A primitive cell origin for B-cell precursor ALL?

A stem cell origin has been described for both acute and chronic myelogenous leukemias. In contrast, childhood B-cell precursor acute lymphoblastic leukemia (ALL) is thought to arise in committed B-lineage cells. Recently described in vitro and in vivo model systems that support the proliferation and expansion of ALL cells have provided new tools to investigate the cellular targets for the origin of this malignancy. Evidence suggests that some subtypes of childhood ALL have a primitive cell origin and share many immunophenotypic characteristics with normal progenitor cells. These leukemic stem cells may be resistant to current therapeutic strategies designed to kill the bulk ALL cell population and subsequent relapses may arise from this population. More precise definition of these ALL stem cells through combined analyses of antigen expression, genetic lesions, and functionality is essential for the development of more effective, targeted therapeutic strategies.

In vitro proliferation and differentiation of megakaryocytic progenitors in patients with aplastic anemia, paroxysmal nocturnal hemoglobinuria, and the myelodysplastic syndromes.

It has previously been shown that patients with aplastic anemia (AA) have a stem cell defect both of proliferation and differentiation. This has been shown by long-term bone marrow (BM) culture, long-term initiating cell assays, and committed progenitor assays. We present, for the first time, data on megakaryocyte (Mk) colony formation from purified BM CD34(+) cells from patients with AA. The results are compared with those from normal controls and from patients with paroxysmal nocturnal hemoglobinuria (PNH) and the myelodysplastic syndromes (MDSs). Those treated for AA had previously received immunosuppression (antithymocyte globulin and/or cyclosporin). No patients had received bone marrow transplantation. A total of 13 AA patients (five untreated, eight treated), six PNH, six MDS, and 13 normal donors were studied. BM CD34(+) cells were purified by indirect labeling and then cultured in a collagen-based Mk assay kit (MegaCult-C, StemCell Technologies). The cultures were fixed on day 12, and the Mk colonies were identified by the alkaline phosphatase anti-alkaline phosphatase technique using the monoclonal antibody CD41 (GP IIb/IIIa). The slides were scored for Mk colony-forming units (CFU-Mks) (3-20 and >20 cells), Mk burst-forming units (BFU-Mks) (>50 cells), and mixed colonies. The results show that total Mk colony formation in AA was significantly lower than in normal donors (p<0.0001), both in untreated patients/nonresponders to treatment (p = 0.0001) and in complete/partial responders (p<0.002). There was no significant difference in Mk colony formation in treated and untreated patients (p = 0.05). Patients with AA had a lower total colony formation than PNH patients (p = 0.0002). PNH patients exhibited lower colony formation than normal controls (p = 0.03), as shown by MDS patients, although the considerable number of variables resulted in a lack of statistically significant difference from normal controls (p = 0.2). We have now shown that Mk colony formation from purified BM CD34(+) cells is significantly reduced, supporting previous evidence that AA results from a stem cell defect.

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.

Interleukin-10-induced CD8 cell proliferation.

Interleukin (IL)-10, a product of T helper 2 (Th2) lymphocytes, has been shown to be an important regulator of lymphoid and myeloid cells, inhibiting mitogen, peptide and alloantigen-induced T-cell proliferation and IL-2 production. The microenvironment at the time of cell activation, notably the presence or absence of cytokines such as IL-10, interferon-gamma (IFN-gamma) and IL-2, is believed to determine the lineage and magnitude of cell-mediated responses. In this study, we show that recombinant human IL-10 (rhIL-10) exerts a dose-dependent inhibitory effect on human peripheral blood mononuclear cells stimulated in vitro, when these cells have not previously been exposed to rhIL-10. Furthermore, incubation of these cells with high doses of rhIL-10, either before or at the time of activation, results in inhibition which is followed several days later by the emergence of a population of CD8 positive cells. This rhIL-10-responsive CD8, positive cell population still emerges even when the cells are washed following incubation with rhIL-10 prior to cell activation. Using purified CD8 populations this was shown to be a direct action of rhIL-10 on CD8 cells and not via CD4 positive cells and monocytes. This finding was only observed when cells were activated with a cross-linking anti-CD3 antibody and not when activated with phorbol-12-mystrate-13-acetate (PMA) and calcium ionophore (CaIon), suggesting that the effect is mediated through cell-surface receptors. Analysis of CD8 positive clones reveal production of Tc2 patterns of cytokines and reduced cell cytotoxicity to allogeneic, natural killer and lymphokine activated cell targets.

Transarticular cross-screw fixation. A technique of ankle arthrodesis.

Many techniques of ankle arthrodesis have been described. Failure rates of up to 40% have been reported in the past. In this study, a technique for internal fixation of ankle fusions was employed using transarticular crossed-screw fixation. This provides bony coaptation, compression, and immobilization necessary for reliable union. Thirty-five patients had ankle arthrodesis with this technique of internal fixation and 12 patients had ankle fusions with Charnley compression arthrodesis. Follow-up evaluation averaged two years. The fusion rate was 100% (35 fusions of 35 attempts) with the transarticular crossed-screw technique and 83% (ten fusions of 12 attempts) with compression arthrodesis.