Stem cell transplantation in adult ALL patients

Less than 40% of adult acute lymphocytic leukaemia (ALL) patients will still be alive at 5 years post-diagnosis. Ways to improve patients' outcome, using high-dose therapy followed by autologous/allogeneic stem cell transplantation (SCT) in first complete remission (CR1) rather than consolidation/maintenance chemotherapy, have been investigated. However, prospective studies are small and results are inconclusive. The largest prospective trial ever being performed in adult ALL patients, the ongoing UKALL 12/ECOG 2993 trial, is assigning all patients who have a sibling donor to receive allogeneic SCT (alloSCT) in CR1, whereas all other patients are randomized to continue chemotherapy versus autologous SCT. An interim analysis of this trial seems to support an alloSCT in first CR in adult ALL patients (reflected by a significantly reduced relapse rate with an improved disease-free survival). However, less than 30% of the patients have a matched sibling donor, the majority of the patients are over 40 years old, which makes them less suitable for conventional allograft, and even in those who have a matched sibling donor and are young and fit enough to receive it the treatment-related mortality (TRM) is about 20%. Strategies for expanding donor availability, meanwhile, to reduce the TRM, remain challenges. Data regarding the efficacy of reduced-intensity regimens in ALL patients are still scanty. Another way of improving patient outcome is to select patients for allograft more carefully. There are enough data to suggest now that children who achieved a clinical remission but failed to obtain a molecular/immunological remission are more prone to relapse. Similar data have recently been published for adult ALL. However, data are still limited, and the significance of minimal residual disease (MRD) has never been studied prospectively in adult ALL patients. A reasonable approach is to assign all patients with a matched related donor who has failed to achieve a molecular/immunological remission to receive a conventional alloSCT, whereas all others might be randomized to receive alloSCT versus chemotherapy/autologousSCT. However, patients with Ph(+) ALL who have a donor should receive an alloSCT in CR1, regardless of their MRD results.It appears that alloSCT provides the best chance for cure. However, by improving our ability to select those who have the highest risk for relapse, unnecessary toxicity/mortality might be prevented and the general outcome might improve.     

下调GPM6B基因抑制平滑肌表达特异性标记蛋白

目的 研究糖蛋白M6B（Glycoprotein M6B,GPM6B）基因对间充质细胞C3H10T1/2向平滑肌细胞分化的影响。 方法 将慢病毒感染后的sh-GPM6B细胞作为处理组,慢病毒感染后的sh-Scramble细胞作为对照组进行后续实验。用sh-GPM6B质粒和工具质粒在293T细胞系包装慢病毒。用病毒液感染C3H10T1/2小鼠间充质细胞系制备sh-GPM6B稳转细胞系。荧光显微镜观察病毒转染效率,Western法检测GPM6B基因的干涉效率。慢病毒shRNA干涉抑制GPM6B基因表达后,通过TGF-β1诱导对照组和sh-GPM6B细胞向平滑肌分化。Western blot法检测平滑肌特异标记蛋白SM-MHC和α-SMA表达水平。q-PCR法检测SM-MHC和α-SMA的mRNA转录水平。 结果 慢病毒感染后sh-GPM6B干涉细胞GPM6B基因蛋白表达降低至对照组（40~70）%水平。Sh-GPM6B处理组细胞与sh-Scramble对照组细胞相比,SM-MHC蛋白表达水平显著下调（P<0.05）;SM-MHC和α-SMA mRNA转录水平明显降低（P<0.01）。 结论 下调GPM6B基因可抑制TGF-β1诱导的间充质细胞向平滑肌细胞分化。     

Kinetics of erythropoiesis in the liver induced in adult mice by phenylhydrazine

Phenylhydrazine treatment of normal mice elicited a rise in the numbers of CFU-S in blood, spleen and liver. High numbers of CFU-S were found in blood and liver 4 d after the first phenylhydrazine injection. CFU-S in the liver decreased slowly and were absent after 2 weeks. Blood CFU-S returned to normal levels by day 6, whereas spleen CFU-S numbers remained high upto day 12 with a 20-fold increase being apparent between days 5 and 8. Bone marrow CFU-S numbers were relatively unaffected except for a dip between days 4 and 7 with a nadir at day 5 where numbers decreased to 50% of the control levels. Approximately 40% of liver, spleen and blood CFU-S present on the 4th d after initiation of phenylhydrazine treatment, were killed with a single dose of hydroxyurea whereas bone marrow CFU-S numbers were not significantly reduced by the drug. Splenectomy performed before (21 d) or during phenylhydrazine treatment did not diminish the number of CFU-S found in theliver on day 4. A 3 d interval was observed between peak numbers of CFS-U and erythroblasts in the liver which suggests that hepatic CFU-S are able to undergo differentiation along the erythroid pathway. The presence of maceophages was correlated with that of erythroblasts in the hepatic central veins. These macrophages may be essential to the liver environment for induction of erythropoiesis.     

Identification of mast cell progenitors in adult mice

It is well known that mast cells are derived from hematopoietic stem cells. However, in adult hematopoiesis, a committed mast cell progenitor has not yet been identified in any species, nor is it clear at what point during adult hematopoiesis commitment to the mast cell lineage occurs. We identified a cell population in adult mouse bone marrow, characterized as Lin(-)c-Kit(+)Sca-1(-)-Ly6c(-)FcepsilonRIalpha(-)CD27(-)beta7(+)T1/ST2+, that gives rise only to mast cells in culture and that can reconstitute the mast cell compartment when transferred into c-kit mutant mast cell-deficient mice. In addition, our experiments strongly suggest that these adult mast cell progenitors are derived directly from multipotential progenitors instead of, as previously proposed, common myeloid progenitors or granulocyte/macrophage progenitors.     

Stem cell expression of the AML1/ETO fusion protein induces a myeloproliferative disorder in mice

The t(8;21)(q22;q22) translocation, present in 10-15% of acute myeloid leukemia (AML) cases, generates the AML1/ETO fusion protein. To study the role of AML1/ETO in the pathogenesis of AML, we used the Ly6A locus that encodes the well characterized hematopoietic stem cell marker, Sca1, to target expression of AML1/ETO to the hematopoietic stem cell compartment in mice. Whereas germ-line expression of AML1/ETO from the AML1 promoter results in embryonic lethality, heterozygous Sca1(+/AML1-ETO ires EGFP) (abbreviated Sca(+/AE)) mutant mice are born in Mendelian ratios with no apparent abnormalities in growth or fertility. Hematopoietic cells from Sca(+/AE) mice have markedly extended survival in vitro and increasing myeloid clonogenic progenitor output over time. Sca(+/AE) mice develop a spontaneous myeloproliferative disorder with a latency of 6 months and a penetrance of 82% at 14 months. These results reinforce the notion that the phenotype of murine transgenic models of human leukemia is critically dependent on the cellular compartment targeted by the transgene. This model should provide a useful platform to analyze the effect of AML1/ETO on hematopoiesis and its potential cooperation with other mutations in the pathogenesis of leukemia.     

Stem cell factor increases the expression of FLIP that inhibits IFNgamma -induced apoptosis in human erythroid progenitor cells

Interferon gamma (IFNgamma) acts on human erythroid colony-forming cells (ECFCs) to up-regulate Fas, without a demonstrable change of Fas ligand (FasL) or Fas-associated DD-containing protein (FADD) expression and activates caspase-8 plus caspase-3, which produce apoptosis. Our previous data showed that stem cell factor (SCF) reduced the inhibitory effect of IFNgamma on human ECFCs when both factors were present in the cultures. However, the mechanism by which SCF prevents IFNgamma-induced apoptosis in ECFCs is unclear. In this study we used highly purified human ECFCs to investigate the mechanism of the effect of SCF on IFNgamma-induced apoptosis. Because the binding of FasL to Fas is the first step of the apoptosis cascade and IFNgamma strongly up-regulates Fas expression, we added FasL (50 ng/mL) to the cultures with IFNgamma to accentuate the IFNgamma-induced activation of caspase-8 and caspase-3 plus subsequent apoptosis. SCF (100 ng/mL) clearly inhibited the activation of caspase-8 and caspase-3 induced by IFNgamma and/or FasL, and it also reduced apoptosis as measured by the terminal dUTP nick-end labeling (TUNEL) assay. SCF did not decrease the surface expression of Fas on the ECFCs. FADD-like interleukin 1 beta (IL-1beta)-converting enzyme (FLICE)-inhibitory protein (FLIP) has been reported to interact with FADD and/or caspase-8 at the death-inducing signaling complex (DISC) level following Fas stimulation and acts as a dominant-negative caspase-8. SCF increased FLIP mRNA and protein expression, concomitant with reduced apoptosis, whereas IFNgamma and/or FasL did not change FLIP expression. Reduction of FLIP expression with antisense oligonucleotides decreased the capacity of SCF to inhibit IFNgamma-induced apoptosis, demonstrating a definite role for FLIP in the SCF-induced protection of ECFCs from IFNgamma-initiated apoptosis.     

Quality of life in adult patients after stem cell transplantation

Many articles pertaining to quality of life (QOL) following stem cell transplantation have been published in the US and western Europe. However, since the actions of health insurance systems and overall cultural aspects are strongly associated with QOL, investigations into QOL should be carried out within all countries. Therefore, we have investigated the QOL of adult patients following stem cell transplantation at 31 hospitals in Japan. The survivors, who were surveyed by mail questionnaire, were 20 years or older at the time of this study. The underlying diseases were acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, myelodysplastic syndrome, and multiple myeloma. Median age at the time of the study was 36 years, and median interval after transplantation was 35.3 months. Of 383 patients surveyed, 282 (73.6%) responded to the questionnaire. One hundred and ninety-two patients were treated with an allogeneic-related transplantation, 52 with allogeneic-unrelated, and 38 with an autologous transplantation. Our data revealed that the length of time since transplantation and the diagnosis of chronic GVHD were associated with QOL. When unrelated and related transplantation recipients were compared, ratings on relief from pain, stability in weight, and confidence in dealing with daily life were lower among unrelated transplantation patients.     

The histone deacetylase inhibitor,trichostatin A,reactivates the developmentally silenced gamma globin expression in somatic cell hybrids and induces gamma gene expression in adult BFUe cultures

Somatic cell hybrids that have undergone globin gene switching and developmental silencing of gamma globin expression were treated with the histone deacetylase inhibitor trichostatin A (TSA). Culture of the post-switch hybrids in the presence of TSA reactivated gamma globin expression and concommitantly downregulated beta globin expression, as determined by both mRNA quantitation and immunofluorescent quantitation of gamma globin expressing cells. In contrast, similar treatment of pre-switch hybrids, which were expressing predominantly gamma globin and only small levels of beta globin, had no effect on the relative gamma or beta globin gene expression. In addition, trichostatin A induced gamma gene expression in adult BFUe cultures in a maturation-independent fashion. The results provide direct evidence that inhibition of HDAC activity can alter expression from the human beta globin locus in the adult stage of development.     

Amelioration of severe hereditary spherocytosis in nonablated adult mice by marrow transplantation

Human severe hereditary spherocytosis (sHS) is life threatening and transfusion dependent. sHS is lethal within 6 days of birth for 99% of jaundiced (ja/ja) mice, making these mice excellent models for early therapeutic interventions. Nonablated ja/ja neonates simultaneously transfused and given intravenous injections of normal marrow become chimeric for donor cells. Significant improvement of red blood cell parameters occurs but is temporary because the donor marrow-derived cells gradually disappear from the circulation. The average lifespan, however, is increased to 8.7 months. We postulate that donor cells are diluted by rapidly proliferating host cells during postnatal growth. Here, we test this hypothesis by determining whether treatment of adults improves long-term therapy. Nonablated ja/ja adults rescued by a single neonatal transfusion were injected intravenously with 1 x 10(10) normal, genetically marked donor marrow cells/kg body weight. Donor cell implantation and blood parameters were monitored periodically and tissue histopathology was determined at necropsy.sHS recipients with 100% donor erythroid cells have significantly improved red blood cell counts throughout life when compared with ja/ja controls transfused once at birth. Total serum iron and bilirubin levels are corrected in ja/ja marrow recipients. Donor-implanted HS mice necropsied at 16 to 21 months of age have normal mean cell hemoglobin concentration and dramatically decreased tissue iron deposits. Reticulocyte counts but not red cell counts normalize, suggesting the HS mice reset their response to hypoxia.Nonablative transplantation performed after cessation of host postnatal red blood cell amplification can be therapeutic long term for transfusion-dependent hemolytic anemias.     

The expression of human blood group antigens during erythropoiesis in a cell culture system.

Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3.     

Hemoglobin C in transgenic mice: effect of HbC expression from founders to full mouse globin knockouts

When present in the homozygous form, hemoglobin C (HbC, CC disease) increases red cell density, a feature that is the major factor underlying the pathology in patients with SC disease (Fabry et al., JCI 70, 1315, 1982). The basis for the increased red cell density has not yet been fully defined. We have generated a HbC mouse in which the most successful founder expresses 56% human alpha and 34% human beta(C). We introduced knockouts (KO) of mouse alpha- and beta-globins in various combinations. In contrast to many KO mice, all partial KOs have normal MCH. Full KOs that express exclusively HbC and no mouse globins have minimally reduced MCH (13. 7 +/- 0.3 pg/cell vs 14.5 +/- 1.0 for C57BL/6) and a ratio of beta- to alpha-globin chains of 0.88 determined by chain synthesis; hence, these mice are not thalassemic. Mice with beta(C) > 30% have increased MCHC, dense reticulocytes, and increased K:Cl cotransport. Red cell morphology studied by SEM is strikingly similar to that of human CC cells with bizarre folded cells. We conclude that red cells of these mice have many properties that closely parallel the pathology of human disease in which HbC is the major determinant of pathogenesis. These studies also establish the existence of the interactions with other gene products that are necessary for pleiotropic effects (red cell dehydration, elevated K:Cl cotransport, morphological changes) that are also present in these transgenic mice, validating their usefulness in the analysis of pathophysiological events induced by HbC in red cells.