and decrease, respectively, of IL-6 mRNA levels. At both IL-3 concentrations 32Dcl.23 cells remained in a fully viable and proliferative state. The influence of IL-3 on IL-6 release could be specifically counteracted by anti-IL-3-antiserum. IL-6 added alone or in concert with IL-3 did not stimulate 32Dcl.23 proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

C-myc and c-myb oncoproteins during induced maturation of human myeloid and erythroid leukaemic lines.

c-myc and c-myb mRNA have been found to be tightly regulated during haemopoietic differentiation [1]. While the expression of both oncogenes is induced in cells stimulated to proliferate, c-myc in the transition from G0 to G1 [2], and c-myb in the transition from G1 to S phase [3], expression ceases in terminally differentiating haemopoietic cell lines. This takes the form of a progressive monotonic mRNA decline during macrophage (MΦ) and granulocyte (GN) differentiation and a biphasic mRNA decline during erythroid (E) differentiation [4–6]. Although this decline may reflect the cessation of proliferation which accompanies the differentiation process, recent reports that constitutive expression of either oncogene inhibits differentiation in murine [7] and human [8] leukaemic lines imply a causative role for each in haemopoietic maturation.KeywordsCell Cycle StatusHuman Promyelocytic Leukemic Cell LineLeukaemic Cell LinePromyelocytic Leukemic Cell Line HL60Relative P62cThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The development of cell lineages: A sequential model

Abstract The concept of cell lineage and the empirical characterization of specific lineages provide valuable insight into the problems of developmental biology. Of central interest is the decision-making process that results in the diversification of cell lines. Studies of the haemopoietic system, in which stem cells can be committed to one of at least six pathways of differentiation, have suggested that the restriction of differentiation potentials is a progressive and stochastic process. We have recently proposed an alternative model which hypothesizes that lineage potentials during haemopoiesis are expressed individually and in a predetermined sequence as progenitor cells mature. The model first arises from experimental studies which show that both normal myeloid progenitor cells and a human promyeloid cell line, which are able to differentiate towards either neutrophils or monocytes, express these potentials sequentially in culture. The close linear relationship between other haemopoietic progenitor cells is inferred from collective data from studies of bipotent progenitor cells and of haemopoietic proliferative disorders. If the development of haemopoietic cell lineages shows a tendency to follow a particular program, such a mechanism is likely to operate throughout development. In this paper we consider the evidence in favour of programmed events within progenitor cells implementing diversification, and the implications of predetermined and restricted pathways of embryonic development.

Role of disulfide bridges in determining the biological activity of interleukin 3.

Total chemical synthesis of analog proteins was used to examine the requirement for specific disulfide bridges for the biological activity of interleukin 3 (IL-3), a growth factor that stimulates multiple lineages of hemopoietic cells. Four structural analogs of the mature, 140 amino acid murine IL-3 molecule were synthesized in which specific cysteine residues were replaced by alanines. In a quantitative IL-3 assay, based on [3H]thymidine incorporation into factor-dependent cells, the IL-3 analog with alanines substituted for all four cysteines--i.e., [Ala17,79,80,140]IL-3--had 1/500th as much activity as the molecule synthesized according to the native sequence. The two analogs [Cys17,79,Ala80,140]IL-3 and [Cys17,140,Ala79,80]IL-3 had similarly low activity, whereas the [Cys17,80,Ala79,140]IL-3 analog had 2000-fold higher activity than these three analogs, and 3-fold higher than the molecule with the native sequence. This shows that in IL-3 a single disulfide bridge, between cysteines 17 and 80, is required for biological activity that approximates physiological levels. This disulfide probably stabilizes the tertiary structure of the protein to give a conformation that is optimal for function.

The role of antilymphocyte globulin in the treatment of chronic acquired bone marrow failure

Antilymphocyte globulin is an immunoglobulin preparation prepared from heterologous serum after the animal (horse or rabbit) has been immunised with human lymphocytes, obtained from the thymus (antithymocyte globulin, ATG) or thoracic duct (antilymphocyte globulin, ALG). The rationale for the use of ALG in the treatment of chronic acquired marrow failure is based on its immunosuppressive activity and the fact that a proportion of cases of bone marrow failure, whether affecting single or multiple haemopoietic cell lines are due to immune-mediated suppression of haemopoiesis. In addition, in vitro studies have shown that ALG also has an immunostimulatory effect on lymphokine and haemopoietic growth factor production, and may therefore directly stimulate haemopoietic progenitor cells. ALG has been used for the treatment of aplastic anaemia and acquired chronic marrow failure affecting single cell lines namely pure red cell aplasia (PRCA), amegakaryocytic thrombocytopenia and chronic neutropenia due to immune inhibition of granulopoiesis (‘acquired white cell aplasia’). ALG is used for treatment of non-severe aplastic anaemia (NSAA) and in those cases of severe aplastic anaemia (SAA) where allogeneic transplantation is not possible or is not indicated. Treatment with ALG results in 75% long term survival for NSAA and 40–50% for SAA although there is a very severe subgroup of SAA defined by peripheral blood neutrophils of <0.2 × 10 9/I who rarely benefit from ALG therapy. For those patients who do not respond a second course of ALG can be given later using ALG from a different animal source. A proportion of cases of acquired PRCA are due to immune suppression of stem cell function with normal numbers of erythroid progenitors present in the marrow and these cases respond well to ALG compared with those associated with a primary stem cell defect. Some cases of PRCA respond much more rapidly to ALG than in aplastic anaemia suggesting a different mechanism of action of ALG or a different target cell. Amegakaryocytic thrombocytopenia and ‘acquired white cell aplasia’ are both very rare but there have been anecdotal reports of successful ALG therapy for these conditions.