John Scopes

Haemopoietic progenitor cells are reduced in aplastic anaemia

Summary We investigated the frequencies of early populations of progenitors in aplastic anaemia (AA) bone marrow, from patients with a range of disease severity, compared with normal. Double‐colour immunofluorescent staining for CD34 and CD33 was carried out on bone marrow mononuclear cells (BMMC) and analysed using fluorescence activated cell sorting (FACS), AA CD34+ cells were reduced by 68% compared to normal. In addition, AA CD33+ cells and the three progenitor subsets (CD34+/CD33–, CD34+/CD33+ and CD34–/CD33+) were reduced by 44–80%. Our data lend further support for an early stem cell deficiency in AA.

Aplastic anaemia following exposure to 3,4‐methylenedioxymethamphetamine (‘Ecstasy’)

Summary. We report two cases of aplastic anaemia following exposure to ‘Ecstasy’ (MDMA, 3,4‐methylenedioxymethamphetamine). In both cases the aplastic anaemia resolved spontaneously 7–9 weeks after presentation. Long‐term bone marrow culture study of one patient demonstrated complete normalization of haemopoiesis at time of haematological recovery, suggesting either that damage to the haemopoietic stem cell had been only transient, or that a more mature, committed progenitor cell was the target. Because MDMA may have been a factor in the aetiology of the bone marrow suppression in these two cases, we recommend close haematological monitoring of young adults presenting with toxicity from MDMA, and a detailed history of exposure to recreational drugs in all new patients presenting with aplastic anaemia.

Correction of stromal cell defect after bone marrow transplantation in aplastic anaemia

Defects in stromal cell function have been demonstrated in a number of aplastic anaemia (AA) patients. Here we have studied a patient with severe AA and abnormal stromal cell function who underwent bone marrow transplantation (BMT). The objective of this study was to investigate the timing and the mechanism of correction of the stromal defect after transplantation. The patient, a 25‐year‐old woman with severe AA, underwent BMT from her brother. BM was obtained from the patient on five occasions: 2 weeks pre BMT, and 3, 8, 16 and 21 months post BMT. Stromal cells were grown to confluence and recharged with purified CD34+ cells from normal donors. The support of such cells, as assessed by weekly colony‐forming assay (CFU) of non‐adherent cells, was compared with that of stromal layers grown from normal BM. A novel technique of combined fluorescence in situ hybridization (FISH) and immunocytochemistry was used to determine the origin of specific stromal cell types on cytospins of stroma post BMT. Stromal function was defective at 2 weeks pre BMT and at 3 months post BMT, but returned to normal at 8 and 16 months post BMT. At 21 months post BMT, stromal fibroblasts and endothelial cells were shown to be of recipient origin, and macrophages and T cells were of donor origin. We present here evidence in a case of severe AA for defective stromal function before BMT and delayed normalization of function after BMT. This correlated with engraftment of donor macrophages and T cells, but not fibroblasts and endothelial cells.

Haemopoietic growth factor production by normal and aplastic anaemia stroma in long‐term bone marrow culture

Summary. Defective marrow stroma, or microenvironment, have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA). This could involve defects in positive‐ or negative‐acting haemopoietic regulator expression by AA stroma, or alteration of normal stroma‐stem cell interactions.

Human Immunodeficiency Virus Infection Impairs Hemopoiesis in Long-Term Bone Marrow Cultures: Nonreversal by Nucleoside Analogues

Hematologic abnormalities are often seen in patients infected with human immunodeficiency virus (HIV). The effect of HIV infection of bone marrow stroma on support of uninfected CD34 progenitor cells in long-term bone marrow culture (LTBMC) was investigated. Results show that HIV-infected bone marrow stroma was unable to adequately support CD34 progenitor cells in vitro. Zidovudine or didanosine was added to cultures in an attempt to reverse the suppressive effects exerted by HIV and to determine whether such suppression was mediated by transfer of HIV infection to progenitor cells. Didanosine failed to reduce the suppressive effects of HIV, whereas zidovudine compounded the observed suppression. HIV infection of bone marrow stroma, while reducing the production of nonadherent cells, did not increase apoptosis and cell death in such cells. In contrast, zidovudine enhanced apoptosis and cell death in nonadherent cells produced by both HIV-infected and control LTBMC.

IL-3 is produced by normal stroma in leng-term bone marrow cultures

Summary. Interleukin‐3 (IL‐3) has been shown to have significant effects on haemopoiesis in vitro, but early investigations of normal human long‐term bone marrow cultures (LTBMC) have failed to demonstrate LL‐3 production by stromal cells, either by Northern blotting for mRNA, or assaying for bioactivity in culture supernatants. One recent report, using reverse transcription‐polymerase chain reaction (RT‐PCR), demonstrated IL‐3 expression in only one of eight cultures.

The effect of human flt-3 ligand on committed progenitor cell production from normal, aplastic anaemia and Diamond-Blackfan anaemia bone marrow.

Summary. We investigated the effect of the human ligand for flt‐3 (FL) on the committed progenitor colony formation of normal bone marrow (BM) (n = 9) and BM from four aplastic anaemia (AA) and three Diamond‐Blackfan anaemia (DBA) patients. Methylcellulose committed progenitor cell assays were carried out using FL alone and in combinations with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). interleukin‐3 (IL‐3) and c‐kit ligand (KL). FL alone had a limited, though significant, effect on the production of granulocyte‐macrophage colony‐forming unit (CFU‐GM) colonies from normal BM and showed an additive effect with IL‐ 3 and GM‐CSF separately, but not in combination. FL did not increase the stimulation of KL and did not have an effect on the production of erythroid progenitor colonies. FL had no effect on the AA and DBA BMs studied.