Investigation of patient factors associated with the number of transfusions required during chemotherapy for high-risk neuroblastoma.

BACKGROUND: Blood transfusion is an important supportive care for high-risk neuroblastoma. When the number of transfusions increases, transfusion-associated adverse reactions may be more problematic. However, the factors determining the degree of myelosuppression and the number of transfusions during chemotherapy for high-risk neuroblastoma remain unclear. MATERIALS AND METHODS: We investigated patient factors determining the number of required transfusions in 15 high-risk neuroblastoma patients who received five courses of chemotherapy. Clinical data, cytokine profile and colony-forming assay with bone marrow samples at diagnosis were analysed. RESULTS: The required number of transfusions of both platelets and erythrocytes decreased once in the second course and then increased as the course progressed. The variability among cases increased as the chemotherapy course progressed. In cases of low peripheral blood platelet count and lower fibrinogen level at diagnosis, the number of platelet transfusions was higher during chemotherapy. In contrast, there was a negative correlation between the forming ability of granulocyte-macrophage or erythroid colonies and the number of erythrocyte transfusions in the latter period. CONCLUSION: In the early stages of chemotherapy, bone marrow infiltration in neuroblastoma and/or coagulopathy complication may cause thrombocytopenia and requirement of platelet transfusion; conversely, in the later stages, the number of erythrocyte transfusions may be defined by the patient's inherent hematopoietic ability. These factors may be useful in predicting the required number of transfusions.

Expression of gilt acts as a positive regulator of mouse hematopoietic progenitor cells.

Gamma interferon inducible lysosomal thiol reductase (GILT), is known to be involved in immunity, but its role in hematopoiesis has not been previously reported. Herein, we demonstrate using gilt knockout (-/-) mice that loss of gilt associates with decreased numbers and cycling status of femoral hematopoietic progenitor cells (CFU-GM, BFU-E, and CFU-GEMM) with more modest effects on splenic progenitor cells. Thus, GILT is associated with positive regulation of hematopoietic progenitor cells in mice, mainly in bone marrow.

The effects of selective beta-adrenergic blockade on bone marrow dysfunction following severe trauma and chronic stress.

INTRODUCTION: Propranolol has been shown to improve erythroid progenitor cell growth and anemia following trauma and this study sought to investigate the mechanisms involved by evaluating the effects of selective beta blockade. METHODS: Male Sprague-Dawley rats were subjected to lung contusion, hemorrhagic shock and chronic stress (LCHS/CS) ± daily selective beta-1, beta-2, or beta-3 blockade (B1B, B2B, B3B). Bone marrow cellularity and growth of erythroid progenitor colonies, hemoglobin, plasma granulocyte colony-stimulating factor (G-CSF), hematopoietic progenitor cell mobilization, and daily weight were assessed. RESULTS: Selective beta-2 and beta-3 blockade improved bone marrow cellularity, erythroid progenitor colony growth and hemoglobin levels, while decreasing plasma G-CSF, progenitor cell mobilization and weight loss following LCHS/CS. CONCLUSIONS: Attenuating the neuroendocrine stress response with the use of selective beta-2 and 3 adrenergic blockade may be an alternative to improve bone marrow erythroid function following trauma.

Effects of iron and vitamin B12 deficiencies on peripheral blood colony-forming unit capacity.

Iron and vitamin B12 deficiencies are two of the most common diseases in the childhood group. Deficiencies of iron and vitamin B12 affect many systems in the body. In this study, to discover the effects of iron and vitamin B12 deficiencies on the hematopoietic stem cells, we studied CFU assay from peripheral blood. One hundred and two children were included in our study and were evaluated in five categories: iron deficiency, iron deficiency anemia, vitamin B12 deficiency, iron and vitamin B12 deficiency, and controls. As a result of statistical analysis, no significant difference was detected between five groups in terms of CFU assays. The results of our study suggest that, in emergent situations, stem cell samples can be collected before treatment with B12 or iron which are common deficiencies in donors of hematopoietic stem cell transplantation. We conclude that we could reach more accurate results by designing a study which contains more patients and includes in vivo results.

Evaluation of drug-induced hematotoxicity using novel in vitro monkey CFU-GM and BFU-E colony assays.

In order to evaluate drug-induced hematotoxicity in monkey cells in vitro, colony-forming unit-granulocyte, macrophage (CFU-GM), and burst-forming unit-erythroid (BFU-E) colony assays were established using mononuclear cells in the bone marrow collected from male cynomolgus monkeys. Furthermore, the effects of doxorubicin, chloramphenicol, and linezolid on CFU-GM and BFU-E colony formation were investigated using established monkey CFU-GM and BFU-E colony assays in comparison with those on human CFU-GM and BFU-E colonies acquired from human umbilical cord blood cells. Bone marrow mononuclear cells were collected from the ischial or iliac bone of male cynomolgus monkeys. The cells were subsequently processed by density gradient separation at 1.067, 1.070, or 1.077 g/mL for CFU-GM or 1.077 g/mL for BFU-E, and then cultured in methylcellulose medium for 9 or 13 days, respectively. A sufficient number of CFU-GM colonies were formed from mononuclear cells processed at a density of 1.070 g/mL. Moreover, the number of BFU-E colonies from the cells processed at a density of 1.077 g/mL was sufficient for the colony assay. The number of CFU-GM or BFU-E colonies decreased after treatment with the drugs of interest in a concentration-dependent manner. Compared with human CFU-GM, monkey CFU-GM were more sensitive to chloramphenicol and resistant to doxorubicin, whereas monkey BFU-E were more sensitive to all compounds in comparison to the sensitivity of human BFU-E. In conclusion, monkey CFU-GM and BFU-E colony assays were established and considered useful tools to evaluate the differences in drug-induced hematotoxicity between species.

Cyclin A2 regulates erythrocyte morphology and numbers.

Cyclin A2 is an essential gene for development and in haematopoietic stem cells and therefore its functions in definitive erythropoiesis have not been investigated. We have ablated cyclin A2 in committed erythroid progenitors in vivo using erythropoietin receptor promoter-driven Cre, which revealed its critical role in regulating erythrocyte morphology and numbers. Erythroid-specific cyclin A2 knockout mice are viable but displayed increased mean erythrocyte volume and reduced erythrocyte counts, as well as increased frequency of erythrocytes containing Howell-Jolly bodies. Erythroblasts lacking cyclin A2 displayed defective enucleation, resulting in reduced production of enucleated erythrocytes and increased frequencies of erythrocytes containing nuclear remnants. Deletion of the Cdk inhibitor p27Kip1 but not Cdk2, ameliorated the erythroid defects resulting from deficiency of cyclin A2, confirming the critical role of cyclin A2/Cdk activity in erythroid development. Loss of cyclin A2 in bone marrow cells in semisolid culture prevented the formation of BFU-E but not CFU-E colonies, uncovering its essential role in BFU-E function. Our data unveils the critical functions of cyclin A2 in regulating mammalian erythropoiesis.

Enforced expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo.

Hoxa5 is preferentially expressed in haematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs), and is more highly expressed in expanding HSCs. To date, little is known regarding the role of Hoxa5 in HSCs and downstream progenitor cells in vivo. In this study, we show that increased expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo. Hoxa5 differentially modifies the cell cycle of HSCs and lineage committed progenitor cells, depending on the cellular context. Hoxa5 drives HSCs, but not MPPs, through the cell cycle and arrests erythroid progenitor cells in G0 phase. Although the HSC pool shrinks after overexpression of Hoxa5, HSCs sustain the abilities of self-renewal and multipotency. In vivo, Hoxa5 has two effects on erythropoiesis: it causes a predominance of mature erythroid lineage cells and the partial apoptosis of erythroid progenitors. RNA-seq indicates that multiple biological processes, including erythrocyte homeostasis, cell metabolism, and apoptosis, are modified by Hoxa5. The results of this study indicate that Hoxa5 is a key regulator of the HSC cell cycle, and the inappropriate expression of Hoxa5 in lineage-committed progenitor cells leads to aberrant erythropoiesis.

Erythro-myeloid progenitors: "definitive" hematopoiesis in the conceptus prior to the emergence of hematopoietic stem cells.

Erythro-myeloid progenitors (EMP) serve as a major source of hematopoiesis in the developing conceptus prior to the formation of a permanent blood system. In this review, we summarize the current knowledge regarding the emergence, fate, and potential of this hematopoietic stem cell (HSC)-independent wave of hematopoietic progenitors, focusing on the murine embryo as a model system. A better understanding of the temporal and spatial control of hematopoietic emergence in the embryo will ultimately improve our ability to derive hematopoietic stem and progenitor cells from embryonic stem cells and induced pluripotent stem cells to serve therapeutic purposes.

Combined inhibition of PI3K and activation of MAPK p38 signaling pathways trigger erythroid alternative splicing switch of 4.1R pre-mRNA in DMSO-induced erythroleukemia cells.

There is increasing evidence showing that many extracellular cues modulate pre-mRNA alternative splicing, through different signaling pathways. We here show that 4.1R exon 16 splicing is altered in response to specific signals. The switch from erythroblastic isoform lacking exon 16 to mature erythrocytic isoform containing this exon is tightly regulated during late erythroid differentiation, and blocage of this splicing switch in erythroleukemia cells is seen as a consequence of the deregulation of important regulatory pathways. We support that combined inhibition of PI3K and activation of p38 signaling pathways impinge on erythroid 4.1R pre-mRNA alternative splicing switch, and on cell differentiation as witnessed by hemoglobin production. By contrast, MEK/ERK signaling appeared not to affect neither cell hemoglobin production nor erythroid 4.1R pre-mRNA splicing. We also found that the signal-induced alternative splicing is not typically distinctive of EPO-non-responsive cells, but operates in EPO-responsive cells as well. Pre-mRNA splicing is a major regulatory mechanism at the crossroad between transcription and translation. We here provide evidence that inhibition of PI3K activates the splicing switch in a promoter-dependent manner, whereas p38 activation induces this event in a promoter-independent fashion. Our data further support that constitutive activation of EPO-R by the viral protein gp55 and the short form of the tyrosine kinase receptor Stk, transduces PI3K proliferation signal, but not MAPK p38 differentiation signal. Concurrently, this work lend credence to the concept that DMSO triggers transient activation of p38 signaling and irreversible inhibition of PI3K/AKT signaling pathway, hence uncovering an old conundrum regarding the mechanism by which DMSO induces erythroleukemia cell differentiation.

Changes in DNA methylation of erythroid-specific genes in K562 cells exposed to phenol and hydroquinone.

Benzene is a common occupational hazard as well as a widespread pollutant. Its metabolites play important roles in its toxicity to the hematopoietic system, but little is known about how benzene metabolites affect erythropoiesis. Our previous study demonstrated that benzene metabolites, including phenol and hydroquinone, inhibited hemin-induced erythroid differentiation of K562 cells. In present study, to elucidate the role of DNA methylation in benzene metabolites-induced inhibition on erythroid differentiation, it was investigated whether DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-CdR), was able to prevent benzene metabolites inhibiting hemin-induced erythroid differentiation in K562 cells, and the methylation levels of erythroid-specific genes in benzene metabolites-treated K562 cells were analyzed by Quantitative MassARRAY methylation analysis platform. It was found that treatment of K562 cells with 5-aza-CdR completely prevented phenol and hydroquinone inhibiting hemin-induced hemoglobin synthesis and hemin-induced expression of erythroid specific genes, including α- and ß-globin, erythroid porphobilinogen deaminase and GATA binding protein 1 (GATA-1). Consistently, the exposure to benzene metabolites caused an increase in DNA methylation levels at a few CpG sites in some erythroid specific genes, including α-globin gene and α-cluster HS40 element, ß-globin gene and HS core sequence in LCR of ß-globin gene cluster, erythroid porphobilinogen deaminase gene, and GATA-1 gene. These results indicated that DNA methylation played a role in benzene metabolites inhibiting hemin-induced erythroid differentiation of K562 cells via down-regulating transcription of some erythroid related genes.

Molecular defects identified by whole exome sequencing in a child with Fanconi anemia.

Fanconi anemia is a rare genetic disease characterized by bone marrow failure, multiple congenital malformations, and an increased susceptibility to malignancy. At least 15 genes have been identified that are involved in the pathogenesis of Fanconi anemia. However, it is still a challenge to assign the complementation group and to characterize the molecular defects in patients with Fanconi anemia. In the current study, whole exome sequencing was used to identify the affected gene(s) in a boy with Fanconi anemia. A recurring, non-synonymous mutation was found (c.3971C>T, p.P1324L) as well as a novel frameshift mutation (c.989_995del, p.H330LfsX2) in FANCA gene. Our results indicate that whole exome sequencing may be useful in clinical settings for rapid identification of disease-causing mutations in rare genetic disorders such as Fanconi anemia.

Ectopic bone formation in severely combat-injured orthopedic patients -- a hematopoietic niche.

Combat-related heterotopic ossification (HO) has emerged as a common and problematic complication of modern wartime extremity injuries, contributing to substantial patient morbidity and loss of function. We have previously reported that HO-forming patients exhibit a more pronounced systemic and local inflammatory response very early in the wound healing process. Moreover, traumatized muscle-derived mesenchymal progenitor cells from these patients have a skewed differentiation potential toward bone. Here, we demonstrate that HO lesions excised from this patient population contain highly vascularized, mature, cancellous bone containing adipogenic marrow. Histologic analysis showed immature hematopoietic cells located within distinct foci in perivascular regions. The adipogenic marrow often contained low numbers of functional erythroid (BFU-E), myeloid (CFU-GM, CFU-M) and multilineage (CFU-GEMM) colony-forming hematopoietic progenitor cells (HPCs). Conversely, tissue from control muscle and non-HO traumatic wound granulation tissue showed no evidence of hematopoietic progenitor cell activity. In summary, our findings suggest that ectopic bone can provide an appropriate hematopoietic microenvironment for supporting the proliferation and differentiation of HPCs. This reactive and vibrant cell population may help maintain normal hematopoietic function, particularly in those with major extremity amputations who have sustained both massive blood loss, prompting systemic marrow stimulation, as well as loss of available native active marrow space. These findings begin to characterize the functional biology of ectopic bone and elucidate the interactions between HPC and non-hematopoietic cell types within the ectopic intramedullary hematopoietic microenvironmental niche identified.

Idiopathic bone marrow dysplasia of unknown significance (IDUS): definition, pathogenesis, follow up, and prognosis.

Minimal diagnostic criteria for myelodysplastic syndromes (MDS) include constant cytopenia recorded for at least 6 months, dysplasia, and exclusion of other causes of cytopenia and dysplasia. However, there are patients with dysplastic bone marrow features with or without a karyotype, who have only mild if any cytopenia. This condition has been termed idiopathic dysplasia of unknown significance (IDUS). Out of a series of 1,363 patients with suspected MDS or mild cytopenia seen between 1997 and 2010, we have identified 10 patients with IDUS, and analyzed their clinical course and outcome as well as features potentially involved in disease-evolution. Follow-up ranged between 2 and 13 years. Progression to an overt myeloid neoplasm was observed in 4 patients: two progressed to frank MDS, one to chronic myelomonocytic leukemia, and one to a myelodysplastic/myeloproliferative neoplasm exhibiting 5q-and JAK2 V617F. Consecutive studies revealed that most IDUS patients have an adequate production of erythropoietin (EPO) and sufficient numbers of EPO-responsive erythroid progenitors, features rarely seen in MDS. The erythropoiesis-promoting JAK2 mutation V617F was only detectable in one case. We hypothesize that the dysplastic clone in IDUS cannot manifest as frank MDS because i) the clone retains responsiveness against EPO, and ii) an adequate EPO-production counteracts anemia. Evolution of IDUS to low risk MDS may thus depend on the biological properties of the clone as well as patient-related factors such as EPO production. The latter often decreases with age and may thus explain why MDS often manifests in the elderly.