Cutaneous Mycobacterium chelonae infection following autologous peripheral blood stem cell transplantation for POEMS syndrome

Although hematopoietic stem cell transplantation (HSCT) may increase the curability of refractory hematologic diseases, it requires complication management due to a long-term immunocompromised state. We experienced a case who received an autologous peripheral blood stem cell transplantation (Auto-PBSCT) for POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes) and developed cutaneous Mycobacterium chelonae infection. It is clear that attention needs to be paid to prevent bacterial, fungal and viral infection after HSCT. It is also important to keep in mind that tuberculous and nontuberculous mycobacteria (NTM), in rare cases, lead to lethal complications.     

Lineage-related and particle size-dependent cytotoxicity of chitosan nanoparticles on mouse bone marrow-derived hematopoietic stem and progenitor cells

Chitosan nanoparticles (CSNPs) have potential applications in stem cell research. In this study, ex vivo cytotoxicity of CSNPs on mouse bone marrow-derived (MBMCs) hematopoietic stem and progenitor cells (HSPCs) was determined. MBMCs were exposed to CSNPs of different particle sizes at various concentrations for up to 72 h. Cytotoxicity effect of CSNPs on MBMCs was determined using MTT, Live/Dead Viability/Cytotoxicity assays and flow cytometry analysis of surface antigens on HSCs (Sca-1⁺), myeloid-committed progenitors (CD11b⁺, Gr-1⁺), and lymphoid-committed progenitors (CD45⁺, CD3e⁺). At 24 h incubation, MBMCs' viability was not affected by CSNPs. At 48 and 72 h, significant reduction was detected at higher CSNPs concentrations. Small CSNPs (200 nm) significantly reduced MBMCs' viability while medium-sized particle (∼400 nm) selectively promoted MBMCs growth. Surface antigen assessment demonstrated lineage-dependent effect. Significant decrease in Sca-1⁺ cells percentage was observed for medium-sized particle at the lowest CSNPs concentration. Meanwhile, reduction of CD11b⁺ and Gr-1⁺ cells percentage was detected at high and intermediate concentrations of medium-sized and large CSNPs. Percentage of CD45⁺ and CD3e⁺ cells along with ROS levels were not significantly affected by CSNPs. In conclusion, medium-sized and large CSNPs were relatively non-toxic at lower concentrations. However, further investigations are necessary for therapeutic applications.     

Sensitivity of Ph1+ CFU-GM to human recombinant interferon α and γ alone and in combination

Summary The in vitro effect of human recombinant interferon (IFN) alone and in combination were studied on granulomonocytic colony forming units (CFU-GM) from the peripheral blood of 10 Ph1+ chronic myeloid leukemia (CML) patients and from the marrow of 5 normal or non-leukemic subjects. - and -IFN alone determined a slight inhibition on colony growth with a preferential effect on pure macrophagic colonies. At maximum concentration (10⁴ U/ml) leukemic colony inhibition was 46±34% for IFN and 43±19% for IFN. Culture growth with + IFN in combination were significantly inhibited (up to 96±4%) with a concentration-related effect. Similar results were obtained with normal CFU-GM. The synergism that was found in vitro is probably relevant for the in vivo therapeutic effects of these compounds in CML and suggest that the combination is worth testing in vivo.This work was supported by CNR, Oncology Finalized Project, contract n. 8701169.44. and n. 8600603.44. and by AIRC     

The in vitro effects of stem cell factor, interleukin 3 and granulocyte-macrophage colony stimulating factor on haemopoietic progenitor cells from premature infants

Summary. Circulating haemopoietic progenitor cells from premature infants were assessed for their ability to respond to interleukin 3, granulocyte-macrophage colony stimulating factor and stem cell factor (SCF) in vitro. All three cytokines increased the number of colonies derived from burst forming units erythroid (BFU-E), colony forming units granulocyte-macrophage (CFU-GM) and multi-lineage progenitors (CFU-Mix) grown in the presence of erythropoietin (Epo). The size and haemoglobin content of BFU-E derived colonies also increased in the presence of the cytokines. Of those tested, SCF was found to be the most potent additive to Epo for the enhanced growth of BFU-E and CFU-Mix. In short-term liquid cultures without Epo, SCF alone induced globin synthesizing cells. Progenitors from premature infants were at least as responsive to all three cytokines as those from healthy adults. The use of SCF in combination with Epo in the prevention or treatment of anaemia in premature infants warrants further investigation.     

Growth stimulatory effect of thrombopoietin on the blast cells of acute myelogenous leukaemia

Thrombopoietin stimulated blast colony formation in 11/20 acute myelogenous leukaemia (AML) patients studied. The FAB subtypes of the blasts responding to thrombopoietin were not restricted to those of the megakaryocyte lineage, but also included M1–M5 AML blasts. The morphology of colony cells produced by megakaryocytic blasts showed megakaryocytoid features, whereas colony cells produced by M1–M5 AML blasts remained myeloblasts. An increase in CD41 was observed in the cells of colonies produced by blasts from the megakaryocyte lineage involving leukaemia and chronic myeloid leukaemia in blastic crisis. Thrombopoietin receptor was observed on leukaemic blasts which formed colonies following incubation with thrombopoietin.     

Molecular Genetics of Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by the clonal expansion of glycosylphosphatidylinositol (GPI)-deficient cells that leads to complement-mediated hemolysis. A somatic mutation in the PIG-A gene involved in GPI biosynthesis causes a deficiency of GPI-anchored proteins. However, it is evident that the clonal expansion of GPI-deficient cells is not caused by only the PIG-A mutation and that other changes should be involved in the development of PNH. Some patients with aplastic anemia (AA) develop PNH. Furthermore, it has been reported that most patients with AA and refractory anemia (RA) who carry HLA-DRB1*15 and show a good response to immunosuppressive therapies have an expanded population of GPI-deficient clones. This finding, together with recent data showing resistance of GPI-deficient cells to cytotoxic cells, suggests that GPI-deficient cells escape immunologic attack and are positively selected in the autoimmune environment. However, GPI-deficient clones found in AA and RA are generally small and do not increase to near-complete dominance. Therefore, 1 or more additional genetic abnormalities that confer the growth phenotype on GPI-deficient cells are probably required for fully developed PNH or so-called florid PNH. The next 10 years should witness the discovery of the molecular mechanisms of immunologic selection and the identification of abnormalities involved in the further clonal expansion of PNH cells.     

异基因外周血干细胞移植后淋巴细胞和粒细胞嵌合体的动态改变

目的：探讨异基因外周血干细胞移植(allo-PBSCT)后T细胞、粒细胞嵌合体的动态改变及临床价值。方法：将多重PCR扩增短串联重复序列(STR—PCR)的法医试剂进行条件优化，间隔短时间抽取9例恶性血液病患者(5例清髓性PBSCT，4例非清髓性PBSCT)的外周血样，STR—PCR定量分析T细胞和粒细胞的嵌合体，并观察其对移植后应用免疫抑制剂的指导作用。结果：STR—PCR定量分析嵌合体的敏感性为5％，并具高度可重复性。清髓性PBSCT后10d( 10d)，5／5例患者的粒细胞迅速演变为完全供者嵌合体(CDC)， 14d，4，／5例患者的T细胞获得CDC。非清髓性PBSCT， 7～ 14d，供者T细胞信号的植入速度快于粒细胞；随后，供者粒细胞的比例突然增加，并迅速获得CDC，T细胞的植入却渐缓慢，最后，T细胞取得CDC的时间迟于粒细胞。依据供者T细胞信号的植入程度，及时调整非清髓性PBSCT后环孢素A(CsA)的用量，移植早期T细胞即获CDC，随访2～16个月，T细胞和粒细胞均呈稳定的供者植入状态。结论：供者T细胞的完全植入迟于粒细胞．动态监测T细胞嵌合体，可能有助于免疫抑制剂的调整。     

In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells

Aims/hypothesis We recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors. Methods Mouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice.Results The protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols.Conclusions/interpretation These methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.Abbreviations Anti-Shh antibody against sonic hedgehog - D3 undifferentiated D3 stem cell line - EB embryoid bodies - ES embryonic stem - FBS fetal bovine serum - LIF leukaemia inhibitory factor - mES mouse embryonic stem - Ngn3 neurogenin 3 - P gelatine-coated plates - Pdx-1 pancreatic duodenum homeobox 1     

High cyclin-dependent kinase inhibitors in Bcl-2 and Bcl-xL-expressing CD34+-proliferating haematopoietic progenitors

We have previously described the isolation of primitive, slow-proliferating progenitors from normal, circulating CD34+ cells by using the fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). CFDA-SE(bright) (primitive) and CFDA-SE(dim) (differentiating) cells were isolated following cytokine stimulation on the basis of their different proliferation rates. In the present work we analysed the expression levels of a number of proteins involved with differentiation, proliferation and survival/apoptosis in CFDA-SE(bright)/CD34+/slow-proliferating cells that were previously defined as progenitors capable of differentiating into different lineages. The aim of this work was to gain a better understanding of our model system in order to define some of the important parameters that regulate differentiation in haematopoietic progenitors. GATA-1 and PU.1 RNA levels were similar in freshly isolated (d 0) CD34+ and in CFDA-SE(bright) (bright) cells, whereas they increased in CFDA-SE(dim) (dim) cells. Accordingly, Nm23 was expressed at higher levels in bright cells. Moreover, bright cells had higher p21WAF1/CIP1, p27KIP1 and p16Ink4 protein levels than dim cells. Consistently, Cdc2 and Cdk2 kinase activity was much higher in the dim than in the slower proliferating bright cells. C-myc and p53 levels were higher in bright cells than in d 0 CD34+ and dim cells, and so was Bcl-xL, which followed the trend we have previously described for Bcl-2. Thus, bright cells, despite having a higher proliferation rate than the starting d 0 CD34+ population, have strikingly elevated levels of cyclin-dependent kinase inhibitors, which are likely to also act as inhibitors of differentiation.     

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G₀/G₁ phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.