Cultures Of Erythroid Progenitors Research Articles

Effect of the NAMPT Activator P7C3-A20 on γ-Globin Expression in Baboon CD34+ Erythroid Cell Cultures

Increased levels of Fetal Hemoglobin (HbF) reduce the symptoms of sickle cell disease (SCD) and lengthen the life span of patients. New, more effective pharmacological agents that can be safely administered long term to increase HbF levels in SCD patients are highly sought. Expression of the γ-globin gene in adult erythroid cells is normally repressed by the recruitment of multi-protein co-repressor complexes to the γ-globin promoter by sequence-specific DNA binding proteins including BCL11A, LRF1 and TR2/TR4. Enzymes contained within these co-repressor complexes, such as DNMT1, LSD1, G9A, and HDACs, modify the chromatin surrounding the γ-globin promoter by catalyzing repressive epigenetic modifications to both histones and DNA. Small molecule pharmacological inhibitors of these enzymes are potent inducers of HbF in various in cell culture and animal models and in SCD patients, but the use of these drugs in patients has been hindered by their dose-dependent effects on hematopoietic differentiation. An alternative strategy to the use of these pharmacological inhibitors to increase HbF would be to employ pharmacological activators that increase the activity of proteins that positively promote γ-globin expression. Previous studies have shown that pharmacological activators of the Sirtuin 1 protein deacetylase increased γ-globin expression in cultured human CD34+ erythroid progenitor cell cultures (Dai et al; Am J Hematol 92:1177-1186, 2017). Because Sirtuin deacetylase activity is dependent upon nicotinamide adenine dinucleotide (NAD) as a co-factor, we tested the hypothesis that increased concentrations of nicotinamide, an NAD precursor, would also increase γ-globin expression. Baboon bone marrow derived CD34+ erythroid progenitor cells from 4 individual baboons were cultured on AFT024 monolayers for 14 days in the presence and absence of varying concentrations of nicotinamide. Globin chain expression was measured in cell lysates by high performance liquid chromatography (HPLC). Nicotinamide (500μM) appeared to increase γ-globin 2 fold (0.015±0.098 γ/γ+β) compared to untreated controls (0.072±0.04 γ/γ+β; n=4; p<0.08). Because the nicotinamide levels used in this experiments are higher than can be easily achieved by dietary supplementation, additional experiments were performed to test the effect of P7C3-A20, an allosteric activator of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD synthesis, on γ-globin expression. Addition of P7C3-A20 (2.5μM) to CD34+ erythroid progenitor cultures on d1, 4, 7, and 10 increased γ-globin 2.7 fold (0.247±0.10 γ/γ+β) compared to vehicle-treated controls (0.090±0.06 γ/γ+β; n=5; p<0.01). P7C3-A20 treatment did not affect cell viability or growth at concentration< 2.5μM and dose-response experiments showed increased γ-globin in cultures treated with submicromolar concentrations of the drug. Addition of P7C3-A20 to cultures on days 1 and 4 resulted in near maximal stimulation of γ-globin expression with lesser effects when the drug was added on later days (d4 and7 or d7 and 10) strongly suggesting that the drug targets cells at an early stage of differentiation. Additional experiments showed that the effect of P7C3-A20 (2.5μM) in combination with either the DNMT1 inhibitor decitabine (DAC) or the LSD1 inhibitor tranylcypromine (TCP) resulted in a greater than additive effects on γ-globin expression in the absence of cytotoxicity (Figure 1). In conclusion, the NAMPT activator P7C3-A20 increased γ-globin expression in baboon CD34+ erythroid progenitor cells with greater than additive effects in combination with DAC or TCP. P7C3-A20 has potent in vivo effects as a neuroprotective drug in mouse models and non-human primates. Therefore, the potential of this drug for in vivo HbF induction warrants further investigation. [Display omitted] DisclosuresSaunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Lesional Mechanism of Alpha Thalassemias : Towards a Common Mechanism Alpha Thal / Beta Thal

Context and Objectives: Erythropoiesis is a complex process based on the balance between proliferation / maturation and apoptosis, which produces more than 109 red blood cells per day. The lesional mechanism of thalassemia (thal) appears during the synthesis of hemoglobin (Hb) and the classical hypothesis proposes that the clinical severity is proportional to the imbalance of production of alpha / non-alpha-globin chains. However, the severe alpha thal (HbH) phenotypic expression does not adhere to this model since the HbH linked to the association of deletions (alpha0 / alpha +) are less severe than HbH associating deletion and point mutants (alpha0 / alphaCS alpha) while the deficiency is less important, and that there are severe alpha thal linked to homozygosity for an alpha variant without deletion. In order to understand the lesional mechanisms involved, and in light of recent work on beta thal pathophysiology, we have carried out cultures of erythroid progenitors from patients with these two types of « deletional » HbH (alpha0/ -3,7) and « non deletional » HbH (alpha0/ CS) as well as from a homozygous patient of an Agrinio variant (alpha2 : p29 Leu®Pro). We have characterized the amount of alpha globin aggregates in the progenitors of the different patients. In addition, the Agrinio mutation was produced in E. coli to study the consequences of the mutation on interactions between α-HbAgrinio and the alpha-Hemoglobin Stabilizing Protein (AHSP) chaperone.Materials and Methods: Erythroid progenitors were analyzed by multiparametric cytometry (Amnis®) and confocal microscopy to quantify and characterize cytoplasmic aggregates. “Non-thalassemic red cells” (csp) and progenitors from a beta thal major patient were used as negative and positive controls, respectively. Normal α-Hb proteins (α-HbWT) and α-HbAgrinio were produced as glutathione S transferase (GST) fusion proteins associated with their chaperones (GST-AHSP / GST-α -Hb). After cleavage of the GST moiety, the characterization of the AHSP / α-HbAgrinio complex was carried out by fluorescence spectroscopy.Results: The proportion of cells with cellular aggregates analyzed by multiparametric cytometry is proportional to the clinical severity of the patient with an order csp < alpha°/ alpha+ < alpha°/alphaCS < alphaAgrinio alpha / alphaAgrinio alpha < beta thal. The expression in E.coli of α-HbAgrinio is similar to that observed for α-HbWT, but the amount of α-HbAgrinio obtained after purification is greatly decreased indicating a significant instability of α-HbAgrinio. The absorbance spectroscopy shows that the amplitude of the Soret band, characteristic of the heme molecule, is highly decreased for the AHSP / α-HbAgrinio complex compared to that observed for the AHSP / α-HbWT complex. The fluorescence emission spectrum of the AHSP / α-HbAgrinio complex is increased compared to that of the normal complex indicating that the tryptophan AHSP fluorescence signal is less attenuated since fewer heme molecules are present. These results show that α-HbAgrinio is unstable probably due to an alteration of interaction with its chaperone and to a lack of insertion of heme in the globin for this mutant.Conclusion: Recent work has shown that one of the mechanisms responsible for dyserythropoiesis in beta thal major progenitors is the sequestration of the HSP70 chaperone in the cytosol by alpha globin precipitates. HSP70 can therefore no longer protect the nuclear GATA1 factor from cleavage by the activated caspase3 at the beginning of terminal differentiation. This leads to inachieved differentiation and apoptosis. Our results indicate that in HbH and non-deletional alpha thal, a comparable mechanism would be an important element in the pathophysiology unifying the lesional mechanism of two apparently opposite pathologies. Studies in the bacterial model confirm the strong instability of α-HbAgrinio and in vitro studies show that this mutant is partially heminized. DisclosuresNo relevant conflicts of interest to declare.

Proteomic Studies for the Investigation of γ-Globin Induction by Decitabine in Human Primary Erythroid Progenitor Cultures.

Reactivation of γ-globin is considered a promising approach for the treatment of β-thalassemia and sickle cell disease. Therapeutic induction of γ-globin expression, however, is fraught with lack of suitable therapeutic targets. The aim of this study was to investigate the effects that treatment with decitabine has on the proteome of human primary erythroid cells from healthy and thalassemic volunteers, as a means of identifying new potential pharmacological targets. Decitabine is a known γ-globin inducer, which is not, however, safe enough for clinical use. A proteomic approach utilizing isobaric tags for relative and absolute quantitation (iTRAQ) analysis, in combination with high-pH reverse phase peptide fractionation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), was employed to investigate the effects of decitabine treatment. Bioinformatics analysis making use of the Database for Annotation, Visualization and Integrated Discovery (DAVID) was employed for functional annotation of the 192 differentially expressed proteins identified. The data are available via ProteomeXchange with identifier PXD006889. The proteins fall into various biological pathways, such as the NF-κB signaling pathway, and into many functional categories including regulation of cell proliferation, transcription factor and DNA binding, protein stabilization, chromatin modification and organization, and oxidative stress proteins.

Ex vivo culture of adult CD34+ stem cells using functional highly porous polymer scaffolds to establish biomimicry of the bone marrow niche

Haematopoiesis, the process of blood production, occurs from a tiny contingent of haematopoietic stem cells (HSC) in highly specialised three-dimensional niches located within the bone marrow. When haematopoiesis is replicated using in vitro two-dimensional culture, HSCs rapidly differentiate, limiting self-renewal. Emulsion-templated highly porous polyHIPE foam scaffolds were chosen to mimic the honeycomb architecture of human bone. The unmodified polyHIPE material supports haematopoietic stem and progenitor cell (HSPC) culture, with successful culture of erythroid progenitors and neutrophils within the scaffolds. Using erythroid culture methodology, the CD34+ population was maintained for 28 days with continual release of erythroid progenitors. These cells are shown to spontaneously repopulate the scaffolds, and the accumulated egress can be expanded and grown at large scale to reticulocytes. We next show that the polyHIPE scaffolds can be successfully functionalised using activated BM(PEG)2 (1,8-bismaleimido-diethyleneglycol) and then a Jagged-1 peptide attached in an attempt to facilitate notch signalling. Although Jagged-1 peptide had no detectable effect, the BM(PEG)2 alone significantly increased cell egress when compared to controls, without depleting the scaffold population. This work highlights polyHIPE as a novel functionalisable material for mimicking the bone marrow, and also that PEG can influence HSPC behaviour within scaffolds.

Cooperation of germ line JAK2 mutations E846D and R1063H in hereditary erythrocytosis with megakaryocytic atypia

AbstractThe role of somatic JAK2 mutations in clonal myeloproliferative neoplasms (MPNs) is well established. Recently, germ line JAK2 mutations were associated with polyclonal hereditary thrombocytosis and triple-negative MPNs. We studied a patient who inherited 2 heterozygous JAK2 mutations, E846D from the mother and R1063H from the father, and exhibited erythrocytosis and megakaryocytic atypia but normal platelet number. Culture of erythroid progenitors from the patient and his parents revealed hypersensitivity to erythropoietin (EPO). Using cellular models, we show that both E846D and R1063H variants lead to constitutive signaling (albeit much weaker than JAK2 V617F), and both weakly hyperactivate JAK2/STAT5 signaling only in the specific context of the EPO receptor (EPOR). JAK2 E846D exhibited slightly stronger effects than JAK2 R1063H and caused prolonged EPO-induced phosphorylation of JAK2/STAT5 via EPOR. We propose that JAK2 E846D predominantly contributes to erythrocytosis, but is not sufficient for the full pathological phenotype to develop. JAK2 R1063H, with very weak effect on JAK2/STAT5 signaling, is necessary to augment JAK2 activity caused by E846D above a threshold level leading to erythrocytosis with megakaryocyte abnormalities. Both mutations were detected in the germ line of rare polycythemia vera, as well as certain leukemia patients, suggesting that they might predispose to hematological malignancy.

The Tet Dioxygenase Co-Factor Ascorbic Acid Reduces DNA Methylation and Increases Expression of the γ-Globin Gene and Acts in a Combinatorial Manner with HbF-Inducing Drugs Targeting Repressive Epigenetic Modifications

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). Differences in DNA methylation of the γ-globin gene promoter between adult and fetal liver erythroid cells are highly associated with developmental differences in γ-globin expression. Mechanisms that establish and/or modulate DNA methylation of the γ-globin promoter during adult and fetal erythroid differentiation are important in the regulation of γ-globin expression. Pharmacological manipulation of DNA methylation increases HbF in nonhuman primates and SCD patients. Decitabine, a DNA methyltransferase inhibitor that demethylates DNA and increases HbF, is currently in clinical trials. Recent studies have shown that 5-hydroxymethylcytosine (5-hmC), an oxidative product of 5-methylcytosine (5-mC) catalyzed by activity of the TET dioxygenase family, is an intermediate in developmental processes that demethylate DNA. Previously we showed that the γ-globin gene promoter was demethylated during fetal liver erythroid differentiation and to a lesser extent during adult bone marrow (BM) erythroid differentiation. We have investigated the role of 5-hmC in the mechanism of γ-globin gene demethylation by analyzing 5-hmC levels at the HpaII site located at position -51 5’ to the γ-globin transcription start site using a T4-MspI assay in DNA isolated from FACS-purified subpopulations of baboon BM cells enriched for different stages of erythroid lineage differentiation. Levels of 5-hmC were >3 fold higher (p<0.001) in the CD117+CD36+ subpopulation enriched in CFUe (7.15+1.34%) compared to the terminal erythroid precursors (2.33+0.84%) showing that 5-hmC levels are dynamically regulated during erythroid differentiation. Although baboon BM erythroid subpopulations express both TET2 and TET3, higher levels of TET3 were observed in terminal erythroid precursors than in the more primitive CD117+CD36+ subpopulation. High levels of TET3 were also observed in FACS-purified erythroid cells derived from cultured CD34+ baboon BM, human peripheral blood, and human cord blood cells suggesting a role for TET3 in erythroid differentiation. To investigate the relationship between 5-hmC, 5-mC, and γ-globin expression, levels of γ-globin promoter 5-hmC and 5-mC were determined in purified erythroid cells derived from baboon BM CD34+ erythroid progenitors grown in culture conditions resulting in either high (liquid culture) or low (AFT024 murine fetal liver stromal cell line co-culture) levels of γ-globin expression. Levels of γ-globin promoter 5-hmC (mean difference 4.93% total cytosine; p<0.005) and γ-globin chain expression (mean difference γ/γ+β=0.44; p<0.001) were higher and γ-globin promoter 5-mC levels lower (mean difference -25.2% total cytosine; p<0.01) in erythroid progenitors grown in liquid cultures compared to stromal cell line co-cultures. Supplementation of culture media with ascorbic acid, a co-factor of the TET dioxygenases, increased γ-globin expression (mean difference γ/γ+β=0.12; p<0.005) and reduced the level of γ-globin promoter DNA methylation (mean difference -29.0% total cytosine; p<0.001) in baboon BM erythroid progenitors grown in both liquid and co-cultures compared to untreated controls. Ascorbic acid also increased γ-globin expression in cultures derived from human peripheral blood CD34+ progenitors (mean difference γ/γ+β=0.08; p<0.05). In addition, in baboon BM erythroid progenitor cultures ascorbic acid increased γ-globin expression in an additive manner in combination with either the DNA methyltransferase inhibitor decitabine (p<0.001) or the LSD1 inhibitor tranylcypromine (p<0.001) compared to either drug alone, while no combinatorial effects on γ-globin expression were observed with hydroxyurea. These results demonstrate that ascorbic acid is a DNA hypomethylating agent that increases γ-globin gene expression and are consistent with a role for the TET-mediated 5-hmC pathway in the regulation of DNA methylation and expression of the γ-globin gene. Furthermore, these results suggest that vitamin C deficiency, observed in approximately 50% of patients with sickle cell disease, may limit HbF induction by drugs that target epigenetic silencing mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Aconitase Regulation of Erythropoiesis Correlates with a Novel Licensing Function in Erythropoietin-Induced ERK Signaling

BackgroundErythroid development requires the action of erythropoietin (EPO) on committed progenitors to match red cell output to demand. In this process, iron acts as a critical cofactor, with iron deficiency blunting EPO-responsiveness of erythroid progenitors. Aconitase enzymes have recently been identified as possible signal integration elements that couple erythropoiesis with iron availability. In the current study, a regulatory role for aconitase during erythropoiesis was ascertained using a direct inhibitory strategy.Methodology/Principal FindingsIn C57BL/6 mice, infusion of an aconitase active-site inhibitor caused a hypoplastic anemia and suppressed responsiveness to hemolytic challenge. In a murine model of polycythemia vera, aconitase inhibition rapidly normalized red cell counts, but did not perturb other lineages. In primary erythroid progenitor cultures, aconitase inhibition impaired proliferation and maturation but had no effect on viability or ATP levels. This inhibition correlated with a blockade in EPO signal transmission specifically via ERK, with preservation of JAK2-STAT5 and Akt activation. Correspondingly, a physical interaction between ERK and mitochondrial aconitase was identified and found to be sensitive to aconitase inhibition.Conclusions/SignificanceDirect aconitase inhibition interferes with erythropoiesis in vivo and in vitro, confirming a lineage-selective regulatory role involving its enzymatic activity. This inhibition spares metabolic function but impedes EPO-induced ERK signaling and disturbs a newly identified ERK-aconitase physical interaction. We propose a model in which aconitase functions as a licensing factor in ERK-dependent proliferation and differentiation, thereby providing a regulatory input for iron in EPO-dependent erythropoiesis. Directly targeting aconitase may provide an alternative to phlebotomy in the treatment of polycythemia vera.

Modulation of erythroid adhesion receptor expression by hydroxyurea in children with sickle cell disease

We investigated adhesion receptor levels on red blood cells, reticulocytes and erythroid progenitors from children with sickle cell disease treated or not with hydroxyurea. Four groups of patients were investigated: (i) children receiving hydroxyurea for severe vaso-occlusive events (n=26); (ii) untreated children with a history of vaso-occlusive events (n=20); (iii) children with no history of vaso-occlusive events (n=28); and (iv) healthy African controls (n=27). Expression of adhesion receptors was analyzed by flow cytometry with specific mono-clonal antibodies. Reticulocytes and/or red blood cells from the children with sickle cell disease showed significantly higher expression of CD36, alpha 4beta 1, Lu/BCAM than those from controls, whatever the severity of the disease, as well as less marked increases in expression of ICAM-4, CD47 and CD147. Under hydroxyurea treatment, the expression of CD36, alpha 4beta 1 and ICAM-4 (to a lesser extent) was decreased, but surprisingly the expression of Lu/BCAM (and also CD47 and CD147 to a lesser extent) was significantly increased. Alterations of levels of adhesion receptors could be recapitulated in two-phase liquid cultures of erythroid progenitors from controls and untreated children with a history of vaso-occlusive disease, grown in the absence or presence of hydroxyurea. Our results suggest that hydroxyurea acts during erythroid development and modulates adhesion receptor expression and function differently, possibly by acting on gene expression and the signaling cascade leading to receptor activation.

Expression of γ-Globin Is Reactivated by a Novel Mechanism in Baboon CD34+ Erythroid Progenitor Cell Cultures.

Elevated levels of fetal hemoglobin (HbF) reduce the symptoms of sickle cell disease and increase the life span of patients. Future pharmacologic therapies to increase HbF will depend on increased knowledge of the mechanism(s) regulating γ-globin gene expression. To investigate the relationship between DNA methylation, chromatin structure, and γ-globin gene regulation, DNA methylation of 5 CpG sites within γ-globin gene promoter, levels of acetyl-histone H3 and H4 and histone H3 lys4 trimethyl throughout the β-globin gene complex, and the pattern of γ-globin polypeptide chain synthesis were analyzed in1.primary baboon fetal liver (FL) erythroid cells,2.primary baboon bone marrow (ABM) erythroid cells from phlebotomized adults expressing low levels of HbF,3.ABM erythroid cells from adults expressing high levels of HbF following treatment in vivo with the DNA demethylating drug decitabine, and4.erythroid cells expressing high levels of HbF generated from CD34+ baboon BM erythroid progenitors in a liquid culture system.High levels of histone acetylation were associated with the ε- and γ-globin genes and low levels with the β-globin gene in FL erythroid cells while in ABM erythroid cells expressing low levels of HbF (6.69±1.93%) high levels were associated with the β-globin gene and low levels with the γ-globin gene. Histone H3 lys 4 trimethyl was enriched near the γ-globin gene in FL and the β-globin gene in ABM. The γ-globin gene was not methylated in FL. The level of methylation was similar in bled (75.1±8.26%) and normal (82.1±7.51%) ABM erythroid cells. The ratio of expression of 5′ Iγ- and 3′ Vγ-globin genes in the fetus (1.85) differed from bled adults (0.65). Expression of γ-globin was reactivated to similar levels in decitabine-treated baboons (51.5±4.50%) and in erythroid progenitor cell cultures (45.3±12.1%; d14). The Iγ- and Vγ-globin chains were expressed at the characteristic fetal ratio in erythroid progenitor cell cultures (1.76±0.21), but not in decitabine-treated baboons (0.76±0.32). Distribution of histone acetylation and histone H3 lys4 trimethyl was nearly identical in erythroid cells expressing high levels of HbF from decitabine-treated animals and from erythroid progenitor cultures and was characterized by high levels of histone H3 lys4 trimethyl associated with both the active γ- and β-globin genes and enrichment of histone H3 and H4 acetylation near the ε-, γ-, and β-globin genes. HbF reactivation in decitabine-treated baboons was associated with a reduction in the level of γ-globin gene methylation (33.5±9.43% dmC). Surprisingly, the level of γ-globin gene methylation in erythroid cells purified from erythroid progenitor cell cultures (79.5±9.80% dmC; d11) synthesizing high levels of γ-globin was not significantly different than in ABM erythroid cells expressing <2% HbF. These results demonstrate that reactivation of γ-globin expression in erythroid progenitor cell cultures is uniquely characterized by the Iγ/Vγ-globin chain ratio of fetal development. In contrast to the absence of γ-globin gene methylation in FL and reduced levels associated with reactivation of HbF following decitabine treatment, the level of γ-globin gene methylation in erythroid progenitor cell cultures was the same as in ABM. Our data strongly suggests that reactivation of γ-globin gene expression in erythroid progenitor cell cultures is achieved through a novel mechanism not dependent on loss of DNA methylation.

Fetal Hemoglobin Induction in Baboons (P. Anubis) Following Admistration of a Novel Decitabine Dinucleotide (S110) Compound.

Increased fetal hemoglobin (HbF) levels can ameliorate the symptoms of patients with sickle cell disease and increase their life span. The DNA methyltransferase (DNMT) inhibitor decitabine increases HbF in non-human primates (baboons) and sickle cell patients and offers great promise as an effective therapeutic agent for the treatment of this disease. The goal of this investigation was to develop and test new compounds that inhibit DNMT, increase HbF, and offer significant advantages compared to decitabine such as increased stablity, reduced cytotoxicity, and a lower potential for mutagenicity. S110 is decitabine-guanine dinucleotide developed by Supergen, Inc., that is cytosine deaminase resistant and more stable in plasma than decitabine. Experiments were performed to compare the effect of S110 and decitabine on γ-globin expression in human erythroid progenitor cell cultures derived from CD34+ peripheral blood cells. Cultures were treated with decitabine (1 X 10−6 M) or S110 (1 and 5 X 10−6 M) on day 8. Forty eight hours later, RNA was purified for real time PCR analysis of ε- and γ-globin gene expression by the ΔΔCT method using α-globin as a control. Effects on globin polypeptide chain expression was determined by HPLC analysis of freeze-thaw lysates prepared 72 hours following drug addition. Both S110 and decitabine increased expression of ε-globin and γ-globin mRNA and the γ/γ + β polypeptide chain ratio (see Table). Effect of S110 on ε-and γ-globin mRNA and the γ / γ + β chain ratio in Human Erythroid Progenitor Cultures (n=4)DRUGCONCENTRATIONε-globin mRNA (Fold increase)γ-globin mRNA (Fold increase)Globin chain ratio (γ/γ + β)Untreated3 +/− 3.3Decitabine1 X 10-6 M3 +/− 24.82 +/− 2.88 +/− 3.2S1101 x 10-6 M5 +/− 4.24 +/− 1.18 +/− 1.9S1105 X 10−69 +/− 34.535 +/− 1.52 +/− 2.9The effect of S110 on HbF levels in vivo was then tested in non-human primates. Two baboons (PA 7256; PA 7470, P. anubis) were phlebotomized for eight days to achieve a HCT of 20 followed by treatment with S110 (1mg/kg/d; sc) for ten days. Pretreatment HbF levels were 3.1% (PA 7256) and 3.5% (PA 7470). Following S110 treatment, peak HbF levels of 46.1% (PA 7256) and 75.5% (PA 7470) were attained that were similar to levels observed in animals treated with equivalent molar doses of decitabine. Bisulfite sequence analysis showed that the level of DNA methylation of 5 CpG sites within the γ-globin promoter in purified BM erythroblasts following S110 administration was significantly lower (41.5%, PA 7256; 16.3%, PA 7470) compared to erythroblasts from bled, untreated baboons (70.8%; n=4). Pharmacokinetics was also evaluated in parallel to assess systemic exposure. These data demonstrate that S110, a newly developed decitabine-guanine dinucleotide, effectively increased HbF and reduced DNA methylation in cultured human erythroid progenitor cells and in experimental non-human primates.

Hypoxia alters progression of the erythroid program

Hypoxia can induce erythropoiesis through regulated increase of erythropoietin (Epo) production. We investigated the direct influence of oxygen tension (pO(2)) in the physiologic range (2-8%) on erythroid progenitor cell differentiation using cultures of adult human hematopoietic progenitor cells exposed to decreasing (20% to 2%) pO(2) and independent of variation in Epo levels. Decreases in hemoglobin (Hb)-containing cells were observed at the end of the culture period with decreasing pO(2). This is due, in part, to a reduction in cell growth and, at 2% O(2), a marked increase in cell toxicity. Analysis of the kinetics of cell differentiation showed an increase in the proportion of cells with glycophorin-A expression and Hb accumulation at physiologic pO(2). Cells were characterized by an early induction of gamma-globin expression and a delay and reduction in peak levels of beta-globin expression. Overall, fetal Hb and gamma-globin expression were increased at physiologic pO(2), but these increases were reduced at 2% O(2) as cultures become cytotoxic. At reduced pO(2), induction of Epo-receptor (Epo-R) by Epo was decreased and delayed, analogous to the delay in beta-globin induction. The oxygen-dependent reduction of Epo-R can account for the associated cytotoxicity at 2% O(2). Epo induction of erythroid transcription factors, EKLF, GATA-1, and SCL/Tal-1, was also delayed and decreased at reduced pO(2), consistent with lower levels of Epo-R and resultant Epo signaling. These changes in Epo-R and globin gene expression raise the possibility that the early increase of gamma-globin is a consequence of reduced Epo signaling and a delay in induction of erythroid transcription factors.

Everolimus Is a Potent Inducer of Erythroid Differentiation and γ-Globin Gene Expression in Human Erythroid Cells

We studied the effects of everolimus on the erythroid differentiation of human leukaemic K562 cells and on the cultures of erythroid progenitors derived from the peripheral blood of β-thalassaemia patients. A quantitative real-time reverse-transcription polymerase chain reaction assay was employed for the quantification of the accumulation of globin mRNAs. The results obtained demonstrate that everolimus is a potent inducer of the erythroid differentiation of K562 cells. Erythroid induction is associated with an increase in α- and γ-globin mRNAs. In erythroid precursor cells from 4 β-thalassaemia patients, everolimus stimulated a preferential increase (ranging from 1.8- to 7.2-fold) in γ-globin mRNA. Only minor effects were observed on the expression of α-globin genes. These results, in our opinion, are of interest as this compound is already employed in clinical trials as an anti-rejection agent following kidney transplantation. These data suggest that everolimus warrants further evaluation as a potential therapeutic drug in the treatment of β-thalassaemia.

Reduced-Intensity Transplants - Red Cell Antigens, Antibodies, and Blood Product Usage.

Introduction Reduced-intensity conditioning (RIC) transplants currently comprise 25–30% of allogeneic transplants in Europe. Previously reported data suggested delayed donor erythroid engraftment and isohaemaglutinin disappearance in RIC transplants, Additionally, reduced blood product usage has been reported. We prospectively studiedthe kinetics of donor red cell engraftment;kinetics of recipient- and donor-type isohaemaglutinnins, andusage of blood products, in 100 consecutive patients undergoing haemopoietic cell transplantation (HCT) for haematological malignancy at a single centre:Material and Methods 59 patients had RIC conditioning (37 FB8C (Fludarabine Busulphan 8mg/kg Campath1H), 22 FMC, Melphalan 140mg/m2), and 41 standard (Std) conditioning (13, TBI 14.4Gy BuCyCampath, 4 BuCy200, 16 BEAM-Campath1H, 8 FB16C, Busulphan 16mg/kg). Emergence of donor red cells and isohaemaglutinine titres were detected by Diamed columns (sensitivity approx. 5%). Bone marrow samples on days 28, 56, and 100 post transplant were used for semisolid culture of erythroid progenitors (BFU-E).Results. In 50/100 patients (26 RIC and 24 Std) emergence of donor red cell antigens was detectable by serology (19 major and 8 bidirectional ABO mismatches; 22 patients were informative due to a Rh antigen mismatch (10 D, 8 E, 2 C, and 2 c); One had a M-mismatch). Median time for the appearance of donor red cells was 66 days for Std conditioning and 56 days for RIC (not significant). Within the RIC group, there was no significant difference between ABO vs. Rh - mismatched transplants. Similarly, the kinetics of disappearance of recipient-type isohaemaglutinin was not different between RIC vs. Std conditioning (median 82 days for RIC and 69 days for Std). Appearance of donor-type isohaemaglutinin, followed in patients with minor or bidirectional ABO mismatch, was delayed regardless of the conditioning regime: only 3/24 patients had detectable donor-derived anti-A/B after a median of 336 days post transplant.Patients receiving a RIC regime required significantly less platelet transfusions: 14.4 vs 7.8 units (P= 0.02). There was a trend toward a higher number of RBC units transfused to patients receiving Std than RIC regimes: 14 vs 9 units (P= 0.06), but with substantial variation between different standard regimes.BFU counts showed a linear increase but the mean count was still subnormal by day 100. within the RIC group, BFU-E counts were not significantly different between ABO identical vs. non-ABO identical transplants. DNA from BFU-E colonies from 87 patient- points has been stored, to be compared with serology and blood lymphoid/myeloid chimerism data.There were no cases of Pure Red Cell Aplasia (PRCA) in the RIC patients with major ABO mismatch. Only one case of autoimmune haemolytic anaemia was identified in the entire study group, after a median follow up of 345 days.Conclusions Donor red cell emergence and disappearance of recipient-type anti-A/B are not delayed wit RIC regimes that we use. In keeping with this is the low incidence of post-transplant PRCA. Donor-type anti-A/B production is suppressed, probably due to the effect of Campath 1H, accounting for the absence of severe “passenger lymphocyte” haemolysis. Red cell and platelet usage are markedly decreased in RIC transplants due to reduced myelosuppression.

Recurrent De Novo Mutations of EPOR Gene in Primary Congenital Polycythemia.

Primary familial and congenital polycythemia (PFCP) is a rare inherited disorder presenting with elevated red blood cell mass, elevated hemoglobin concentration and low levels of erythropoietin. Ten mutations in the erythropoietin receptor (EPOR) gene to date have been associated with PFCP. All of these mutations result in deletion of 59 to 82 amino acids from the carboxy terminal of EpoR which has been shown to contain a negative regulatory domain. Here, we describe a 2-year old boy of French-Canadian descent presenting with polycythemia and splenomegaly. Sequencing of the EPOR gene showed the proband to be heterozygous for a G to A transition in nucleotide 6002 (G6002A). The mutation generates a stop codon instead of tryptophan at amino acid 439, leading to a truncated EpoR. The association of the G6002A mutation in the EPOR gene with PFCP has been previously described in a large Finnish family (dela Chapelle et al., Proc Natl Acad Sci USA 1993; 90: 4495) and in a 16-year old boy of English descent (Percy et al., Br J Hematol 1998; 100:407). The G6002A mutation in both cases was considered to have arisen independently based on differences in a microsatellite polymorphism in the 5′UT of EPOR and the absence of the mutation in the immediate family of the English boy. We studied our proband's parents for the G6002A EPOR mutation and did not find it. Their parentage was confirmed using 24 different microsatellite markers. This indicates that the G6002A mutation in the proband arose de novo. Since the mutation arose de novo, in vitro methycellulose cultures of erythroid progenitors isolated from peripheral blood of the proband were grown in the presence of increasing concentrations of Epo to rule out genetic mosaicism. The erythroid progenitors showed hypersensitivity to Epo as is characteristic of PFCP. However, we did not find evidence supportive of genetic mosaicism as all 70 BFU-E colonies analyzed were heterozygous for the G6002A mutation. Previously, two other polycythemia-associated EPOR mutations, 5974insG (Sokol et al., Blood 1995; 86:15) and 5959G>T (Kralovics et al., Am J Hematol 2001; 68:115) were shown to have arisen de novo. This case is thus the fourth instance out of 13 reported cases of polycythemia-associated EPOR mutations that has arisen de novo. Because of the rarity of polycythemia-associated EPOR mutations, their frequent de novo occurrence suggests that these mutations do not have a selective advantage but are detrimental. Their possible association with increased risk of thromboembolic and atherosclerotic disease due to chronically augmented Epo signaling is being explored by ongoing clinical studies.

Effects of rapamycin on accumulation of α‐, β‐ and γ‐globin mRNAs in erythroid precursor cells from β‐thalassaemia patients

We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 beta-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the beta-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to gamma-globin mRNA accumulation, being only minor for beta-globin and none for alpha-globin mRNAs. The ability of rapamycin to preferentially increase gamma-globin mRNA content and production of HbF in erythroid precursor cells from beta-thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti-rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in beta-thalassaemia and sickle cell anaemia.

Maladie inflammatoire cryptogénétique de l’intestin et thrombocytémie latente : nouvelle étiologie de thrombose des veines hépatiques ?

It has been suggested in previous studies, that inflammatory bowel disease can induce hepatic vein thrombosis. However, the main weakness of those studies was the lack of extensive screening of prothrombotic factors. We report an unusual association of severe inflammatory bowel disease, hepatic vein thrombosis and latent platelet proliferation disorder. Early treatment with heparin and cyclosporin administered intravenously induce a remission of intestinal disease and total disappearance of hepatic vein thrombosis. Extensive screening excluded the classical causes of hepatic vein thrombosis, inherited coagulation disorders and latent polycythemia by culture of erythroid progenitors without added erythropoietin. However, using recent bone marrow culture conditions, we observed spontaneous colony formation of megakaryocyte progenitors revealing latent thrombocythemia. In summary, progress in the diagnosis of haemostasis disorders, will probably confirm that one or more additional predisposing prothrombotic factors are needed to the development of hepatic vein thrombosis in inflammatory bowel disease. This hypothesis could explain the scarcity of this complication in inflammatory bowel disease. Moreover, this case-report suggests that administration of heparin at the onset of the thrombosis may induce a complete regression of the thrombus in hepatic veins.

Maturation and developmental stage-related changes in fetal globin gene expression are reproduced in transiently transfected primary adult human erythroblasts

A novel system is described, which uses transfection of primary human erythroblasts for the study of gene regulation in differentiating human red cells. This system includes a protocol for liquid culture of erythroid progenitors, which reproduces developmental differences in globin gene expression found between adult and cord blood as well as the maturation-related changes in fetal globin levels observed in adult cells. Reporter constructs driven by globin gene promoters were electroporated into adult and cord blood-derived erythroblasts at different time points during culture. Both the developmental stage and maturation-related differences in endogenous fetal and adult globin gene expression could be reproduced by the transiently transfected reporter constructs. Transfection of primary human erythroblasts during differentiation provides a previously unavailable opportunity to study dynamic aspects of erythropoiesis.

The glucocorticoid receptor is a key regulator of the decision between self-renewal and differentiation in erythroid progenitors.

During development and in regenerating tissues such as the bone marrow, progenitor cells constantly need to make decisions between proliferation and differentiation. We have used a model system, normal erythroid progenitors of the chicken, to determine the molecular players involved in making this decision. The molecules identified comprised receptor tyrosine kinases (c-Kit and c-ErbB) and members of the nuclear hormone receptor superfamily (thyroid hormone receptor and estrogen receptor). Here we identify the glucocorticoid receptor (GR) as a key regulator of erythroid progenitor self-renewal (i.e. continuous proliferation in the absence of differentiation). In media lacking a GR ligand or containing a GR antagonist, erythroid progenitors failed to self-renew, even if c-Kit, c-ErbB and the estrogen receptor were activated simultaneously. To induce self-renewal, the GR required the continuous presence of an activated receptor tyrosine kinase and had to cooperate with the estrogen receptor for full activity. Mutant analysis showed that DNA binding and a functional AF-2 transactivation domain are required for proliferation stimulation and differentiation arrest. c-myb was identified as a potential target gene of the GR in erythroblasts. It could be demonstrated that delta c-Myb, an activated c-Myb protein, can functionally replace the GR.

Fetal hemoglobin induction by acetate, a product of butyrate catabolism [see comments

Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma- chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF- containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two- carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

Fetal Hemoglobin Induction by Acetate, a Product of Butyrate Catabolism

Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma-chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF-containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two-carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.