Frances M. Gibson

Serum thrombopoietin levels in patients with aplastic anaemia

Endogenous serum thrombopoietin (TPO) levels were measured in 31 patients with aplastic anaemia (AA) using an enzyme immunoassay with a sensitivity of 20 pg/ml. The median platelet count for all AA patients was 30 ± 29 × 109/l (range 5–102) compared with a median of 284 ± 59 × 109/l (range 148–538) for normal controls. Serum TPO levels were significantly elevated in all patients compared with normals (1706 ± 1114.2, range 375–5000 v 78 ± 54, range 16.5–312.9, P < 0.0001). There was no correlation between serum TPO levels and the degree of thrombocytopenia in AA patients, but TPO levels were significantly higher in patients who were platelet transfusion dependent than in patients who were transfusion independent (P < 0.01). There was a trend for higher TPO levels in patients with severe AA compared with non‐severe AA patients. Clinical trials of TPO and a related truncated, pegylated molecule, megakaryocyte growth and development factor (PEG‐rHuMGDF), are awaited to determine whether treatment with these drugs will result in increased platelet counts in patients with AA.

Increased apoptosis of bone marrow CD34+ cells and impaired function of bone marrow stromal cells in patients with systemic lupus erythematosus

The changes in bone marrow (BM) stem cell reserve and function and stromal cell function in patients with active systemic lupus erythematosus (SLE) were investigated. The study was carried out on seven SLE patients and 28 healthy controls using flow cytometry and in vitro cell culture assays. We found that patients had low CD34+ cells, compared with the control group, reflecting the decrease of both CD34+/CD38− and CD34+/CD38+ cells. Patient CD34+/Fas+ but not CD34−/Fas+ cells were significantly increased. Apoptotic (7AADdim) cells were higher among CD34+/Fas+ than among CD34+/Fas− cells, and individual values of apoptotic CD34+ cells strongly correlated with the number of CD34+/Fas+ cells. These findings are suggestive of a Fas‐mediated apoptosis accounting for the low CD34+ cells in SLE patients. Moreover, we found that patients had low numbers of granulocyte‐macrophage colony‐forming units (CFU‐GM) and erythroid burst‐forming units (BFU‐E), compared with the control group, and that the generation of colony‐forming cells in long‐term BM cultures was significantly reduced. Patient BM stroma failed to support allogeneic progenitor cell growth. In one patient, CD34+ cells were increased, apoptotic CD34+/Fas+ cells were normalized and defective stromal cell function was restored after autologous stem cell transplantation. We concluded that defective haemopoiesis in SLE patients is probably caused, at least in part, to the presence of autoreactive lymphocytes in BM.

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.

Mechanisms of bone marrow progenitor cell apoptosis in aplastic anaemia and the effect of anti‐thymocyte globulin: examination of the role of the Fas–Fas‐L interaction

The mechanism of action of anti‐thymocyte globulin (ATG) in aplastic anaemia (AA) is complex. Bone marrow (BM) CD34+ cells in AA have been shown to be more apoptotic and have a higher expression of Fas antigen (Fas‐ag) than in normal donors. The aims of this study were to delineate further the mechanism for increased bone marrow progenitor cell apoptosis in AA and investigate the effects of ATG on apoptosis and Fas‐ag expression. BM was obtained from six normal donors and 10 untreated AA patients. We confirmed that AA BM CD34+ cells were more apoptotic than normal donor cells (P = 0·002). Following treatment with ATG, the mean percentage reduction of apoptosis was 34% (9·2–65·9%). BM from 30 AA and 10 normal donors was then stained for CD34, Fas‐ag and 7‐AminoActinomycin D. The proportion of CD34+ Fas+ cells was higher in untreated AA (P = 0·0001) than in normal donors. Results also showed that the majority of CD34+ Fas+ cells were apoptotic/dead in normal donors (mean 81%) and AA (88%), indicating that Fas is involved in apoptosis of CD34+ cells. In contrast, the majority of CD34+ Fas− cells in normal donors were live (mean 91%), while two patterns emerged in untreated AA. In seven patients, the majority of cells were live, however, in the remaining eight patients, the majority of cells were apoptotic/dead, suggesting an alternative mechanism for apoptosis in addition to Fas‐ag. Finally, we have shown that in vivo ATG treatment reduced the expression of Fas‐ag on AA BM CD34+ cells.

Long-term culture of aplastic anaemia bone marrow

Summary. Long‐term bone marrow cultures (LTBMC) were established with marrow from 11 patients with aplastic anaemia (AA). Bone marrow from five patients, with low numbers of committed progenitor cells, exhibited an increase in committed progenitor cell production to normal levels in the first week of LTBMC. None of 44 haematologically normal marrow cultures showed this increase. Mature and committed progenitor cell production in all cultures from aplastic anaemia bone marrow, declined faster than in normal cultures. This study indicates that short‐term culture for committed progenitor cells is an underestimate of the proliferative capacity of bone marrow from some patients with AA. LTBMC may provide a useful system for further studies into the mechanisms responsible for this increased growth in some patients with AA.

Acute myeloid leukaemia blast cells bind to human endothelium in vitro utilizing E‐selectin and vascular cell adhesion molecule‐1 (VCAM‐1)

Summary The adhesion of acute myeloid leukaemia (AML) blast cells to human umbilical vein endothelial cells (HUVECs) was investigated in vitro. Adhesion of blast cells from 10 cases of AML to unstimulated and interleukin‐1β (IL‐1) stimulated HUVECs was similar to or greater than that of control neutrophils. The extent to which endothelial E‐selectin and vascular cell adhesion molecule‐1 (VCAM‐1) were involved in this adhesive process was investigated using blocking monoclonal antibodies to these proteins. In the majority of cases studied (7/8), anti‐E‐selectin significantly inhibited adhesion to IL‐1 stimulated endothelium (26–65% inhibition) and in 5/8 cases so did anti‐VCAM‐1 (maximum of 31% inhibition). All cases were found to express the sialylated Lewis x antigen and very late activation antigen‐4, ligands for E‐selectin and VCAM‐1 respectively. Our results indicate that leukaemic blast cells adhere to human endothelium and that there are E‐selectin and, to a lesser extent. VCAM‐1‐dependent components to this process. Such adhesive interactions are likely to confer on AML blast cells the ability to migrate across the vascular wall and so to establish extravascular disease.

Effects of antithymocyte globulin on bone marrow CD34 + cells in aplastic anaemia and myelodysplasia

The mechanism of action of antithymocyte globulin (ATG) in the treatment of aplastic anaemia (AA) and myelodysplastic syndromes (MDS) is poorly understood and may involve many different mechanisms. The aim of this in vitro study was to investigate further the effect of ATG on haemopoietic progenitor cells. A total of 16 patients (10 AA and 6 MDS) and 12 normal control subjects were studied. Purified bone marrow (BM) CD34+ cells were cultured in committed progenitor assay in the presence of ATG and autologous serum, then scored on day 14 for granulocyte–monocyte colony‐forming units (CFU‐GM) and erythroid colonies. ATG was found to be inhibitory to haemopoietic progenitor cells at high concentrations (1000 μg/ml and 100 μg/ml). This was confirmed by CD34‐FITC and 7AAD staining of purified normal CD34+ cells after overnight incubation with ATG. In contrast, at lower doses (0.1–10 μg/ml), ATG produced an increase in colony growth in most normal, MDS and AA BM CD34+ cells. The greatest effect was in patients with non‐severe AA, in whom the greatest increase in CFU‐GM was seen at 0.5 μg/ml (P < 0.02) and 0.1 μg/ml (P = 0.02) and erythroid colonies at 0.1 μg/ml (P < 0.05). Serum ATG levels peaked during infusion to levels that were found to be toxic to haemopoietic progenitor cells in vitro and fell thereafter to levels that were associated with the highest colony numbers (0.1 and 0.5 μg/ml) in vitro. These results suggest that an increase in haemopoietic progenitor cells by ATG may be one of several important mechanisms for haematological recovery in AA and MDS.

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.

Bcl‐2 and Bcl‐x expression in the CD34+ cells of aplastic anaemia patients: relationship with increased apoptosis and upregulation of Fas antigen

Aplastic anaemia (AA) is a syndrome of haemopoietic failure involving increased apoptosis in stem cells. AA CD34+ cells often have upregulated Fas antigen, but this does not explain the increased apoptosis in all patients. To examine whether abnormal expression of the apoptotic modulators Bcl‐2 and Bcl‐x is involved in increased apoptosis in the CD34+ cells of patients, we examined cells from 19 AA patients and 18 normal controls by triple staining for CD34, Bcl‐2 or Bcl‐x, together with 7‐amino actinomycin D to determine viability or with staining for Fas antigen. We confirmed increased apoptosis of CD34+ cells in patients. All CD34+ cells in patients and controls expressed Bcl‐2 and Bcl‐x with no significant difference between the groups. In patients, viability of CD34+/Bcl‐2hi cells was similar to that of CD34+/Bcl‐2lo cells, but CD34+/Bcl‐xhi cells were significantly more viable than CD34+/Bcl‐xlo cells. CD34+ cells from AA patients expressed upregulated Fas antigen, but this did not correlate with Bcl‐2 or Bcl‐x expression. These results suggest a more significant role for Bcl‐x as an anti‐apoptotic regulator in CD34+ cells in AA than Bcl‐2. The induction of death by Fas antigen may bypass the anti‐apoptotic effect of Bcl‐2 and Bcl‐x in CD34+ cells in AA.

G-CSF-mobilized CD34+ peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34+ cells: role of G-CSF as survival factor

The mechanism of release of CD34+ cells into the peripheral blood (PB) after mobilization treatment with chemotherapy and/or growth factors is not clearly understood. Growth factors may induce increased proliferation and self renewal within the stem cell compartment. It is possible that they alter adhesion molecule profiles or other progenitor:stroma interactions, to allow release of these cells into the periphery. However, CD34+ cells are present in the PB under steady‐state conditions, albeit in low number. Growth factors such as granulocyte colony‐stimulating factor (G‐CSF) may promote the survival of CD34+ cells in the PB by suppressing apoptosis. In order to test this hypothesis, we have quantitated apoptotic cells in the CD34+ fraction of peripheral blood stem cell (PBSC) collections, using two‐colour flow cytometry, after staining with anti‐CD34 antibody and the fluorescent DNA binding agent, 7‐amino actinomycin D (7AAD). 7AAD differentially stains live, apoptotic and dead cells, due to the altered accessibility of DNA in each subpopulation.