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PubMed2000

Effects of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus infection on hematopoietic precursors of the zebrafish.

The zebrafish Danio rerio is a new model system for studying the genetics of hematopoiesis. To define naturally occurring viruses which could infect and replicate within hematopoietic precursors of the zebrafish, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) were studied. Infection of whole fish with viral supernatants demonstrated infectious replicants for both viruses, indicating that the virus host range includes the zebrafish. In other species, infection with these viruses leads to prominent hematopoietic necrosis of the head kidney, the major site of adult hematopoiesis. We detected a transient toxicity of the virus to hematopoietic precursors and terminally differentiated red cells after viral infections. The kinetics of hematopoietic defects between IHNV and IPNV infection differed; fish infected with either virus, however, recovered by 6 days postinfection. In contrast to other fish infected with the virus, hematocrit did not change appreciably during this time. These studies are the first to demonstrate IHNV and IPNV infection of the zebrafish and reveal the potential for use of such viruses for gene transfer experiments to infect zebrafish hematopoietic cells.

Animals↗
PubMed2000

Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells.

Ferrochelatase, the last enzyme in the heme pathway, chelates protoporphyrin IX and iron to form heme and is mutated in protoporphyria. The ferrochelatase gene is expressed in all tissues at low levels to provide heme for essential heme-containing proteins and is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF-E2 sites, all of which bind their cognate trans-acting factors in vitro. To investigate the role of these elements during erythropoiesis, we introduced expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells. EGFP expression was monitored during hematopoietic differentiation in vitro using flow cytometry. We show that a promoter fragment containing the Sp1 sites, the NF-E2 and GATA elements, was sufficient to confer developmental-specific expression of the EGFP transgene, with an expression profile identical to that of the endogenous gene. In this system the -0.275 kb NF-E2 cis-element is required for erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between -0.375kb and -1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria. There was no effect of the G haplotype on transcriptional activity of the -1.1 kb transgene.

Animals↗
PubMed1998

Erythroid maturation and globin gene expression in mice with combined deficiency of NF-E2 and nrf-2.

NF-E2 binding sites, located in distant regulatory sequences, may be important for high level alpha- and beta-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2-related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2-deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of alpha- and beta-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2-deficient mice is not the result of compensation by Nrf-2.

Amino Acid Sequence↗
PubMed1994

Differential regulation of the two xGATA-1 genes during Xenopus development.

Xenopus laevis provides an established developmental system to study the regulation of cell lineage establishment and the generation of tissue-specific patterns of gene expression. We have isolated from Xenopus erythroid cell RNA two distinct cDNA clones encoding the xGATA-1 transcription factor. We have identified, in erythroid nuclear extracts, the sequence-specific DNA-binding protein they encode. By characterizing the expression patterns for RNA derived from two distinct homologues, we find that the two xGATA-1 genes are differentially regulated. The xGATA-1a mRNA predominates in embryos prior to terminal differentiation of erythroid cells, while the differentiated cells contain RNA derived predominantly from the xGATA-1b gene. Both proteins activate a target globin promoter in transient transfection assays. During early development, GATA-1 transcripts are localized to ventral regions of the embryo. GATA-1 should therefore provide a useful early marker for studying signalling pathways which result in the generation of ventral mesoderm. The differentially regulated genes may be distinct markers for targets of ventral mesoderm induction.

Animals↗
PubMed1991

Aplastic anaemia in systemic lupus erythematosus: a cellular immune mechanism?

Aplastic anaemia is a rare complication of systemic lupus erythematosus (SLE). The mechanism is unclear but is thought to be related to an autoantibody to bone marrow precursors of haematopoiesis. We report a case of SLE related aplastic anaemia in which therapy with methylprednisolone and high dose cyclophosphamide followed by prednisolone and azathioprine resulted in complete clinical and haematological remission. Bone marrow cultures showed inhibition of erythropoiesis when incubated with acute and remission serum. Myeloid colony growth was not affected by either serum. The serum inhibitor we demonstrated was only active in vitro, and we postulate that the mechanism for marrow aplasia may have been an autoimmune cellular process.

Adult↗
PubMed1995

Separation of myeloid and erythroid progenitors based on expression of CD34 and c-kit.

In this report, a novel approach is described to physically separate erythroid progenitors from monocyte and granulocyte progenitors, based on the expression of CD34 and Kit. Using biotin-labeled human Kit ligand (KL) and flow cytometry, Kit was detectable on 2% to 3% of the nucleated cells in rhesus monkey bone marrow. Combination of biotin-KL with CD34 monoclonal antibodies (MoAb) showed that Kit was expressed on subsets of CD34low and CD34pos cells. Our data clearly demonstrate that CD34pos cells are more heterogeneous with respect to Kit expression than observed in studies using Kit MoAb. A small cluster, approximately 7% of the CD34pos cells, expressed CD34 at submaximal levels and stained brightly with biotinylated KL. This CD34pos/kithi fraction contained predominantly erythroid progenitors (burst-forming units-erythroid; BFU-E). The majority of the granulocytic and monocytic progenitors (colony-forming units-granulocyte/macrophage; CFU-GM) were CD34pos/kitmed. Some BFU-E were also detected in the CD34pos/kitmed and CD34low/kitpos fractions at low frequency. In the latter subset, most erythroid colony-forming units (CFU-E) were recovered. Using three-color flow cytometry, we analyzed expression of Kit in relation to that of CD34 and the class II major histocompatibility antigen, RhLA-DR. The most immature bone marrow cells that can be identified in vitro, ie, CD34pos/RhLA-DRlow cells, were kitmed. The CD34pos/kithi and CD34pos/kitneg subsets predominantly contained the more mature RhLA-DRbright cells. Our results demonstrate that erythroid precursors express c-kit at much higher levels than monomyeloid precursors and pluripotent progenitors. The difference in expression levels of CD34 and c-kit can be exploited to isolate BFU-E populations that are virtually devoid of nonerythroid cells.

Animals↗
PubMed1996

Circulating megakaryocytes and their progenitors in early thrombocytopenia in preterm neonates.

Thrombocytopenia is common in sick preterm babies. Despite this, platelet production in thrombocytopenic preterm babies has rarely been assessed. To address this problem we have developed miniaturized assays to study circulating megakaryocyte (MK) progenitors [burst-forming unit (BFU)-MK and colony-forming unit (CFU)-MK], total cultured MK precursors and mature MK, by culturing mononuclear cells purified from 0.5-1 mL of preterm peripheral blood. MK lineage colonies and cells are identified by an anti-IIb/IIIa antibody (CD61). We prospectively studied circulating BFU-MK/CFU-MK, total cultured MK precursors and mature MK in 63 preterm babies (gestational age 24-34 wk). Twenty-six developed early thrombocytopenia (platelets < 150 x 10(9)/L by 48 h), whereas the remaining 37 babies maintained normal platelet counts. Twenty-one of the 26 thrombocytopenic babies were born to mothers with pregnancy-induced hypertension or were growth retarded. At birth, thrombocytopenic babies had severely reduced numbers of all MK precursors compared with nonthrombocytopenic babies: BFU-MK 82 +/- 50 versus 663 +/- 174 colonies/mL, mean +/- SEM; CFU-MK 596 +/- 196 versus 3267 +/- 530 colonies/mL; total MK precursors 97 +/- 30 versus 301 +/- 49 x 10(3) cells/mL and mature MK 8 +/- 2 versus 37 +/- 8 x 10(3) cells/mL, respectively. Thrombocytopenia resolved by d 10 in all babies accompanied or preceded by a recovery to normal numbers of circulating MK progenitors. Eighteen (69%) of the thrombocytopenic babies were also neutropenic (neutrophils < 2 x 10(9)/L); in these babies neutrophil progenitor cells (CFU-granulocyte/monocyte) were also severely reduced compared with the nonthrombocytopenic babies (539 +/- 280 versus 1937 +/- 348 colonies/mL, mean +/- SEM). This indicates that the principal cause of the thrombocytopenia and neutropenia is reduced platelet and neutrophil production occurring as a consequence of reduced numbers of MK and CFU-granulocyte/monocyte progenitors, respectively. Taken together these data suggest the hematologic abnormalities characteristic of newborns born to mothers with pregnancy-induced hypertension or with intrauterine growth retardation are a consequence of dysregulation of fetal hemopoiesis occurring proximal to committed MK and neutrophil progenitors, most likely at the level of the primitive multipotent hemopoietic stem cell.

Case-Control Studies↗
PubMed2002

Development of myelofibrosis in mice genetically impaired for GATA-1 expression (GATA-1(low) mice).

The phenotype induced by the GATA-1(low) (neodeltaHS) mutation is here further characterized by analyzing the hemopoietic system during the aging (up to 20 months) of a GATA-1(low) colony (135 mutants and 40 normal littermates). Mutants expressed normal hematocrit values (Hct = 45.9 +/- 4.0) until 12 months but became anemic from 15 months on (Hct = 30.9 +/- 3.9; P <.05). Anemia was associated with several markers of myelofibrosis such as the presence of tear-drop poikilocytes and progenitor cells in the blood, collagen fibers in the marrow and in the spleen, and hemopoietic foci in the liver. Semiquantitative reverse transcription-polymerase chain reaction showed that growth factor genes implicated in the development of myelofibrosis (such as osteocalcin, transforming growth factor-beta1, platelet-derived growth factor, and vascular endothelial growth factor) were all expressed in the marrow from the mutants at higher levels than in corresponding normal tissues. The GATA-1(low) mutants experienced a slow progression of the disease because the final exitus was not observed until at least 15 months with a probability of survival more favorable than that of W/Wv mice concurrently kept in the animal facility (P <.001, by Kaplan-Meier analysis). In conclusion, impaired GATA-1 expression may contribute to the development of myelofibrosis, and the GATA-1(low) mutants may represent a suitable animal model for the human disease that may shed light on its pathogenesis.

Anemia↗
PubMed1998

Deficient proliferation of myeloid, erythroid, and multipotent progenitor cells in long-term marrow cultures from patients with aplastic anemia treated with immunosuppressive therapy.

By using Dexter-type long-term marrow cultures (D-LTMC), it has been shown previously that hematopoietic progenitor cells (HPC) from patients with aplastic anemia (AA) have a deficient proliferation in vitro. The studies reported to date, however, have focused exclusively on granulomonocytic progenitors and no information exists on erythroid or multipotent progenitor cells. On the other hand, in such studies, the input progenitor cell numbers were significantly below normal levels, thus suggesting that the rapid disappearance of myeloid progenitor cells from AA D-LTMC could also be due, at least in part, to their reduced number at culture onset. In the present study, we have followed the kinetics of myeloid, erythroid, and multipotent progenitors, from 24 AA patients subjected to immunosuppressive therapy (including patients that achieved complete, partial, or no remission at all), throughout a seven-week culture period. For analysis, we grouped all the patients based on their initial content of all three types of progenitors. Thus, we were able to evaluate separately the kinetics of these cells in D-LTMC from patients with normal and subnormal levels of progenitor cells. At the time of marrow sampling, most patients showed decreased levels of HPC; in fact, only 21%, 8%, and 16% of them showed normal levels of myeloid, erythroid, and multipotent progenitors, respectively. When cultured in D-LTMC, HPC from all AA patients analyzed showed a relatively fast disappearance from the cultures. Indeed, myeloid progenitors could be detected for only six weeks, whereas erythroid and multipotent progenitors disappeared from the cultures after two and one weeks of culture, respectively. In contrast, in normal marrow D-LTMC, myeloid, erythroid, and multipotent progenitors were detected for at least seven, five, and three weeks, respectively. Such a deficient proliferation was observed even in cultures of AA patients that contained normal levels of HPC at culture onset. Interestingly, no correlation was found between HPC proliferation in D-LTMC and response to treatment. Thus, the results of this study indicate the presence of a functional in vitro deficiency in the hematopoietic system of patients with AA, including those that achieved partial or complete remission after immunosuppressive treatment. Furthermore, this work suggests that such a proliferation deficiency is more pronounced in erythroid and multipotent progenitors than in their myeloid counterparts.

Adult↗
PubMed1999

Selective modulation of specific protein kinase C (PKC) isoforms in primary human megakaryocytic vs. erythroid cells.

We have investigated the pattern of expression of classical (alpha, betaI, betaII, gamma), novel (delta) and atypical (zeta) protein kinase C (PKC) isoforms during the course of human hematopoietic differentiation along the closely related megakaryocytic and erythroid lineages. Using in situ immunofluorescence analysis, freshly isolated human pluripotent CD34+ hematopoietic progenitor cells expressed detectable amounts of all the PKC isoforms investigated. On the other hand, clear-cut differences in terms of PKC staining were noticed between cells belonging to the erythroid and megakaryocytic lineages, obtained after 9 days of serum-free liquid culture in the presence of specific growth factors. Specifically, 1) erythroid cells showed a very weak expression of PKC-alpha, -betaI, -betaII, and -gamma, while megakaryocytes showed an enhanced expression of all classical PKC isoforms, predominantly confined to the cytoplasm; 2) the expression of PKC-delta increased in the cytoplasmic and nuclear compartments of both erythroid and megakaryocytic cells with respect to CD34+ cells; and 3) atypical PKC-zeta isoform showed a striking accumulation in the nucleus during both erythroid and megakaryocytic differentiation.

Cell Differentiation↗
PubMed1996

Identification of a human erythroid progenitor cell population which expresses the CD34 antigen and binds the plant lectin Ulex europaeus I.

Two and three color flow cytometry of normal human bone marrow was used to identify CD34+ progenitor cells and examine their binding to the plant lectin Ulex europaeus I (Ulex). In normal bone marrow, 48.48 +/- 17.4% of the CD34+ cells bind to Ulex. Two color flow cytometry was used to sort CD34 + cells, and subsets of CD34+ cells, CD34+ Ulex+ and CD34+ Ulex-. These populations were sorted into colony assays to assess myeloid (CFU-GM) and erythroid (BFU-E) progenitors. The CD34+ Ulex+ subset was 84 +/- 14% BFU-E colonies (mean +/- S.D.) and had the highest cloning efficiency of 28 +/- 13%. Three color analysis of CD34+ Ulex+ cells showed staining with other erythroid (CD71, GlyA) antibodies and lack of stain. ing with myeloid (CD13, CD45RA) antibodies. These studies confirmed the erythroid characteristics of this subpopulation.

Antibodies↗
PubMed1996

Effect of the interaction between transforming growth factor beta and erythropoietin on the proliferation of normal erythroid progenitors and leukemic UT-7 cells: action of transforming growth factor beta on the erythropoietin receptor.

The actions of transforming growth factor beta (TGF beta) and erythropoietin (Epo) were studied using normal erythroid progenitors from fetal rat liver and spleen at 18, 19 and 20 days. rhTGF beta 1 inhibited the growth of late BFUe colonies significantly at each age and in both organs in methylcellulose cultures containing 2 U/ml rhEpo. There was no significant inhibition of CFUe proliferation, except for spleen CFUe at 18 days, suggesting different CFUe sensitivities to growth factors at a given fetal age, 18 days, in liver and spleen. The colorimetric MTT assay was used to examine the inhibition of the growth of human leukemic UT-7 cells by TGF beta 1. TGF beta 1 inhibited the proliferation of UT-7 cells in cultures without Epo at 24 h and in cultures with Epo at 24 and 72 h. The specific binding of [125I]Epo to UT-7 surface was decreased by TGF beta 1 without any change in non-specific binding. TGF beta 1 also inhibited the expression of Epo-receptors on UT-7 cells, without changing receptor affinity. The inhibition of hematopoietic progenitor cell growth by TGF beta could involve altering the cell surface expression of growth factor receptors.

Animals↗
PubMed1990

Establishment of an erythroid cell line (JK-1) that spontaneously differentiates to red cells.

The authors established a new hemopoietic cell line (JK-1) from a patient with chronic myelogenous leukemia in erythroid crisis. This JK-1 line predominantly consists of immature cells, but a small number of mature erythroblasts and red cells can be consistently seen without any specific differentiation inducer. The JK-1 cells grow in suspension culture supplemented with human plasma and carry double Philadelphia chromosomes. Hemoglobin staining with benzidine was positive for about 20% of cells and the type of the hemoglobin was for the most part HbF. Surface-marker analysis revealed JK-1 cells positive for glycophorin A, EP-1, and HAE9. The proportion of mature cells was elevated by the addition of delta-aminolevulinic acid. Erythropoietin (EPO) enhanced the growth of JK-1 cells either in the suspension or in methylcellulose semisolid culture. The total number of EPO receptors was 940 per cell, of which 220 sites had an affinity higher than the other 720 sites. This is the first report of an established human erythroid cell line which spontaneously undergoes terminal differentiation.

Antigens, Surface↗
PubMed1990

Rapid reduction of methemoglobin in rat bone marrow erythroid cells.

Methemoglobin reduction was shown to proceed much more rapidly in erythroid cells from rat bone marrow than in rat erythrocytes. Methemoglobin reduction in suspensions of intact, nitrite-treated bone marrow cells does not depend on the presence of glucose in the incubation mixture, even after the cells have been stored in substrate-free medium. 2-Deoxyglucose and iodoacetate prevent the reduction from proceeding to completion. The results suggest that, relative to erythrocytes, immature erythroid cells more efficiently catalyze methemoglobin reduction and more effectively store metabolites which provide electrons for this reaction.

Animals↗
PubMed1994

Spontaneous remission of polycythemia vera: clinical and cell culture characteristics.

A 20-year-old woman presented with polycythemia vera and was treated with phlebotomy alone for eleven years, following which all clinical manifestations of the disease disappeared. The clinical remission with normal physical findings and normal peripheral blood counts has persisted for a further 11 years. Erythroid colony culture results have paralleled the clinical state. Initial bone marrow cultures revealed spontaneous growth of erythroid burst-forming units (BFU-E). Subsequent cultures of peripheral blood cells throughout most of the period of spontaneous clinical remission have revealed little or no spontaneous growth of BFU-E. This suggests a suppression of the abnormal stem cell clone during the period of remission.

Adult↗
PubMed1994

Regulation of erythropoietin production in a case of congenital erythropoietin-dependent pure erythrocytosis.

In a patient with congenital erythropoietin-dependent pure erythrocytosis (EDPE) associated with hypersensitivity of erythroid progenitor cells to erythropoietin (Epo), the investigations planned to elucidate the mechanism responsible for hormone hyperproduction revealed that Epo synthesis was (1) independent of normal oxygen-mediated feedback induced by phlebotomy; (2) not modulated by adenosine as a second messenger (the treatment with the adenosine antagonist theophylline in fact left unchanged the serum Epo levels); and (3) uninfluenced by iron therapy. The Epo dose-response curve for growth of erythroid progenitor was similar to that of three age-matched thalassemia patients with increased serum Epo levels, (sEpo) suggesting that the observed erythroid progenitors hypersensitivity to Epo could represent an ex vivo artifact induced by the increased sEpo levels.

Bloodletting↗