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Recent studies in erythroid cells have shown that autophagy is an important process for the physiological clearance of mitochondria during terminal differentiation. However, autophagy also plays an important role in removing damaged and dysfunctional mitochondria. Defective mitochondria and impaired erythroid maturation are important characteristics of low-risk myelodysplasia. In this study we therefore questioned whether the autophagic clearance of mitochondria might be altered in erythroblasts from patients with refractory anemia (RA, n=3) and RA with ringed sideroblasts (RARS, n=6). Ultrastructurally, abnormal and iron-laden mitochondria were abundant, especially in RARS patients. A large proportion (52+/-16%) of immature and mature myelodysplastic syndrome (MDS) erythroblasts contained cytoplasmic vacuoles, partly double membraned and positive for lysosomal marker LAMP-2 and mitochondrial markers, findings compatible with autophagic removal of dysfunctional mitochondria. In healthy controls only mature erythroblasts comprised these vacuoles (12+/-3%). These findings were confirmed morphometrically showing an increased vacuolar surface in MDS erythroblasts compared to controls (P<0.0001). In summary, these data indicate that MDS erythroblasts show features of enhanced autophagy at an earlier stage of erythroid differentiation than in normal controls. The enhanced autophagy might be a cell protective mechanism to remove defective iron-laden mitochondria.

Platelet production requires compartmentalized caspase activation within megakaryocytes. This eventually results in platelet release in conjunction with apoptosis of the remaining megakaryocyte. Recent studies have indicated that in low-risk myelodysplastic syndromes (MDS) and idiopathic thrombocytopenic purpura (ITP), premature cell death of megakaryocytes may contribute to thrombocytopenia. Different cell death patterns have been identified in megakaryocytes in these disorders. Growing evidence suggests that, besides apoptosis, necrosis and autophagic cell death, may also be programmed. Therefore, programmed cell death (PCD) can be classified in apoptosis, a caspase-dependent process, apoptosis-like, autophagic and necrosis-like PCD, which are predominantly caspase-independent processes. In MDS, megakaryocytes show features of necrosis-like PCD, whereas ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like PCD (para-apoptosis). Triggers for these death pathways are largely unknown. In MDS, the interaction of Fas/Fas-ligand might be of importance, whereas in ITP antiplatelet autoantibodies recognizing common antigens on megakaryocytes and platelets might be involved. These findings illustrate that cellular death pathways in megakaryocytes are recruited in both physiological and pathological settings, and that different forms of cell death can occur in the same cell depending on the stimulus and the cellular context. Elucidation of the underlying mechanisms might lead to novel therapeutic interventions.

Myelodysplasia (MDS) is mostly characterized by a normal or increased number of normoblasts in the bone marrow and an impaired in vitro colony formation. In the present study we analyzed whether this might be due to a disconnection between proliferation and differentiation. CD34+/CD36- sorted bone marrow cells of 18 MDS patients were cultured in a clonogenic and suspension culture assay in the presence of erythropoietin (Epo) and mast cell growth factor (MGF). Burst-forming units erythroid (BFU-E, 75 +/- 88/10(4) CD34+ cells, X +/- s.d.) and colony-forming units E (CFU-E) were observed in eight of the 13 cases (62%) with refractory anemia with or without ring sideroblasts (RA and RARS) and one of the five cases with RA with excess of blasts or in transformation (RAEB and RAEB-T). Suspension cultures with CD34+/CD36- sorted cells with Epo plus MGF demonstrated an 8.9 +/- 6.5-fold expansion after 7 days in cases with >10 BFU-E/10(4) CD34+/CD36- cells while cases with <10 BFU-E/10(4) CD34+/CD36- cells demonstrated 1.0 +/- 0.8-fold expansion especially in cases with RAEB/RAEB-T. FACS and morphology analysis after 7 days of suspension culture demonstrated partial differentiation along the erythroid lineage in cases with RA/RARS (75%) and RAEB/RAEB-T (66%) reflected by the presence of erythroblasts and normoblasts with variable expression of CD34, CD36 and Glycophorin A. In cases with erythroid colony formation 69 +/- 24% of the cells were CD34-/CD36+ and in cases with <10 BFU-E/10(4) CD34+ cells 18 +/- 16% of cells were CD34-/CD36+. Iron staining showed the presence of ring sideroblasts in two cases with RARS indicating that the cells originate from the abnormal erythroid clone. Finally, it was shown that cases with an impaired proliferative response demonstrate an enhanced binding of Annexin-V on CD34+ cells during the first days of the cell suspension culture phase. These results suggest that a defect in the proliferative response is most pronouncedly expressed in MDS whereas a subpopulation of cells retain the capacity to differentiate between transition to a terminated stage.

In refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS) a discrepancy is observed between the decreased in vitro erythroid colony formation and the normal or increased number of normoblasts in the bone marrow. To study the in vivo and in vitro erythropoiesis in more detail erythron transferrin uptake (ETU), soluble transferrin receptor (sTfR) and erythroid in vitro colony formation were performed in 24 patients with RA and five patients with RARS. These results were correlated with bone marrow morphology and transfusion dependency. Increased (mean, 124.9; range, 74-225 micromol/l blood/day) and normal (mean, 60.6; range, 50-71) ETU values were observed in 51% and 28% of the cases, whereas 21% of the cases demonstrated a diminished ETU value (mean, 35.8; range, 28-46), which correlated significantly with sTfR in cases with RA (P < 0.05, r = 0.64). A significant difference in ETU values was observed between RA (mean, 77.6; range, 28-189) and RARS (mean, 144.0; range, 59-225, P < 0.05). Most of the cases (73%) with increased ETU values showed an augmented percentage of erythroblasts in the bone marrow, which was inversely related with the serum Epo levels (P < 0.05, r = 0.51). However no correlation was found between the ETU values and the in vitro erythroid colony formation. Transfusion dependency was associated with normal to increased ETU levels (P < 0.05) and cytogenetic abnormalities (P < 0.05). These observations demonstrate that different patterns of defects can be observed in the erythropoiesis of RA and RARS patients whereby normal to increased ETU levels and the presence of cytogenetic abnormalities differentiate between cases of RA with ineffective erythropoiesis associated with regular transfusions and cases who are relatively transfusion independent.

Since 1989, granulocyte-macrophage and granulocyte colony-stimulating factors (GM-CSF, G-CSF) have been increasingly applied in the treatment of drug-induced agranulocytosis. In order to evaluate the effectiveness of GM-CSF and G-CSF in the treatment of drug-induced agranulocytosis, we have studied all reported cases (n = 70) treated with GM-CSF and G-CSF, including ten patients treated during the last 2 years in The Netherlands. The results demonstrate that patients with a severe granulocytopenia (< 0.1 x 10(9)/l) treated with hematopoietic growth factors had a significantly faster recovery of the peripheral blood granulocytes compared to previous published studies. At the same time, a significantly lower mortality rate was observed. In patients with a severe granulocytopenia treated with GM-CSF or G-CSF a mortality rate of 5% was noted. No difference in granulocyte recovery was observed in patients treated with GM-CSF or G-CSF. The results of this review indicate that G-CSF and GM-CSF enhance the recovery of the myeloid lineage, resulting in a faster normalization of the peripheral blood granulocyte count and a reduced incidence of fatal complications.

A patient with large granular lymphocyte (LGL) expansion (T-gamma lymphocytosis), neutropenia and thrombocytopenia was studied longitudinally. Analysis of peripheral blood mononuclear cells (PBMC) demonstrated an unusual large proportion of CD3+ T-lymphocytes expressing a gamma delta T-cell receptor (TcR-gamma delta). Immunofluorescence (IF) stainings with subset-specific monoclonal antibodies showed a fluctuating expansion of TcR-gamma delta+ T-cells expressing V gamma 9 and V delta 2 variable (V) gene segments. Biochemical characterization of PBMC showed the presence of a disulphide-linked TcR-gamma delta. TcR gene rearrangement studies on sorted TcR-gamma delta+ T-cells showed rearrangements of V gamma 9-J gamma 1.2 and V delta 2-J delta 1 V and joining (J) gene segments, thereby confirming the IF staining results. These data alone did not allow us to determine whether the TcR-gamma delta+ LGL expansion represented a polyclonal or monoclonal proliferation, because the combinatorial repertoire of TcR-gamma delta receptors is limited due to the availability of only a few V and J segments within the TcR-gamma and TcR-delta genes and because of the preferential usage of V gamma 9-J gamma 1.2 and V delta 2-J delta 1 rearrangements by TcR-gamma delta+ T-cells in blood of healthy individuals. We therefore used polymerase chain reaction (PCR)-mediated amplification of the TcR-gamma delta rearrangements, using specific V gamma 9, J gamma 1.2, V delta 2, and J delta 1 oligonucleotides to determine the junctional diversity of the TcR-gamma delta+ T-cell population. Sequence analysis of the PCR products obtained revealed a mixture of different junctional region sequences compatible with a polyclonal expansion. This is in contrast to the few reported TcR-gamma delta+ LGL and the majority of TcR-alpha beta+ LGL expansions, which appeared to consist of monoclonal proliferations.

As anemia is frequently the main problem in myelodysplastic syndromes (MDS), we studied the efficacy of human erythropoietin (rhEpo) in stimulating the erythroid lineage in 14 patients, starting with 40 U/kg three times a week and doubling the dose every 6 weeks until a response was observed. The highest doses administered were 80 (n = 1), 160 (n = 4), 320 (n = 8) and 640 U/kg (n = 1). One patient (refractory anemia with an excess of blasts, RAEB) showed an increase of hemoglobin, white blood cells and platelets with 80 U/kg rhEpo. However, this patient developed acute leukemia while on therapy. Two other patients (RAEB and RAEB in transformation) also transformed to acute leukemia. In the other 11 patients no response was observed. There was no correlation between in vitro culture data and in vivo responsiveness. The treatment was well tolerated and no nonhematological side effects were observed. From this study we conclude that rhEpo, even when given at high doses, has a low response rate in patients with MDS. Further investigation is needed in order to clarify whether rhEpo increases the potential risk of transformation to acute leukemia.