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PubMed2000

Analysis of parameters affecting engraftment in children undergoing autologous peripheral blood stem cell transplants.

Eighty-three pediatric patients underwent autologous peripheral blood stem cell transplants at a single institution and were included in a study evaluating the correlations between five engraftment parameters and the time to both neutrophil and platelet recovery. The parameters included: the number of nucleated cells per kg (TNC/kg), the absolute CD34+ cell content per kg (CD34+/kg), the number of mononuclear cells per kg (MNC/kg), the number of BFU-E/kg, and the number of CFU-GM/kg. A two-tailed Mann-Whitney test (alpha = 0.05) was used to determine if there were significant differences between patients with neuroblastoma (n = 45) and patients with other diagnoses (n = 38). No statistically significant differences existed between neuroblastoma patients and patients with other diagnoses. Therefore, the two groups of patients were pooled together. Data were analyzed using both a univariate and multivariate correlation method and Student's t-test (alpha = 0.05). Two statistically significant logarithmic relationships were found. The first relationship was between MNC/kg and time to ANC reconstitution (P = 0.05). The second relationship was between CFU-GM/kg and time to platelet recovery (P = 0.01). Based on the statistical data, we conclude that there is no correlation between nucleated cell dose, CD34+ cell dose, and BFU-E content with either neutrophil or platelet recovery. Accordingly, in this study MNC cell dose per kilogram was the most important parameter predicting the length of time between graft infusion and neutrophil recovery while CFU-GM content per kilogram was the most important parameter predicting the length of time until platelet recovery.

Adolescent↗
PubMed2006

Erythroid progenitor renewal versus differentiation: genetic evidence for cell autonomous, essential functions of EpoR, Stat5 and the GR.

The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.

Animals↗
PubMed2000

Reversal of aberrant splicing of beta-thalassaemia allele (IVS-2-654 C-->T) by antisense RNA expression vector in cultured human erythroid cells.

The antisense fragment targeting the aberrant splice sites of the beta-thalassaemia allele, IVS-2-654 C-->T (beta654), pretranscript was cloned into the mammalian expression vector, pcDNA3. The recombinant construct, pCMVA, was then used to repair the defective splicing of the beta654 mutant pretranscript in cultured beta654 erythroid cells by the lipofectin-mediated DNA transfection method. The total RNA was extracted at given time points after transfection and the effect of antisense RNA was studied by reverse transcription polymerase chain reaction (RT-PCR)-mediated mRNA quantitative assay, as well as globin chain microbiosynthesis. The antisense fragment transcribed from pCMVA effectively improved the beta654 splicing pattern in cultured erythroid cells. The level of correctly spliced transcript increased from 0.19 (day 0 after transfection) to 0.58 (day 8) in beta654/beta654 homozygous erythroid cells, and from 0.45 (day 0) to 0.83 (day 8) in beta654/betaA heterozygous erythroid cells, as determined by the ratio of normally spliced beta-globin transcript over total beta-globin transcript. Correspondingly, the ratios of globin chain biosynthesis (beta/alpha) increased from 0.16 (day 0) to 0.52 (day 8) in beta654/beta654 erythroid cells, and from 0.39 (day 0) to 0.84 (day 8) in beta654/betaA erythroid cells. Antisense RNA had no significant effect on the splicing pattern in betaA/betaA erythroid cells. The splicing pattern in transfected cells with pCMVA showed significant changes compared with that in untransfected cells and that in transfected cells with the control antisense fragment (human SRY gene sequence). In addition, we did not observe side-effects on cytological features after the introduction of pCMVA. All these results indicated that the antisense RNA transcribed from the mammalian expression vector pCMVA could efficiently and specifically suppress the aberrant splicing pattern of beta654 mutant pretranscript and restore the correct splicing pathway in vivo, leading to the improvement of globin chain biosynthesis in thalassaemic cells.

Adult↗
PubMed2002

Enforced expression of HOX11 is associated with an immature phenotype in J2E erythroid cells.

The HOX11 gene encodes a homeodomain transcription factor that is essential for spleen development during embryogenesis. HOX11 is also leukaemogenic, both through its clinical association with childhood T-cell acute lymphoblastic leukaemia, and its ability to immortalize other haematopoietic cell lineages experimentally. To examine the pathological role of HOX11 in tumorigenesis, we constitutively expressed HOX11 cDNA in J2E murine erythroleukaemic cells, which are capable of terminal differentiation. Enforced HOX11 expression was found to induce a profound alteration in J2E cellular morphology and differentiation status. Our analyses revealed that HOX11 produced clones with a preponderance of less differentiated cells that were highly adherent to plastic. Morphologically, the cells overexpressing HOX11 were larger and had decreased globin levels, as well as a reduction in haemoglobin synthesis in response to erythropoietin (EPO). Immunocytochemical analysis confirmed the immature erythroid phenotype imposed by HOX11, with clones transfected with HOX11 demonstrating expression of the c-Kit stem cell marker, while retaining EPO receptor expression. Taken together, these results show that HOX11 alters erythroid differentiation, favouring a less mature progenitor-like stage. This supports the notion that disrupted haematopoietic cell differentiation is responsible for pre-leukaemic immortalization by the HOX11 oncoprotein.

Animals↗
PubMed1999

Mechanism of regulation of complement receptor type 1 transcription by cytosine arabinoside in a pre-erythroid model.

Binding to erythrocyte complement receptor type 1 (CR1) clears immune complexes from blood and tissues, preventing complement-mediated pathological inflammation in disease. Previous work has demonstrated that Ara-C, a cytosine analogue, induces an 11-fold increase in CR1 mRNA expression in K-562 erythroleukaemia cells. In this work we therefore investigated whether the Ara-C/K-562 system could be used as a model for studying the pre-erythroid regulation of CR1. We demonstrated that increased CR1 expression could be induced independently of increased haemoglobin expression. Increases in CR1 mRNA levels produced by Ara-C treatment were not a function of increased stability of the message. However, Ara-C induced a protein synthesis-dependent increase in transcription initiation rate as early as 12h after treatment. Further data suggest that the effect of Ara-C on transcription is not a result of its direct DNA-damaging or DNA polymerase-inhibition activities. Induction of receptor transcription was inhibited by tyrosine kinase (TK) and protein kinase C (PKC) inhibitors. These data suggest that TK, PKC and dCTP-adducted phospholipid signalling pathways may all play a role in the mechanism of Ara-C-induced CR1 transcription.

Butyrates↗
PubMed1992

Transcription of the hypersensitive site HS2 enhancer in erythroid cells.

In the human genome, the erythroid-specific hypersensitive site HS2 enhancer regulates the transcription of the downstream beta-like globin genes 10-50 kilobases away. The mechanism of HS2 enhancer function is not known. The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids. In erythroid K562 cells in which the HS2 enhancer is active, the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene. In nonerythroid HL-60 cells in which the HS2 enhancer is inactive, long enhancer transcripts are not detectable. Splitting the HS2 enhancer between two tandem Ap1 sites abolishes the synthesis of a group of long enhancer transcripts and results in loss of enhancer function and transcriptional silencing of the cis-linked CAT gene. In directing the synthesis of RNA through the intervening DNA and the gene by a tracking and transcription mechanism, the HS2 enhancer may (i) open up the chromatin structure of a gene domain and (ii) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site.

Base Sequence↗
PubMed1997

Globin gene silencing in primary erythroid cultures. An inhibitory role for interleukin-6.

There are numerous similarities between the erythroid and megakaryocytic lineages which suggest that commitment to either lineage occurs relatively late in hematopoiesis. Commitment toward megakaryocyte development requires obligatory silencing of erythroid-specific genes. Therefore, we investigated the effects of interleukin-6, a known inducer of thrombocyte production, on globin gene expression during erythroid differentiation. Studies in K562 cells demonstrated inhibition of gamma globin gene mRNA production and chain biosynthesis in the presence of exogenous interleukin-6 which was abrogated by anti-interleukin-6 monoclonal antibody. Similar studies in primary erythroid progenitors showed inhibition of burst-forming unit-erythroid colony formation when interleukin-6 was added late in cultures with decreased gamma and beta globin gene mRNA production. Protein binding studies demonstrated an increase in activator protein-1 binding to its consensus sequence by 24 h of interleukin-6 treatment. Inhibition of activator protein-1 gene activity had no effect on gamma gene silencing by interleukin-6. A potential interleukin-6 response element was identified in the gamma globin gene. Interleukin-6 treatment led to a rapid increase in protein binding to the target DNA sequence. These results suggest that interleukin-6 may play an important role in globin gene silencing during megakaryocytic lineage commitment.

Butyrates↗
PubMed2006

Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood.

BACKGROUND: Umbilical cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSC/HPC) for the reconstitution of the hematopoietic system after clinical transplantation. Cryopreservation of these cells is critical for UCB banking and transplantation as well as for research applications by providing readily available specimens. The objective of this study was to optimize cryopreservation conditions for CD34+ HSC/HPC from UCB. METHODS: Cryopreservation of CD34+ HSC/HPC from UCB after mononuclear cell (MNC) preparation was tested in a research-scale setup. Experimental variations were concentration of the cryoprotectant, the protein additive and cell concentration. In addition, protocols involving slow, serial addition and removal of DMSO were compared with standard protocols (fast addition and removal of DMSO) in order to avoid osmotic stress for the cryopreserved cells. Viability and recoveries of MNC, CD34+ cells and total colony-forming units (CFU) were calculated as read-outs. In addition, sterility testing of the collected UCB units before further processing was performed. RESULTS: The optimal conditions for cryopreservation of CD34+ HPC in MNC preparations were 10% DMSO and 2% human albumin at high cell concentrations (5 x 10(7) MNC/mL) with fast addition and removal of DMSO. After cryopreservation using a computer-controlled freezer, high viabilities (89%) and recoveries for CD34+ cells (89%) as well as for CFU (88%) were observed. Microbial contamination of the collected UCB samples was reduced to a rate of 6.4%. DISCUSSION: Optimized cryopreservation conditions were developed for UCB MNC in respect of the composition of the cryosolution. In addition, our results showed that fast addition of DMSO is essential for improved cryopreservation and post-thaw quality assessment results, whereas the speed of DMSO removal after thawing has little influence on the recoveries of CD34+ cells and CFU.

Antigens, CD34↗
PubMed2001

Hydroxyurea promotes the reduction of spontaneous BFU-e to normal levels in SS and S/beta thalassemic patients.

We have studied the effects of hydroxyurea on growth and differentiation of early erythroid progenitor cells (BFU-e) from peripheral blood of sickle cell disease patients (five SS and two Hb S/beta-thalassemia) in the presence or absence of exogenous stimulating factors. When the mononuclear cells from the sickle cell disease patients were cultured at diagnosis (before hydroxyurea treatment), there was an increased number of BFU-e in relation to controls (p < 0.05, Wilcoxon test) when cells were grown in the presence or absence of 5637 conditioned medium and erythropoietin. Colonies that developed in the absence of added growth factors were considered "spontaneous". A significant difference was observed after hydroxyurea treatment in the number of BFU-e obtained in the presence and absence of stimulus, with a higher reduction in the spontaneous BFU-e number. As expected, there was an increased Hb F level in these patients when compared with their pretreatment levels. There was no correlation between spontaneous BFU-e and hemoglobin levels in all patients studied.

Anemia, Sickle Cell↗
PubMed1990

Purification of an inhibitor of erythroid progenitor cell cycling and antagonist to interleukin 3 from mouse marrow cell supernatants and its identification as cytosolic superoxide dismutase.

We have isolated a protein from media conditioned by a murine marrow-derived cell line (PB6) and from mouse marrow supernatants that antagonizes interleukin 3-dependent proliferation of cells in culture and reversibly inhibits DNA synthesis of erythroid progenitor cells (BFU-E) in vitro. This protein, p16 (monomer Mr = 16 kD on SDS-PAGE), was purified to homogeneity and amino acid sequencing of a polypeptide fragment yielded a sequence identical to that of murine cytosolic Cu,Zn-containing superoxide dismutase (SOD). The identification of p16 as SOD was confirmed by the detection of SOD enzymatic activity in pure p16 fractions, and when a commercial human erythrocytic SOD preparation was tested it showed the same cell inhibitory activities as p16. These observations show that superoxide dismutase is able to affect the cycling and growth factor responses of hematopoietic cells, activities that have not previously been associated with this enzyme.

Amino Acid Sequence↗
PubMed1996

Analysis of interleukin 6 receptor and gp130 expressions and proliferative capability of human CD34+ cells.

We recently demonstrated that stimulation of gp130 by a combination of soluble interleukin 6 receptor (sIL-6R) and IL-6 but not IL-6 alone significantly stimulates the ex vivo expansion of primitive hematopoietic progenitors and the generation of erythroid cells from human CD34+ cells in the presence of stem cell factor (SCF). Here, we show that gp130 is found low positively on most CD34+ cells, whereas IL-6R is expressed on only 30-50% of these cells. Although most of the colonies generated from FACS-sorted CD34+IL-6R+ cells were granulocyte/macrophage (GM) colonies, CD34+IL-6R- cells gave rise to various types of colonies, including erythroid bursts, GM, megakaryocytes, and mixed colonies in methylcellulose culture with a combination of IL-6, sIL-6R, and SCF. Similar results were obtained in culture supplemented with a combination of IL-3, IL-6, SCF, granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. A limiting dilution analysis of long-term culture-initiating cells (LTC-IC) showed that the CD34+IL-6R- cells contained a larger number of LTC-IC than did the CD34+IL-6R+ cells. In a serum-free suspension of CD34+IL-6R- cells, the addition of sIL-6R to the combination of IL-6 and SCF dramatically increased the total and multipotential progenitors, whereas CD34+IL-6R+ cells failed to do so under the same conditions. These results indicate that most of the erythroid, megakaryocytic, and primitive human hematopoietic progenitors are included in the IL-6R- populations, and the activation of gp130 on these progenitors can be achieved by a complex of IL-6-sIL-6R, but not by IL-6 alone. The present culture system using IL-6, sIL-6R, and SCF may provide a novel approach for ex vivo expansion of human primitive hematopoietic progenitors.

Adult↗
PubMed2002

High-level erythroid lineage-directed gene expression using globin gene regulatory elements after lentiviral vector-mediated gene transfer into primitive human and murine hematopoietic cells.

Lentiviral vectors efficiently transduce primitive human hematopoietic cells and are capable of transferring complex genomes. Vectors were designed with hypersensitive sites containing regulatory elements from the beta-globin locus control region linked to the beta-globin gene promoter to drive expression of the enhanced green fluorescent protein marker to facilitate analysis of the pattern of gene expression in various hematopoietic lineages. Such vectors gave higher level, induced expression in mouse erythroleukemia cells than a previously described vector that utilized an enhancer from the alpha locus and the ankyrin-1 promoter [Moreau-Gaudry, F., Xia, P., Jiang, G., Perelman, N.P., Bauer, G., Ellis, J., Surinya, K.H., Mavilio, F., Shen, C.K., and Malik, P. (2001). Blood 98, 2664-2672]. The addition of gamma-globin intron sequences further augmented vector expression. Expression was also effectively targeted to the erythroid lineage in cultured human cells from peripheral blood and in mouse red blood cells in vivo, although lower levels of expression were also observed in other lineages. Thus, these newly described vectors provide a means to achieve high-level gene expression, predominantly in erythroid cells, an outcome that may have potential therapeutic application.

Animals↗
PubMed2002

An embryonic/fetal beta-type globin gene repressor contains a nuclear receptor TR2/TR4 heterodimer.

We recently described an erythroid epsilon-globin gene repressor activity, which we named DRED (direct repeat erythroid-definitive). We show that DRED binds with high affinity to DR1 sites in the human embryonic (epsilon-) and fetal (gamma-) globin gene promoters, but the adult beta-globin promoter has no DR1 element. DRED is a 540 kDa complex; sequence determination showed that it contains the nuclear orphan receptors TR2 and TR4. TR2 and TR4 form a heterodimer that binds to the epsilon and gamma promoter DR1 sites. One mutation in a DR1 site causes elevated gamma-globin transcription in human HPFH (hereditary persistence of fetal hemoglobin) syndrome, and we show that this mutation reduces TR2/TR4 binding in vitro. The two receptor mRNAs are expressed at all stages of murine and human erythropoiesis; their forced transgenic expression reduces endogenous embryonic epsilony-globin transcription. These data suggest that TR2/TR4 forms the core of a larger DRED complex that represses embryonic and fetal globin transcription in definitive erythroid cells, and therefore that inhibition of its activity might be an attractive intervention point for treating sickle cell anemia.

Adult↗
PubMedDate not supplied

Culture of fetal cells from maternal blood for prenatal diagnosis.

The isolation and analysis of fetal cells from maternal blood would allow non-invasive prenatal genetic screening and diagnosis. Over the past decade, progress has been made towards this goal using various enrichment strategies and analysis by fluorescence in-situ hybridization with chromosome-specific probes and PCR. One method that is currently being explored involves culturing fetal cells. Developing conditions which allow the number of fetal-derived cells to expand in culture and the number of maternally derived cells to be suppressed in culture may lead to a new selection process for obtaining fetal cells. Culturing of fetal cells from maternal blood could make possible conventional metaphase analysis of fetal cells for diagnosis of chromosomal abnormalities.

Blood Cells↗
PubMed2004

Molecular identification of K-CL cotransporter in dog erythroid progenitor cells.

KCC1 cDNA was cloned in dog erythroblasts that had differentiated from peripheral mononuclear cells. The size of the cDNA was 3,258 bp, the same as in pigs, but 3 bp longer than in humans and rodents. The dog KCC1 cDNA encodes for 1,086 amino acid residues with a calculated molecular mass of 120 kDa. The 560 bp cDNA fragment from position 679 to 1,238 in the full length cDNA from the dog erythroblasts was 100% identical to that in the kidney. Hydropathy analysis showed that the structure of dog KCC1 was similar to in other species; 12 trans membrane domains, four glycosylation sites in loop 5, and 17 consensus phosphorylation sites in the cytosol. However, there were variations in dog KCC1 compared to in other species; there was one CK2 phosphorylation site that was found only in dog KCC1. There were also substitutions of amino acids that affect pH sensitivity (His) and change acidic/basic residues or charged residues. In HEK 293 cells transfected with dog KCC1 cDNA (HEK-dKCC1), the Rb influx, which was ouabain-resistant, Cl-dependent, N-ethyl maleimide (NEM)- stimulative and Na-independent, was measured as for K-Cl cotransport, and the influx was found to be increased approximately 3 fold in HEK-dKCC1 compared to in the control. This ouabain-resistant Cl-dependent Rb influx was also volume-sensitive in hyposmotic medium, and the volume-sensitive component was inhibited by furosemide. Thus, the KCC1 cDNA cloned in dog erythroblasts encodes a volume-sensitive K-Cl cotransporter.

Amino Acid Sequence↗
PubMedDate not supplied

Clinical value of bone marrow cultures in childhood pure red cell aplasia.

PURPOSE: We assessed the value of marrow cultures for defining the pathophysiology, diagnosis, and therapeutic response to immunosuppressive therapy in childhood pure red cell aplasia (PRCA). PATIENTS AND METHODS: Patients were evaluated either at diagnosis (n = 23) or at the time of treatment failure (n = 2). Twelve patients had transient erythroblastopenia of childhood (TEC), 4 had Diamont-Blackfan anemia (DBA), and 9 had acquired sustained PRCA (A-Su-PRCA). Bone marrow mononuclear cells were cultured with combination of human recombinant (rhu) erythropoietin (EPO), granulocyte monocyte colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Interleukin 3 (IL-3), either with or without stem cell factor (SCF), and burst forming unit of erythroid (BFU-E) growth was assessed. RESULTS: The combination of growth factors without SCF failed to induce any erythropoiesis (BFU-E < 10/10(5) mononuclear cells) in 10 patients (2 with TEC, 2 with DBA, and 6 with A-Su-PRCA), although the growth of erythroid colonies was substantially lower in the remaining patients than in controls (45.5 +/- 15.4 versus 91.7 +/- 12.7, p < 0.05). Addition of SCF restored erythropoiesis in all but 6 patients (5 with A-Su-PRCA and 1 with DBA). Five of 6 nonresponders did not respond to any immunomodulating therapy; of the 5, 3 had or developed some evidence of myelodysplasia. CONCLUSION: Our data indicate that in vitro colony studies might prove to be a useful diagnostic tool, because erythropoiesis' poor response to growth factors, including SCF, may suggest the diagnosis of myelodysplasia. Moreover, it may have predictive value; in cases of PRCA, regardless of etiology, poor growth of erythropoietic colonies may predict refractoriness to immunomodulating therapy.

Adolescent↗
PubMed2005

Relevance of the criteria commonly used to diagnose myeloproliferative disorder in patients with splanchnic vein thrombosis.

Myeloproliferative disorders (MPD) are reported in 25-65% of patients with splanchnic vein thrombosis (SVT). Diagnostic criteria for MPD have not been fully established in this context. Using clusters of abnormal megakaryocytes in bone marrow (BM) biopsy as a reference standard for Philadelphia negative MPD, we assessed the relevance of other criteria currently recommended for the diagnosis of MPD in SVT (128 consecutive SVT patients). First, usual criteria were compared with BM results: endogenous erythroid colony formation (EEC) was strongly correlated with BM results; splenomegaly, blood cell count, total red cell volume, erythropoietin level and cytogenetic were much less accurate. Then, patients were assigned to three groups according to the combination of BM and EEC findings (group I: both present; group II: both absent; group III: other patients); clinical presentation and outcome were compared in each group. At a mean follow-up of 6.09 +/- 6.6 years, progression to a severe form of MPD occurred in 7 of 31 group I patients (23%), in 1 of 34 group III patients (3%) and 0 of 63 group II patients. The combination of marked splenomegaly and platelet count >200 x 10(9)/l was restricted to groups I and III. In conclusion, in patients with SVT, BM findings and EEC allowed the diagnosis of MPD at risk of aggravation. Marked splenomegaly in association with platelet counts >200 x 10(9)/l constitute a simple index with high specificity but low sensitivity.

Bone Marrow Cells↗