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Sphingolipids are membrane and bioactive lipids that are required for many aspects of normal mammalian development and physiology. However, the importance of the regulatory mechanisms that control sphingolipid levels in these processes is not well understood. The mammalian ORMDL proteins (ORMDL1, 2 and 3) mediate feedback inhibition of the de novo synthesis pathway of sphingolipids by inhibiting serine palmitoyl transferase in response to elevated ceramide levels. To understand the function of ORMDL proteins in vivo, we studied mouse knockouts (KOs) of the Ormdl genes. We found that Ormdl1 and Ormdl3 function redundantly to suppress the levels of bioactive sphingolipid metabolites during myelination of the sciatic nerve. Without proper ORMDL-mediated regulation of sphingolipid synthesis, severe dysmyelination results. Our data indicate that the Ormdls function to restrain sphingolipid metabolism in order to limit levels of dangerous metabolic intermediates that can interfere with essential physiological processes such as myelination.

Sickle cell disease (SCD) and β-thalassemia are caused by structural abnormality or inadequate production of adult hemoglobin (HbA, α2β2), respectively. Individuals with either disorder are asymptomatic before birth because fetal hemoglobin (HbF, α2γ2) is unaffected. Thus, reversal of the switch from HbF to HbA could reduce or even prevent symptoms these disorders. In this study, we show that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is one factor that could accomplish this goal. IGF2BP1 is a fetal factor that undergoes a transcriptional switch consistent with the transition from HbF to HbA. Lentivirus delivery of IGF2BP1 to CD34+ cells of healthy adult donors reversed hemoglobin production toward the fetal type in culture-differentiated erythroid cells. Analogous studies using patient-derived CD34+ cells revealed that IGF2BP1-dependent HbF induction could ameliorate the chain imbalance in β-thalassemia or potently suppress expression of sickle β-globin in SCD. In all cases, fetal γ-globin mRNA increased and adult β-globin decreased due, in part, to formation of contacts between the locus control region (LCR) and γ-globin genes. We conclude that expression of IGF2BP1 in adult erythroid cells has the potential to maximize HbF expression in patients with severe β-hemoglobin disorders by reversing the developmental γ- to β-globin switch., Graphical Abstract

Here we investigated in primary human erythroid tissues a downstream element of the heterochronic let-7 miRNA pathway, the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), for its potential to affect the hemoglobin profiles in human erythroblasts. Comparison of adult bone marrow to fetal liver lysates demonstrated developmental silencing in IGF2BP1. Erythroid-specific overexpression of IGF2BP1 caused a nearly complete and pancellular reversal of the adult pattern of hemoglobin expression toward a more fetal-like phenotype. The reprogramming of hemoglobin expression was achieved at the transcriptional level by increased gamma-globin combined with decreased beta-globin transcripts resulting in gamma-globin rising to 90% of total beta-like mRNA. Delta-globin mRNA was reduced to barely detectable levels. Alpha-globin levels were not significantly changed. Fetal hemoglobin achieved levels of 68.6 ± 3.9% in the IGF2BP1 overexpression samples compared with 5.0 ± 1.8% in donor matched transduction controls. In part, these changes were mediated by reduced protein expression of the transcription factor BCL11A. mRNA stability and polysome studies suggest IGF2BP1 mediates posttranscriptional loss of BCL11A. These results suggest a mechanism for chronoregulation of fetal and adult hemoglobin expression in humans.

Improvements in ex vivo generation of enucleated red blood cells are being sought for erythroid biology research, toward the ultimate goal of erythrocyte engineering for clinical use. Based upon the high levels of iron-saturated transferrin in plasma serum, it was hypothesized that terminal differentiation in serum-free media may be highly dependent on the concentration of iron. Here adult human CD34(+) cells were cultured in a serum-free medium containing dosed levels of iron-saturated transferrin (holo-Tf, 0.1-1.0 mg/ml). Iron in the culture medium was reduced, but not depleted, with erythroblast differentiation into haemoglobinized cells. At the lowest holo-Tf dose (0.1 mg/ml), terminal differentiation was significantly reduced and the majority of the cells underwent apoptotic death. Cell survival, differentiation and enucleation were enhanced as the holo-Tf dose increased. These data suggest that adequate holo-Tf dosing is critical for terminal differentiation and enucleation of human erythroblasts generated ex vivo in serum-free culture conditions. Published 2013. This article is a US Government work and is in the public domain in the USA.

Erythroid biology research involving rhesus macaques has been applied to several topics including malaria, hemoglobinopathy and gene therapy research. However, analyses of the rhesus red blood cells are limited by the inability to identify and sort those cells in research blood samples using flow cytometry. Here it is reported that the BRIC 6 hybridoma clone raised to the human erythroid surface molecule (referred to as CD233, Band 3, AE1, or SLC4A1) produces cross-reactive and erythroid-specific antibodies for flow cytometric detection and sorting of rhesus macaque erythrocytes.

Iron deficiency (ID) affects billions of persons worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. Iron homeostasis in mammals is largely maintained by expression of a small hepatic protein named hepcidin.1 Hepcidin itself is regulated at the transcriptional and posttranscriptional levels by multiple extracellular signals related to the iron supply and the erythropoietic demands of the host, as well as by inflammatory states. ID results in a significant reduction in serum hepcidin as measured by mass spectroscopy2 and more recently by hepcidin immunoassay.3 Even in the absence of ID, anemia-related hypoxia4 and erythropoietin production inhibit hepcidin expression.5 In addition, in anemic states characterized by ineffective erythropoiesis, such as thalassemia and congenital dyserythropoiesis,6 inappropriate hepcidin down regulation results in severe iron overload. It was recently demonstrated that human erythroblasts secrete a cytokine named growth differentiation factor 15 (GDF15) that contributes to the inhibition of hepcidin in pathologic conditions.7 As a p53 target gene,8 it was proposed that GDF15 is secreted from apoptotic erythroblasts in clinical settings of ineffective erythropoiesis. However, GDF15 involvement in iron regulation in other clinical settings is largely unknown.

In vivo, inhibition of fetal hemoglobin (HbF) expression in humans around the time of birth causes the clinical manifestation of sickle cell and beta-thalassemia syndromes. Inhibition of HbF among cultured cells was recently described by the adenosine derivative molecule named SQ22536. Here, a primary cell culture model was utilized to further explore the inhibition of HbF by adenosine derivative molecules. SQ22536 demonstrated down-regulation of growth and HbF expression among erythroblasts cultured from fetal and adult human blood. The effects upon HbF were noted in a majority of cells, and quantitative PCR analysis demonstrated a transcriptional mechanism. Screening assays demonstrated that two additional molecules named 5′-deoxy adenosine and 2′,3′-dideoxy adenosine had effects on HbF comparable to SQ22536. Other adenosine derivative molecules, adenosine receptor binding ligands, and cAMP-signaling regulators failed to inhibit HbF in matched cultures. These results suggest that structurally related ribofuranose-substituted adenosine analogues act through an unknown mechanism to inhibit HbF expression in fetal and adult human erythroblasts.

In thalassemia, deficient globin-chain production during erythropoiesis results in anemia. Thalassemia may be further complicated by iron overload (frequently exacerbated by blood transfusion), which induces numerous endocrine diseases, hepatic cirrhosis, cardiac failure and even death. Accumulation of iron in the absence of blood transfusions may result from inappropriate suppression of the iron-regulating peptide hepcidin by an erythropoietic mechanism. To test this hypothesis, we examined erythroblast transcriptome profiles from 15 healthy, nonthalassemic donors. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor-beta superfamily, showed increased expression and secretion during erythroblast maturation. Healthy volunteers had mean GDF15 serum concentrations of 450 +/- 50 pg/ml. In comparison, individuals with beta-thalassemia syndromes had elevated GDF15 serum levels (mean 66,000 +/- 9,600 pg/ml; range 4,800-248,000 pg/ml; P < 0.05) that were positively correlated with the levels of soluble transferrin receptor, erythropoietin and ferritin. Serum from thalassemia patients suppressed hepcidin mRNA expression in primary human hepatocytes, and depletion of GDF15 reversed hepcidin suppression. These results suggest that GDF15 overexpression arising from an expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression.

Increases in fetal hemoglobin have been identified after birth in several clinical settings associated with stressed or malignant erythropoiesis. To better understand the relationship between the expression of this fetal protein and growth, donated human erythroid progenitor cells were cultured in the presence of erythropoietin (EPO) plus the growth-modifying cytokine stem cell factor (SCF), and several growth-related signaling pathways were interrogated. Only the MEK1/2 inhibitor (PD98059) demonstrated significant effects on fetal hemoglobin. In the absence of PD98059, levels of fetal hemoglobin averaged 27.4% ± 7.9% in EPO+SCF compared with 1.26% ± 1.7% in EPO alone (P = .02). A linear dose response in levels of fetal hemoglobin to PD98059 was detected (0.16 μM = 27.13%, 0.8 μM = 19.6%, 4 μM = 12.2%, 20 μM = 1.54%). Western blot analyses revealed that SCF was required for phosphorylation of MEK and p44MAPK in this setting, and quantitative polymerase chain reaction demonstrated a significant increase in γ-globin mRNA. Particular perturbations of growth-related signaling may also function to activate tissue-specific genes normally expressed during fetal development. This concept may be relevant for the development of new treatment rationales for beta hemoglobinopathies.

Transcriptional profiles of cultured primary human erythroid cells were examined to identify those genes involved in the control of erythroid growth during the terminal phase of maturation. Our in silico screening strategy indicated that a hypoxia-inducible proapoptotic member of the Bcl-2 gene family called Nix is expressed during erythropoiesis. We next performed Northern blot analyses and determined that the 1.4-kb Nix transcript is expressed at lower levels in erythroleukemia cells than reticulocytes. Polymerase chain reaction (PCR)–based transcriptional patterning confirmed the increased expression of Nix during human erythropoiesis with a pattern similar to that of Bcl-xL and glycophorin A and opposite that of Bcl-2. Western blot analyses revealed Nix protein levels that were lower than expected due to increased proteosomal degradation. The expression of Nix and Bcl-xL proteins decreased relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control on the removal of erythropoietin (EPO) from the culture medium. Immunocytochemical analyses demonstrated a similar perinuclear mitochondrial expression pattern for both proteins in hemoglobinized precursors. On the basis of these data, we propose that the proapoptotic factor Nix is a highly regulated effector of growth during terminal erythroid maturation.

Multiple components of the highly conserved let-7 cascade are developmentally regulated during the fetal-to adult transition of human ontogeny. Previous studies showed the let-7 miRNAs to be correlated with the developmentally regulated expression of fetal hemoglobin (HbF) in humans. Ex vivo manipulation of the main known let-7 regulator (LIN28) and downstream let-7 targets (IGF2BP1, IGF2BP3 and HMGA2) have shown variant increases in HbF levels (ranging from around 15-68% HbF) among cultured adult human erythroid cells. In addition, IGF2BP1 over-expression (OE) in cord blood erythroblasts raised HbF levels to an average of 90% compared to 55% in control transductions. Remarkably, in the fetal environment, the manipulation of the let-7 cascade has profound effects on HbF levels (cord blood control for the OE vectors: 57.4 ± 8.6%; LIN28A-OE: 90.8 ± 1.7%, p = 0.0003; IGF2BP3-OE: 79.0 ± 3.8%, p=0.001; HMGA2-OE: 73.4 ± 3.1, p=0.014; control for the tough decoy (TuD) vector: 51.6 ± 5.7%; let-7a-TuD: 90.3 ± 2.5%, p=0.001). As such, we hypothesized that the variable HbF effects in the adult cells may be due to independent or parallel effects from each component of the let-7 cascade since LIN28, IGF2BP1, IGF2BP3 and HMGA2 are expressed at very low levels or undetectable in the erythroid compartment after the fetal-to-adult transition. For these studies, lentiviral transduction of human CD34+ cells were investigated in erythropoietin-supplemented serum-free cultures for 21 days and compared to donor-matched control transductions. Based upon its important role in globin gene regulation, mRNA and protein expression patterns of the transcription factor BCL11A were investigated by RT-qPCR and Western blot at day 14 as a mechanism for HbF regulation by the let-7 cascade components. BCL11A mRNA was significantly down-regulated after LIN28A-OE (control: 4.1.E+03 ± 8.2.E+02 copies/ng, LIN28A-OE: 1.7.E+03 ± 1.2.E+03 copies/ng, p=0.04) and let-7a -TuD (control: 1.7E+03 ± 4.5E+02 copies/ng, let-7a-TuD: 4.3E+02 ± 1.8E+02 copies/ng, p=0.003), while no significant differences were observed after IGF2BP1-OE (control: 5.42.E+03 ± 1.77.E+03 copies/ng, IGF2BP1-OE: 4.38.E+03 ± 2.57.E+02 copies/ng, p=0.375), IGF2BP3-OE (control: 5.64.E+02 ± 2.68.E+02 copies/ng, IGF2BP3-OE: 6.70.E+02 ± 3.47.E+02 copies/ng, p=0.694) and HMGA2-OE (control: 4.26E+02 ± 8.18E+01 copies/ng, HMGA2-OE: 2.84E+02 ± 1.48E+02 copies/ng, p=0.104). Interestingly, the protein levels of BCL11A were consistently down-regulated only by LIN28A-OE, let-7a -TuD and IGF2BP1-OE, with minimal changes observed after IGF2BP3-OE and HMGA2-OE. To investigate which elements of the let-7 cascade are required for the fetal hemoglobin effects in adult cells, the expression levels of each component were compared with HbF effects after lentiviral-mediated transduction of LIN28A-OE, let-7a -TuD, IGF2BP1-OE, IGF2BP3-OE, or HMGA2-OE. RT-qPCR analyses were performed at day 14 and HPLC at day 21. HbF levels for the control transductions were less than 5%. Following LIN28A-OE (HbF: LIN28A-OE: 34.8 ± 2.7%, p=0.028), both IGF2BP1 and IGF2BP3 mRNA levels remained below the detection limits, while HMGA2 was slightly down-regulated. After let-7a -TuD (HbF: let-7a-TuD: 31.0 ± 5.0%, p=0.09), LIN28A and HMGA2 remained at background levels while IGF2BP1 and IGF2BP3 were below the detection limits. Following IGF2BP1-OE (HbF: IGF2BP1-OE: 64.6 ± 3.4%, p=0.02), LIN28A, IGF2BP3, and HMGA2 remained undetectable or at background levels. After IGF2BP3-OE (HbF: IGF2BP3-OE: 22.8 ± 4.4%, p=0.021), LIN28A, IGF2BP1 and HMGA2 also remained unchanged at background or undetectable levels. Finally, after HMGA2-OE (HbF: HMGA2-OE: 13.5 ± 1.5%, p=0.01), LIN28A, IGF2BP1 and IGF2BP3 remained below the detection limits. These results were confirmed by Western analysis, with the exception of HMGA2, which was consistently increased at the protein level following LIN28A-OE, let-7a -TuD, IGF2BP1-OE and IGF2BP3-OE. In summary, these results demonstrate that several components the let-7 cascade share a downstream mechanism of BCL11A protein suppression in human erythroblasts. However, the variability of BCL11A and HbF effects as well as the ability of LIN28A-OE, let-7a-TuD and HMGA2-OE to increase HbF in the absence of IGF2BP1 and IGF2BP3 suggest both shared and independent mechanisms for hemoglobin regulation by the let-7 cascade during the human fetal-to-adult switch. Disclosures No relevant conflicts of interest to declare.

Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with sickle cell disease (SCD) and the beta-thalassemias. It was recently reported that increased expression of LIN28 proteins or decreased expression of its target let-7 miRNAs enhances HbF levels in cultured primary human erythroblasts from adult healthy donors. Here LIN28A effects were studied further using erythrocytes cultured from peripheral blood progenitor cells of pediatric subjects with SCD. Transgenic expression of LIN28A was accomplished by lentiviral transduction in CD34(+) sickle cells cultivated ex vivo in serum-free medium. LIN28A over-expression (LIN28A-OE) increased HbF, reduced beta (sickle)-globin, and strongly suppressed all members of the let-7 family of miRNAs. LIN28A-OE did not affect erythroblast differentiation or prevent enucleation, but it significantly reduced or ameliorated the sickling morphologies of the enucleated erythrocytes.

OBJECTIVE: Among older children with sickle cell anemia, leukocyte counts, hemoglobin, and reticulocytosis have previously been suggested as disease severity markers. Here we explored whether these blood parameters may be useful to predict early childhood disease severity when tested in early infancy, defined as postnatal ages 60-180 days. STUDY DESIGN: Data from fifty-nine subjects who were followed at Children's National Medical Center's Sickle Cell Program for at least three years was retrospectively analyzed. Comparisons were made between white blood cell counts, hemoglobin and reticulocyte levels measured at ages 60-180 days and the clinical course of sickle cell anemia during infancy and childhood. RESULTS: A majority of subjects had demonstrable anemia with increased reticulocytosis. Only increased absolute reticulocyte levels during early infancy were associated with a significant increase in hospitalization during the first three years of life. Higher absolute reticulocyte counts were also associated with a markedly shorter time to first hospitalizations and a four-fold higher cumulative frequency of clinical manifestations over the first three years of life. No significant increase in white blood cell counts was identified among the infant subjects. CONCLUSIONS: These data suggest that during early infancy, increased reticulocytosis among asymptomatic SCA subjects is associated with increased severity of disease in childhood.

Recent studies demonstrated that IGF2BP1 over-expression (IGF2BP1-OE) in adult erythroblasts has robust effects on fetal hemoglobin (HbF; >65% of the total globin levels), accompanied by reversal of the beta-like globin expression patterns to a fetal-like phenotype. Here we investigated if another member of the insulin-like growth factor 2 mRNA-binding protein family, IGF2BP3, also has potential for HbF regulation that may be useful for therapeutic application among patients with beta-hemoglobin disorders. The developmental pattern and expression levels for IGF2BP3 were initially determined in cord blood versus adult blood CD34(+) samples cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. RNA samples were collected at culture day 14 and expression levels were measured by qRT-PCR. IGF2BP3 showed a developmentally regulated expression pattern similar to IGF2BP1 (IGF2BP1: cord blood: 1.3.E+03 ± 4.3.E+02 and adult blood: below detection limits; IGF2BP3: cord blood: 5.8.E+02 ± 2.4.E+02 and adult blood: below detection limits). These results were confirmed in vivo by comparing human fetal liver to adult bone marrow samples (IGF2BP1: fetal liver: 3.5.E+02 ± 5.7.E+01, adult bone marrow: below detection limits and IGF2BP3: fetal liver: 2.0.E+01 ± 2.7.E+00, adult bone marrow: below detection limits). To investigate the effects of IGF2BP3 upon erythropoiesis and globin expression, a lentiviral construct was designed for expression of IGF2BP3 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (IGF2BP3-OE), with a matched empty vector control. Transductions were performed in CD34(+) cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Over-expression of IGF2BP3 was confirmedby qRT-PCR and Western blot analyses at culture day 14. IGF2BP3-OE cells maintained their ability to differentiate and enucleate ex vivo compared to donor-matched controls. The expression levels of globin genes were evaluated at culture day 14 by qRT-PCR and showed that IGF2BP3-OE caused significantly increased gamma-globin expression levels compared to control transductions (control: 7.7.E+05 ± 1.7.E+05; IGF2BP3-OE: 8.4.E+06 ± 3.2.E+06; p=0.018). Consistent with increased gamma-globin, HbF rose to moderately high levels upon IGF2BP3-OE (control: 4.0 ± 2.1%; IGF2BP3-OE: 18.6 ± 1.0%; p=0.0021). In addition, the expression pattern of the erythroid transcription factor BCL11A was investigated by qRT-PCR at culture day 14 and no significant changes were observed (control: 5.6.E+02 ± 2.7.E+02; IGF2BP3-OE: 6.7.E+02 ± 3.5.E+02; p=0.694). However, minor decreases in BCL11A protein levels were detected by Western analysis. These results demonstrate that IGF2BP3 is developmentally regulated in human erythroid tissues with silencing during the fetal-to-adult transition. However, the effects of IGF2BP3-OE on HbF levels were less robust when compared to IGF2BP1-OE in cultured adult erythroblasts. Disclosures No relevant conflicts of interest to declare.

In thalassemia and other iron loading anemias, ineffective erythropoiesis and erythroid signaling molecules are thought to cause inappropriate suppression of a small peptide produced by hepatocytes named hepcidin. Previously, it was reported that the erythrokine GDF15 is expressed at very high levels in thalassemia and suppresses hepcidin expression. In this study, erythroblast expression of a second molecule named twisted gastrulation (TWSG1) was explored as a potential erythroid regulator of hepcidin. Transcriptome analyses suggest TWSG1 is produced during the earlier stages of erythropoiesis. Hepcidin suppression assays demonstrated inhibition by TWSG1 as measured by quantitative polymerase chain reaction (PCR) in dosed assays (1-1000 ng/mL TWSG1). In human cells, TWSG1 suppressed hepcidin indirectly by inhibiting the signaling effects and associated hepcidin up-regulation by bone morphogenic proteins 2 and 4 (BMP2/BMP4). In murine hepatocytes, hepcidin expression was inhibited by murine Twsg1 in the absence of additional BMP. In vivo studies of Twsg1 expression were performed in healthy and thalassemic mice. Twsg1 expression was significantly increased in the spleen, bone marrow, and liver of the thalassemic animals. These data demonstrate that twisted gastrulation protein interferes with BMP-mediated hepcidin expression and may act with GDF15 to dysregulate iron homeostasis in thalassemia syndromes.

Candidate drugs are being sought for the suppression of human erythropoiesis. Cl-IB-MECA [2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide] is a derivative of adenosine that inhibits the growth of leukaemic cell lines. To determine the effects of Cl-IB-MECA upon erythropoiesis, studies were performed by using an ex vivo culture system of primary human CD34+ cells. Cl-IB-MECA suppressed erythroblast growth and maturation at doses >/=50 mumol/l through a mechanism of cell cycle inhibition and accumulation of cells in the G1/G0 phase. These findings demonstrate that Cl-IB-MECA inhibits human erythropoiesis, and suggest that further consideration of this drug is warranted for patients with erythrocytosis or polycythemia syndromes.

RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets, Affymetrix HG-U133 arrays were hybridized with probe generated from the reticulocyte total RNA (blood obtained from 14 umbilical cords and 14 healthy adult humans). Among the cord and adult reticulocyte profiles, 698 probe sets (488 named genes) were detected in each of the 28 samples. Among the highly expressed genes, promoter analyses revealed a subset of transcription factor binding motifs encoded at higher than expected frequencies including the hypoxia-related arylhydrocarbon receptor repressor family. Over 100 probe sets demonstrated differential expression between the cord and adult reticulocyte samples. For verification, the array expression patterns for 21 genes were confirmed by real-time PCR (correlation coefficient 0.98). Only four transcripts (MAP17, FLJ32009 , ARRB2, and FLJ27365 ) were identified as being upregulated in the adult blood transcriptome. Further analysis revealed that the lipid-regulating protein MAP17 was present in the membrane fraction of adult erythrocytes, but not detected in cord blood erythrocytes. Combined with other clinical and experimental data, these reticulocyte transcriptome profiles should be useful to better understand the molecular bases of terminal erythroid differentiation, hemoglobin switching, iron metabolism and malarial pathogenesis.

Reticulocytosis begins early in life in infants with sickle cell anemia (HbSS, SCA) as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). Almost one quarter of children with SCA will have at least one episode of splenic sequestration before the age of 2 years, and immature (CD36+) reticulocytes released into circulating blood may contribute to this early pathology because of splenic trapping of those cells. Additionally, clinical studies have identified that absolute reticulocyte counts (ARC) greater than 200 K/µL during early infancy (60-196 days of age) may be a useful marker to identify SCA infants who are at the highest risk for SCA-associated events during childhood. The objective of this study was to determine if expression of the adhesion marker named CD36 changes with ARC levels during early childhood in pediatric SCA patients. Infants between the ages of 6 and 12 months were enrolled in a prospective, longitudinal, observational study after written consent was obtained from a parent or legal guardian. Participants were not receiving hydroxyurea or monthly blood transfusions. After consent was provided, peripheral blood was obtained during study visits at steady state and analyzed within 48 hours of collection and storage at 40 C. Steady state was defined as a sample drawn at least 30 days following an acute event and at least 60 days following a blood transfusion. Hematologic data, including ARC and HbF levels, were measured using CLIA approved methods. F-cells were enumerated with flow cytometry following intracellular staining with a HbF fluorescent antibody. Reticulocytes were identified by flow cytometry after thiazole orange staining and were further quantitated with CD36, CD45, CD71 and CD235a staining. CD36+ reticulocytes were defined here by surface CD36 detected at levels greater than two standard deviations above unstained controls. Calculations were made to determine the proportion of total circulating reticulocytes that expressed surface CD36+ as well as the absolute number of circulating CD36+ reticulocytes/microliter of whole blood. Correlations were calculated to determine the relationships of ARC with HbF, F-cells, and other hematologic data, while two-tailed t-tests were used to compare means. Mean age at enrollment was 278±70 days and 62.5% of the participants were male. None of the eight enrolled patients had SCA complications or hospitalizations prior to enrollment. Mean HbF and F-cell levels were 40.8±11.1% and 92.8±8.8%, respectively. Mean hemoglobin was 10±0.6 g/dL and ARC 189.1±69.4 K/µL and mean number of absolute CD36+ red blood cells was 29.3±19.1 K/µL. ARC increased over time in 6 of the 8 (75%) participants for the study observation period. ARC was positively correlated with the proportion of CD36+ reticulocytes (r=0.78, p The incidence of splenic sequestration is highest in the first two years of life. Results from this cohort of SCA infants reveal that both ARC levels and CD36+ reticulocytes increase during the first 2 years of life, and the increases in ARC are positively correlated with the proportion and absolute number of circulating CD36+ reticulocytes in peripheral blood. This relationship provides a potential mechanism for the previously reported relationship of early reticulocytosis with increased risk of future SCA complications and events. As such, longitudinal studies should be pursued to determine if increasing CD36+ reticulocytes during early childhood serves as a marker or contributes to early SCA-related pathologies including splenic sequestration. Disclosures No relevant conflicts of interest to declare.

We systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factorbeta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 ± 1.0%; EPO + TGF-B = 10.8 ± 1.9%; EPO + SCF = 19.1 ± 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 ± 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 ± 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 ± 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation. (Blood. 2005;105:387-393)

Sir, Anaemia is a common haematological abnormality in patients with cancer. The anaemia of chronic disease or inflammation is thought to be a result of induction of the iron-regulatory hormone, hepcidin, by inflammatory cytokines, especially interleukin 6 (IL-6) [1]. Elevation of hepcidin by IL-6 leads to hypoferraemia associated with anaemia and iron-restricted erythropoiesis [2]. A recent study showed that patients with certain early-stage malignancies are less prone to developing anaemia of chronic disease [3]. Among more than 1400 men with prostate cancer, the odds of developing anaemia before diagnosis were not increased. We hypothesized that the paucity of significant anaemia during the early stages of prostate cancer may be due to potentially balanced effects of hepcidin-regulating cytokines produced by prostate cancer cells. Like IL-6, growth differentiation factor 15 (GDF15), also a cytokine, is overexpressed in prostate cancer tissues [4,5]. However, unlike IL-6, GDF15 suppresses hepcidin expression [6]. Hepcidin, IL-6 and GDF15, as well as iron and haematological variables were therefore examined to determine the potential role of iron-restricted anaemia in men with prostate cancer. In all, 29 men with prostate cancer were recruited for this preliminary study and grouped according to the presence or absence of metastatic bone disease. All studies were conducted at the National Institutes of Health (NIH) and were approved by both the Office of Human Subjects Research and the Institutional Review Board. Mild anaemia (defined here as a haemoglobin [Hb] level of 10.0–13.7 g/dL) was detected in >80% of men in both groups (Fig. 1). As expected, Hb levels were lower in men with metastatic bone disease, with a mean (SD) Hb of 11.6 (1.4) g/dL (P = 0.009) and haematocrit (Hct) of 36.5 (4.1)% (P = 0.025), than in men without metastasis, with a mean (SD) Hb of 12.8 (0.8) g/dL and Hct of 39.4 (2.1)%. None of the 29 men had received erythropoietin or transfused blood, and only one had received iron supplements within the 6 weeks prior to the study. The iron parameters were within the normal range in >80% of the men. FIG. 1 Hepcidin, IL-6, and GDF15 levels in men with prostate cancer with or with no bone metastasis. Serum concentrations of (A) hepcidin, (B) IL-6, and (C) GDF15 from men with prostate cancer, without (−, 14 men) or with (+, 15 men) bone metastasis. ... ELISA-based assay systems were used to examine the serum levels of hepcidin, GDF15 and IL-6 [6, 7]. As shown in the attached Figure 1A, 23 of 29 men studied had measurable hepcidin within the assay’s normal range (10–298 ng/mL) [7]. Five men had hepcidin levels at or near the lower limit of the assay (5.0 ng/mL). Serum hepcidin level increased beyond the normal range only in one man. In the absence of bone metastasis, hepcidin levels clustered in the low normal range, with a mean value of 42 ng/mL. Even with bone metastasis, over half of the men maintained hepcidin levels of

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that bind and regulate target messenger RNAs (mRNAs). The let-7 family consists of twelve genes encoding nine highly conserved miRNAs that are involved in developmental timing events in multicellular organisms. Previous studies showed regulation during the fetal-to-adult transition in the erythroid lineage with significant increases in let-7 miRNAs from adult compared to umbilical cord blood reticulocytes (1). Further studies indicated that reduced expression of let-7 in adult CD34+ cells by “sponge” targeting the miRNA family seed region caused increased fetal hemoglobin (HbF), but the mean level of HbF remained less than 20% of the total hemoglobin (2). Increased expression of LIN28A (a major regulator of all let-7 miRNAs) caused greater increases in HbF (greater than 30% of the total) in cultured erythrocytes from pediatric patients with HbSS genotype (3). However, these studies did not address the potential for targeting an individual let-7 miRNA family member to regulate HbF expression. For this purpose, we initially determined the expression levels of mature let-7 family members in purified cell populations sorted from peripheral blood. The total levels of let-7 miRNAs in peripheral blood cells were as follows: reticulocytes: 1.7E+08 ± 1.0E+08 copies/ng; neutrophils: 2.0E+07 ± 1.1E+07 copies/ng; lymphocytes: 1.1E+07 ± 6.2E+06 copies/ng and monocytes: 3.5E+06 ± 2.7E+06 copies/ng. Among the individual species, let-7a was identified as a predominantly expressed let-7 family member in reticulocytes. As such, we hypothesized that specifically targeting let-7a may be sufficient to regulate HbF levels. To study the effects of let-7a miRNAs upon erythropoiesis and globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a was compared with empty vector controls. Transductions were performed in CD34+ cells from five adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Down-regulation of let-7a was confirmed by Q-RT-PCR at day 14 (control: 1.4E+07 ± 2.4E+06 copies/ng; let-7a-TuD: 1.6E+06 ± 4.6E+05 copies/ng; p=0.0003). Cell proliferation and differentiation were comparable in let-7a-TuD versus control transductions. Expression levels of globin genes were evaluated upon let-7a-TuD by Q-RT-PCR. Let-7a-TuD transductions caused significantly increased gamma-globin mRNA expression levels compared to control transductions (control: 1.2E+06 ± 6.8E+05 copies/ng; let-7a-TuD: 1.1E+07 ± 4.5E+06 copies/ng; p=0.004). HPLC analyses at the end of the culture period demonstrated robust increases in HbF levels after let-7a-TuD transduction (HbF control: 4.7 ± 0.6%; let-7a-TuD: 38.2 ± 3.8%; p=0.00003). In addition, the expression patterns of the erythroid transcription factors BCL11A, KLF1 and SOX6 were investigated. Let-7a-TuD decreased BCL11A mRNA expression levels (control: 1.7E+03 ± 4.5E+02 copies/ng; let-7a-TuD: 4.3E+02 ± 1.8E+02 copies/ng; p=0.003), but major changes in KLF1 or SOX6 were not detected. In summary, we report here that the let-7 miRNA family is differentially expressed in purified cell populations from adult human blood, and that let-7a is a predominantly expressed species in reticulocytes. Further, targeted reduction of let-7a in erythroblasts is sufficient to cause robust increases in gamma-globin mRNA expression and HbF to mean levels around 35-40% of the total hemoglobin produced. Targeting of individual let-7 genes or RNA transcripts may be useful for therapeutic induction of HbF expression in patients with sickle cell disease or other beta-hemoglobinopathies. 1) Noh SJ et al. J Transl Med. 7:98 (2009). 2) Lee YT et al. Blood. 122:1034-41 (2013). 3) Vasconcellos JF et al. Blood. 122: Abstract 313 (2013). Disclosures No relevant conflicts of interest to declare.

Hemoglobin switching is largely complete in healthy infants by 6 months of age. In infants with sickle cell anemia (HbSS, SCA), reticulocytosis begins early in life as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). Previous studies demonstrated that patients with an ARC greater than 200 K/uL during early infancy (60-196 days of age) were at the highest risk for SCA-associated events. 1,2 The objective of this study was to determine if ARC is related to HbF levels in a cohort of pediatric SCA patients. A convenience sample of 106 children with SCA between the ages of 1 month and 20 years who were not receiving hydroxyurea or monthly blood transfusions were enrolled in this observational study [42 (39.6%) less than 1 year of age (28-362 days old), 46 (43.4%) between the ages of 1 and 10 years, and 18 (17.0%) between 10 and 20 years old]. After consent and assent were provided, discarded peripheral blood was obtained during routine clinic visits at steady state and analyzed within 48 hours of collection and storage at 40C. Steady state was defined as a sample drawn at least 30 days following an acute event and at least 60 days following a blood transfusion. Hematologic data, including ARC and HbF levels, were measured using CLIA approved methods. F-cells were enumerated by flow cytometry following intracellular staining with a fluorescent antibody directed against HbF. Correlations were calculated to determine the relationships of ARC with HbF, F-cells, and other hematologic data, while two-tailed t tests were used to compare means. Initial studies compared groups based upon ARC greater than or equal to 200 K/uL (ARC≥200) during infancy because of the previously reported utility of this threshold as a predictive marker for SCA severity.1 Over one third of the infants less than 1 year of age (n=16) had an ARC≥200. Mean HbF and F-cell levels were significantly lower in the ARC≥200 group when compared to the ARC Enrolled patients were also grouped according to age and comparisons were made between ARC and HbF or F-cell levels. HbF and F-cell levels were negatively correlated to ARC in the infant subgroup (r=-0.696, p=3.1E-07 and r=-0.795, p=0.000, respectively). HbF and F-cell levels from children between the ages of 1 and 10 years were inversely related to the ARC, but the correlation was less significant (r=-0.626, p=3.3E-06 and r=-0.538, p=1.2E-04, respectively). The inverse relationship was no longer present in the oldest group of patients (HbF vs. ARC r=-0.203, p=0.420 and F-cells vs. ARC, r=-0.258, p=0.302). According to both analytical approaches described here, increased ARC is associated with decreased HbF and F-cell levels in infants with SCA. Less robust negative correlations are maintained through age 10 years, but no significant correlation was identified in adolescence and young adulthood. Overall, the data suggest that increased ARC levels may identify SCA infants who manifest a more rapid or greater loss of fetal hemoglobin during the later stages of the HbF-to-HbS switching phenomenon. Meier ER, Byrnes C, Lee YT, et al. Increased reticulocytosis during infancy is associated with increased hospitalizations in sickle cell anemia patients during the first three years of life. PLoS One 2013; 8(8):e70794. doi: 10.1371/journal.pone.0070794.Meier ER, Wright EC, Miller JL. Reticulocytosis and anemia are associated with an increased risk of death and stroke in the newborn cohort of the Cooperative Study of Sickle Cell Disease. Am J Hematol 2014 May 31; doi: 10.1002/ajh.23777. [Epub ahead of print] Disclosures No relevant conflicts of interest to declare.

Because abnormal erythroid differentiation is the most common manifestation of the myelodysplastic syndromes (MDS), it was hypothesized that erythroid gene expression may be used to illustrate myelodysplastic transcription patterns. Ten normal bone marrow aspirates (NBM) were first analyzed using an erythroid-focused cDNA array to define steady-state transcription levels. Proliferation and differentiation gene subsets were identified by statistically significant differences between NBM and erythroleukemia gene expression. Next, cDNAs from 5 separate MDS aspirates were studied: refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation (RAEB-T), and RAEB-T/secondary MDS. A distinct pattern of significantly increased proliferation-associated and reduced differentiation-associated gene activity was established for MDS.

Highly-conserved LIN28 proteins regulate certain developmentally-timed events in multicellular organisms by decreasing the levels of let-7 miRNAs. It was recently reported that increased expression of LIN28 proteins or decreased expression of the target let-7 miRNAs in human erythroblasts cultured from healthy adult humans causes increased fetal hemoglobin expression. Here LIN28A expression in sickle cell donors’ cells was explored for its potential to regulate fetal and sickle hemoglobin, and to affect the morphological sickling of the mature erythrocytes. After obtaining consent and assent, CD34(+) cells from five pediatric research subjects with HbSS genotype (ages 9-16 years old) were harvested from discarded whole blood following partial manual exchange transfusions. Transgenic expression of LIN28A was accomplished using lentiviral transduction of human CD34(+) sickle cells cultivated ex vivo in serum-free medium for a total of 21 days. On culture day 14, LIN28A over-expression (LIN28A-OE) was confirmed by Q-RT-PCR (control: 8.6E+00 ± 8.1E+00 copies/ng, LIN28A-OE: 2.3E+05 ± 2.1E+05 copies/ng) and Western blot analyses. Erythroblast differentiation and terminal maturation were not affected by LIN28A-OE. Enucleation, as assessed by thiazole orange (TO) staining, was equivalent between the LIN28A-OE cells and control transductions (LIN28A-OE enucleation 40.8 ± 17.0% compared to control 49.9 ± 23.4%, p=0.19). LIN28A-OE strongly suppressed all members of the let-7 family of miRNAs, with average reductions from 66% to 96% for let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. LIN28A-OE caused reduced expression of BCL11A, a known repressor of gamma-globin gene expression. Gamma-, beta (sickle)- and alpha-globin mRNA levels were also investigated by Q-RT-PCR. Gamma-globin mRNA expression levels were significantly increased in LIN28A-OE samples (control: 2.0E+06 ± 7.0E+05 copies/ng, LIN28A-OE: 2.0E+07 ± 6.0E+06 copies/ng, p=0.006), and beta (sickle)-globin mRNA significantly decreased in LIN28A-OE samples (control: 2.0E+07 ± 5.2E+06 copies/ng, LIN28A-OE: 1.6E+07 ± 6.3E+06 copies/ng, p=0.024). Differences in alpha-globin mRNA expression were not statistically significant. Hemoglobin chromatography (HPLC) demonstrated that LIN28A-OE significantly increased the proportion of fetal hemoglobin (HbF control: 10.8 ± 7.1%, LIN28A-OE: 40.1 ± 14.0%; p=0.003) that was balanced by a significant decrease in the proportion of sickle hemoglobin. HbA was not detected. For investigation of the sickling phenotype, enucleated [TO(-)] sickle erythrocytes from LIN28A-OE and control transductions of two subjects’ cells were sorted at the end of the culture period into duplicate tissue culture wells. The sorted erythrocytes were incubated in hypoxia (2% oxygen) for 16 hours, and imaged using inverted microscopy within three minutes after removal from the hypoxia incubator. Four random microscopic field images from each well were acquired. Blinded observers then scored the images from the control and LIN28A-OE transductions according to non-sickled versus sickled morphologies. Cultured erythrocytes from the control transductions demonstrated 86.3 ± 9.5% with sickled morphologies. By comparison, a significant reduction in sickling morphology was observed in the LIN28A-OE cells (56.2 ± 23.1% sickled morphologies; p=0.000009). These results demonstrate that transgenic expression of LIN28A during ex vivo erythropoiesis causes increased gamma-globin gene and protein expression balanced with decreased beta (sickle)-globin at levels that are sufficient to ameliorate hypoxia-related sickling of mature erythrocytes. Disclosures: No relevant conflicts of interest to declare.

Abstract 2131 Apoptotic cell death is a central feature of beta-thalassemia and other anemias caused by ineffective erythropoiesis. In this study, markers of apoptosis were examined using an ex vivo model of human beta-thalassemia to explore the cause(s) and timing of erythroblast cell death. Beta-globin gene and protein expression were knocked down by lentiviral transduction of beta-globin shRNA (beta-KD) in adult human CD34+ cells. Beta-globin mRNA (Control=4.0E+07 ± 1.4E+06 copies/ng, vs. beta-KD=2.5E+06 ± 1.6E+06 copies/ng, p=0.014) and protein were reduced in beta-KD cells by 90% compared to controls while alpha globin expression was maintained. The effects of imbalanced globin chain synthesis were therefore studied according to the stage of erythroblast maturation. Erythroid progenitor cell commitment and proliferation occur over the first two weeks in culture. During the third week of culture (days 14–21), the proerythroblasts undergo terminal differentiation with the characteristic loss of CD71 from the cell surface. Prior to culture day 14, phenotypic analyses demonstrated low levels of apoptosis in beta-KD and control cultures. On culture day 14, a small but significant increase in active caspase 3 was detected in the beta-KD cells compared to controls (beta-KD=4.0±1.0%, Control=0.7±0.3%, p=0.02) suggesting that apoptosis was initiated during the early stages of terminal maturation. Increases in annexin V staining did not achieve statistical significance on day 14 beta-KD cells (beta-KD=13.1±3.9%, Control=7.6±2.2%, p=0.16). By culture day 18, when orthochromic normoblasts are the most prevalent population in control cultures, a large population of apoptotic cells was detected in the beta-KD. The beta-KD erythroblasts demonstrated further increases in active caspase 3 (beta-KD=11.4±2.2% vs. Control=1.1±0.1%, p=0.014), as well as significant increases in surface annexin V (beta-KD=75.8±3.3%, vs. Control=35.9±12.7%, p=0.024). Western analysis of culture day 18 beta-KD membranes demonstrated a marked increase in alpha-chains compared with culture day 14. Since cleaved caspase 3 was increased in beta-KD cells near the beginning of their terminal maturation and prior to the accumulation of alpha chains in the cell membranes, other triggers of apoptosis were investigated. Mitochondrial superoxide is a reactive species that can be generated by iron or other mitochondrial toxins. Increased levels of mitochondrial superoxide cause apoptosis. The cultured erythroblasts were stained with MitoSOX, a cell-permeable dye that specifically detects mitochondrial superoxide. On culture day 11, mitochondrial superoxide was barely detectable in beta-KD and control cell populations (beta-KD=5.2±3.3% vs. Control=3.6±2.9%, p=0.051). Thereafter, the superoxide detection was increased significantly in beta-KD cells on culture day 14 (beta-KD=54.2±6.7% vs. Control=9.1±2.9%, p=0.003), and culture day 18 (beta-KD=81.1±3.2% vs. Control=34.6±3.8%, p=0.007). Oxidation of cellular membranes by hemichromes and free alpha chains damages thalassemic erythrocytes and their precursors. These data suggest that imbalanced globin chain synthesis also triggers apoptosis during the early stages of terminal differentiation by increasing superoxide formation in the mitochondria. Disclosures: No relevant conflicts of interest to declare.

Abstract 683 Mitochondrial specialization in erythroblasts is important for efficient heme synthesis, with defects or reduced expression of several mitochondrial proteins causing anemia. Trafficking kinesin binding 2 (TRAK2) is known to participate in mitochondrial movement along microtubule by interacting with kinesin motor protein and making a complex with Miro that is localized on the mitochondrial outer membrane. Transcriptome data suggest that TRAK2 is highly and specifically expressed in early erythroid cells. Here the role of TRAK2 was studied among human CD34+ cells that were grown in ex vivo serum-free cultures supplemented with erythropoietin (EPO, total culture period 21 days). Quantitative PCR studies indicated that TRAK2 expression is highly regulated during erythropoiesis. Its expression pattern was nearly identical to aminolevulinate synthase 2, the erythroid specific enzyme for the committed step of the heme biosynthetic pathway, and mitoferrin 1, the erythroid specific mitochondrial iron transporter. Western analyses revealed that TRAK2 protein is detected as a doublet band with molecular weights of 130kD and 105kD. Mitochondrial co-localization of TRAK2 was verified by confocal microscopy in TRAK2-overexpressing K562 cells. To study a potential role of TRAK2 in erythropoiesis, TRAK2 expression was reduced in cultured human erythroid cells using lentiviral shRNA transduction. TRAK2 knockdown (TRAK2-KD) was confirmed by Western analysis in K562 cells. In primary erythroblasts, TRAK2-KD caused slight reduction of CD36+ immature erythroblasts at culture day 7 prior to the addition of EPO (CD36+ population 58% in control vs 40% in TRAK2-KD). After the addition of erythropoietin to the culture medium, TRAK2-KD severely restricted erythroblast proliferation (5.0 million cells/ml in control vs 0.25 million cells/ml in TRAK2-KD on culture day 18). Flow cytometric analyses showed that 90% in control cultures. Annexin-V staining indicated that more than 90% of cells had undergone apoptosis by day 14. These data suggest that TRAK2 expression is required for erythroid differentiation. As such, defects in TRAK2 expression should be considered in cases of unexplained anemia. The data also support the notion that mitochondrial location or mobility within erythroblasts may be important for iron trafficking or heme synthesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 1085 Reversal of anemia is the major target of thalassemia research, but studies of the molecular and cellular basis of the ineffective erythropoiesis of thalassemia are limited by access to donor progenitor cells. Here we demonstrate that thalassemic erythropoiesis may be recapitulated ex vivo by reducing the expression of hemoglobin in cultured CD34+ cells. Using lentiviral transduction of progenitor cells obtained from three healthy adult human donors, shRNA molecules were screened for their ability to reduce beta-globin gene and protein expression over 21 days in culture. Cells transduced with a scrambled vector served as donor-matched controls. Among the screened shRNA, one named HBB caused a consistent and significant reduction in beta-globin mRNA and protein. Beta-globin mRNA was reduced to levels Disclosures: No relevant conflicts of interest to declare.

Abstract 1041 In addition to reduced hemoglobin production, iron deficiency causes anemia though poorly understood defects in erythropoiesis that reduce the number of erythroid cells released from the marrow space. To study the negative effects of iron deficiency upon erythropoiesis, CD34+ cells from three healthy human donors were cultured in serum-free media supplemented with transferrin-bound iron. Dosed titrations of holo-transferrin (1.2, 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 mg/ml) were balanced by apo-transferrin supplementation to maintain total transferrin levels of 1.2 mg/ml in all cultures. Analyses were performed over the 21-day culture period that included assessment of proliferation, differentiation, enucleation and surface phosphatidylserine expression. Diminution of holo-transferrin in the culture medium caused a decrease in total cell counts and reduced cell divisions detected by CFSE analysis. During the first two weeks of culture, the progenitor cells differentiated into a glycophorin A positive population at all holo-transferrin doses. However, several iron-dependent changes were detected in the final stages of differentiation during culture days 14–21. Hemoglobin accumulation was reduced by 72% at the lowest holo-transferrin dose. Flow cytometry demonstrated a decrease in the loss of surface transferrin receptor in iron-deficient cultures. Thiazole orange analysis also demonstrated significantly lower levels of enucleation from 25.9±8.2% (1.2 mg/ml) to 4.4±4.2% (0.1mg/ml). Microscopic examination of the enucleated cells demonstrated a hypochromatic phenotype at lower holo-transferrin concentrations. Phosphatidylserine on the surface membrane (annexin V staining) increased with reduction of holo-transferrin from 6.3±2.6% (1.2 mg/ml) to 78.3±9.4% (0.1 mg/ml, p=0.003). The effects of iron on surface phosphatidylserine expression were limited to holo-transferrin concentrations of Disclosures: No relevant conflicts of interest to declare.

Abstract 2128 The first erythropoietic stress in term neonates occurs at the time of the erythrocyte nadir (around three months of age) and is associated with increased reticulocytosis. In infants with sickle cell disease (HbSS, SCD), this nadir generally precedes the onset of clinical signs and symptoms. The objective of this study was to determine if reticulocyte levels and properties during early infancy in SCD patients are useful in disease severity prediction. Peripheral blood from 111 children with SCD who were enrolled in this observational study was analyzed within 48 hours of collection and storage at 4°C. Reticulocytes were quantified by absolute reticulocyte count (ARC) as well as reticulocyte flow cytometry phenotyping and sorting using CD71 (CD71 Hi, CD71 Lo and CD71 Neg) and CD36 markers. Among the entire group, ARC is negatively correlated with HbF (r=−0.72, p Disclosures: No relevant conflicts of interest to declare.

Abstract 2666 Acute erythroid stress causes elevated levels of fetal hemoglobin (HbF) in primates. However, relationships between the chronic erythroid stress in children with Sickle Cell Disease (HbSS) and HbF production are less well understood. For this study, peripheral blood from children with HbSS was analyzed within 72 hours of collection and storage at 4°C. The level of erythroid stress was determined by absolute reticulocyte counts (ARC) as well as reticulocyte flow cytometry phenotyping and sorting using CD71 (HiCD71, LoCD71 and NegCD71) and CD36. Immature reticulocytes were identified by HiCD71 expression and included the CD36+ population. Sorted populations from at least three subjects were utilized to assess fetal globin expression during reticulocyte maturation according to quantitative polymerase chain reaction (qPCR), and high pressure liquid chromatography (HPLC). While the number of gamma-globin mRNA copies/cell decreased with reticulocyte maturation, the ratio of gamma/gamma+beta mRNA remained stable. HbF protein levels in the HiCD71 and LoCD71 populations were equivalent for each patient with average values of 4.3±4.2% HiCD71 vs. 4.4±4.0% LoCD71, p=0.96. By comparison, HbF levels in the NegCD71 were increased in all subjects (14.2±9.5% NegCD71, p300 mean event rate=0.59±0.60 events/year, p=0.0028). These data suggest that increased levels of erythroid stress as measured by reticulocyte production and maturation in pediatric HbSS subjects is not associated with elevated gamma-globin mRNA or HbF levels. Instead, higher levels of reticulocytosis were associated with lower HbF levels and increased disease severity. Disclosures: Noel: NIH: Patents & Royalties.

Abstract 4255 Based upon the importance of iron for erythropoiesis, it was hypothesized that specific molecular and cellular mechanisms may be manifested during early erythropoiesis in preparation for heme and hemoglobin production. Here iron regulation was studied in immature erythroid progenitor cells prior to the increased production of heme or hemoglobin. CD34+ cells were cultured in SCF, IL-3 and FLT-3 ligand for 7 days, and the erythroid progenitor cells were identified and sorted from three donors according to the differential expression of CD36, CD41, and CD71. Among the sorted populations, the CD71(+)CD36(+)CD41(-)GPA(-) cells grew almost exclusively (>95%) into erythroid colonies in methylcellulose culture. Cytospin examination of those erythroid progenitor cells demonstrated large blasts with deep blue cytoplasm and blebbed membranes consistent with pre-proerythroblasts. Furthermore, heme accumulation and hemoglobin were absent. Western analyses revealed that ferritin, transferrin receptor 1 (TfR1), and transferrin receptor 2 (TfR2) expression were regulated by dosed titrations (0, 10, 20, 40, 60, and 100 %) of iron-saturated transferrin in the culture medium. The intracellular iron-storage protein, ferritin, showed increased expression with higher holo-transferrin saturation. Opposite patterns of iron-dependent regulation for the transferrin receptors was identified. TfR1 protein decreased and TfR2 increased with increased transferrin saturation. While TfR1 was detected at high levels on the plasma membranes, immunofluorescence studies showed TfR2 co-localization with lysosomes (correlation with LAMP1, a lysosomal marker, R = 0.53 ± 0.11, n=22). Decreased holo-transferrin in the culture medium resulted in no significant reduction in the proliferation of these cells until hemoglobin production was induced by erythropoietin. Confocal microscopy demonstrated a distinct peri-centrosomal lysosomal compartment surrounded by mitochondria. Coincidentally, upon sulfide-silver staining, it was observed that iron accumulated within the lysosomal compartment in a transferrin-dependent fashion. No similar iron-containing lysosomal-mitochondrial structure was identified in non-erythroid cells in the same cultures. These results suggest that functional, lineage-specific differentiation occurs at the progenitor stage of hematopoiesis, and that iron import and regulation is highly developed during erythropoiesis prior to hemoglobin production. Disclosures: No relevant conflicts of interest to declare.

Abstract 4043 Poster Board III-978 Iron depletion affects billions worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. Hepcidin is a negative regulator of iron absorption in the body. Low serum hepcidin levels provide a physiological response to iron demand in patients with iron deficiency. Based on a recent discovery of elevated growth differentiation factor 15 (GDF15) as a pathological suppressor of hepcidin in cases of thalassemia (Tanno et al. 2007), it was hypothesized that GDF15 may be elevated under physiological circumstances such as iron deficiency, but this area of research remains controversial. Here GDF15 and hepcidin levels were measured in the peripheral blood of subjects with iron-depleted erythropoiesis, and monitored in patients receiving iron replacement therapy. Initially, clinical studies were performed to measure GDF15 in the serum of iron-depleted blood donors (n = 10) and healthy volunteers (n = 10). Iron depletion was defined for this study as serum ferritin level ( Disclosures: No relevant conflicts of interest to declare.

Abstract 4032 Poster Board III-968 Several types of ineffective erythropoiesis (thalassemia, congenital dyserythropoietic anemia type I, and refractory anemia with ringed sideroblasts) develop anemia and eventually manifest secondary hemochromatosis even in the absence of transfusion therapy. The highly elevated levels of GDF15 in those patients may contribute to iron loading due to suppression of hepcidin. However, the underlying mechanism for high-level expression of GDF15 in association with ineffective erythropoiesis remains vague. Phenylhydrazine (PHZ), which causes hemoglobin denaturation and alpha-globin precipitation, was utilized in CD34+ cultures as an ex vivo model of ineffective erythropoiesis. All experiments were performed in triplicate with cells from three separate human donors. Initial studies were performed to determine the effects of phenylhydrazine upon reactive oxygen species (ROS) production and apoptosis in the erythroblasts. In dosed titrations (0-10 uM), PHZ was added to the culture medium before and after erythroblast hemoglobin accumulation on culture days 3-6 and 11-14, respectively. PHZ addition prior to the accumulation of hemoglobin had little effect on ROS production. In contrast, PHZ added to hemoglobinized erythroblasts resulted in significant and dosed increases in ROS production. In addition, apoptosis cascades that are downstream of ROS production were studied including activation of caspase-3, EGR-1 transcription, p53 stabilization, and phosphatidylserine on the cell surface (detected by Annexin V). There was a dose-dependent increase in caspase-3, p53 stabilization, and Annexin V positive cells that mirrored the ROS production after PHZ treatment only among the more mature erythroblasts. However, the PHZ addition did not cause a significant increase in the nuclear localization of EGR-1. Since GDF15 is a known target of p53, PHZ effects upon GDF15 expression were also measured. Production of GDF15 was not observed on culture days 3-6 in the absence or presence of PHZ. In contrast, dosed increases in GDF15 expression were detected among the hemoglobinized erythroblasts (culture days 11-14) in response to phenylhydrazine (PHZ = 0 uM, GDF15 = 16 ± 3 pg/1×104cells; PHZ = 1 uM, GDF15 = 23 ± 5 pg/1×104cells; PHZ = 5 uM, GDF15 = 48 ± 9 pg/1×104cells [p Disclosures: No relevant conflicts of interest to declare.

MicroRNAs are ~22nt-long small noncoding RNAs that negatively regulate expression of target proteins through mRNA degradation or translational repression. They are functionally important in diverse biological processes including development, oncogenesis, and hematopoiesis. To study a potential role of microRNA for regulating cellular changes during erythroid ontogeny, we examined microRNA abundance patterns in circulating erythroid cells during the fetal and adult stages of human development. Expression profiling of microRNA was performed using total RNA from 4 adult peripheral blood samples compared to 4 cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNA was hybridized to custom spotted array containing 474 human microRNAs species (miRBase release 9.1). Total RNA from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines was used as a hybridization reference for all samples to generate microRNA abundance profile for each sample. Among the differentially expressed probe-pairs, 40 microRNAs were significantly up-regulated in adult erythroid cells and only 4 microRNAs were down-regulated (p < 0.01 and fold change > 2). Two microRNAs, miR-144 and miR-451, previously reported as being GATA-1 regulated in erythroid cells, were detected above the dynamic range of the array technology in all eight samples. Quantitative PCR was performed to further quantitate differences in the microRNA abundance profiles of the 10 most differentially expressed microRNAs as well as miR-144 and miR-451. Per published recommendations, miR-103 was used for signal normalization. Among the studied microRNAs, 8 of 12 demonstrated a more than 4 fold increase in abundance in adult erythroid cells compared to cord blood by qPCR. In particular, let-7d and let-7e demonstrated more than 10-fold increased expression in adult cells. Of note, let-7 microRNAs regulate metamorphic processes in model organisms and inhibit ras-associated growth of cancer cells. According to miRBase predictions, over 1200 genes may be targeted by the 8 microRNAs (p < 0.001). Examination of cord and adult reticulocyte mRNA expression profiles (Goh et al. Phy. Gen. 2007) revealed 24 developmentally downregulated genes as predicted targets. Among globin genes, only gamma globin is a predicted target. These findings demonstrate dramatic up-regulation of specific erythroid microRNAs during the fetal-to-adult transition of ontogeny, and support the notion that microRNAs function as developmental regulators of erythropoiesis and globin gene switching.

Iron deficiency affects billions worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. However, iron depleted erythropoiesis is incompletely understood. In this study, an ex vivo model of iron depleted erythropoiesis was developed using dosed titrations of the iron chelator deferoxamine (DFO) in CD34+ cell cultures. All experiments were performed in triplicate with cells from three separate donors. Hemoglobinization and expression patterns of erythroid markers (GPA/CD71) demonstrated minimal changes in DFO supplemented medium. DFO caused dose-related suppression of cell counts after 14 days (20uM DFO: 42% reduction, 30uM DFO: 86% reduction, 40uM DFO: 96% reduction of cell counts compared to 0uM DFO controls). Cultures supplemented with 30uM DFO were used for subsequent studies, and the DFO-mediated effects were completely reversed by addition of 30uM ferric chloride. To investigate whether apoptosis caused the reduction in cell counts, surface Annexin V was measured by flow cytometry. No significant increases in levels of apoptosis among the proliferating erythroblasts were detected (Annexin V positive cells; 0uM DFO 6.2 ± 0.95%, 30uM DFO 9.0 ± 1.55%, p = 0.058). Analyses of p53 protein quantitation and p53 DNA-binding activity further suggested that the suppression of cell counts by iron chelation was not due to p53 related apoptosis. In the absence of apoptosis or maturation arrest, we hypothesized that the growth suppressing effects of DFO were not due to ineffective erythropoiesis. To test this hypothesis, a recently-discovered marker of ineffective erythropoiesis named GDF15 (Tanno et al. Nat. Med. 2007) was measured in the culture supernatants, and no increase was detected (GDF15 concentration per 1×104 cells: 0uM DFO; 79.7 ± 4.4 pg, 30uM; 43.9 ± 4.3 pg). In the clinic, serum GDF15 levels from 17 blood donors with significantly reduced ferritin and iron saturation (279 ± 98 pg/ml) were not increased when compared to serum GDF15 levels in 17 healthy volunteers with normal iron parameters (410 ± 119 pg/ml). To determine if cell cycling, instead of apoptosis, caused the reduction in cell counts, carboxyfluorescein diacetate succinimidyl diester (CFSE) staining studies were performed during the first and second weeks of culture. CFSE is an intracellular fluorescent stain, the intensity of which halves with each cell division. Three or four fewer erythroblast cell divisions were detected in 30uM DFO during the 14-day culture period when compared to controls. Based upon the reduction in cell divisions, it was estimated that the average cell cycle time was prolonged by 40–70% in DFO. Propidium iodide analyses additionally demonstrated changes in the cell cycle kinetics including a significant reduction in the percentage of S-phase erythroblasts at the peak phase of proliferation (30uM DFO: 37.0 ± 1.8% vs. control: 48.7 ± 3.4%, p = 0.0063). These results suggest that iron depletion inhibits the overall growth of erythroblasts by prolonging the cell cycle rather than causing apoptosis or maturation arrest. Due to the large amount of iron required for hemoglobin production, slower cell cycles may facilitate effective erythropoiesis by allowing extra time for uptake when extracellular iron is scarce.

Within the erythroid lineage, erythropoietin (EPO) responsiveness is manifested by cell division and growth as well as specific changes in heme and globin production that ultimately result in the production of erythrocytes. However, the nascent relationship between EPO-associated mitosis and globin gene regulation has not been fully defined. In this study, cultured adult human CD34+ cells from peripheral blood were used to investigate early cellular responses to erythropoietin in the context of mitosis. Matched cultures were performed in replicate using human cells from at least two healthy adults. To detect mitosis, one million cells were labeled with 2uM carboxyfluorescein diacetate succinamidyl ester (CFSE). The CFSE-labeled cells were then cultured in the presence [EPO(+)] or absence [EPO(−)] of 4U/mL EPO, and analyzed using flow cytometry. No cell divisions were detected in either condition during the first 24 hours in culture, and multiple cell divisions were noted on subsequent culture days only in the EPO(+) cultures. Remarkably, dual-staining with CFSE and CD71 revealed a small (90% of those cells. These novel results suggest that the frequency and levels of gamma-globin transcripts are quite low at the earliest stages of an EPO response among adult human CD34+ cells. However, it is clearly demonstrated that the cells can increase their capacity to import iron and transcribe beta-globin mRNA at very high levels prior to their first EPO-dependent cell division.

Iron overload and bony abnormalities cause considerable morbidity among patients with thalassemia syndromes. One possible explanation for this phenomenon is that proteins normally secreted into the marrow microenvironment during erythropoiesis are over-expressed in thalassemia patients due to expanded and ineffective erythropoiesis. We previously discovered that GDF15 is produced at very high levels in thalassemia patients and inhibits hepcidin expression. Transcriptome screens of erythroblasts were utilized here to identify twisted gastrulation (TWSG1) as a second candidate protein for further study. Quantitative PCR using the β-thalassemia murine model (Hbbth3/+ β-thalassemia intermedia mouse model, n=13; Hbbth3/th3 β-thalassemia major mouse model, n=5) revealed that splenic expression levels of Tsg (murine TWSG1) were significantly higher in thalassemia mice (Hbbth3/+, 2.2E02 ± 2.7E01 copies/ng RNA, p

Erythrocyte inclusion bodies that are now commonly referred to as Howell-Jolly Bodies (HJB) were originally discovered over 100 years ago by microscopy. These nuclear fragments are observed in the erythrocytes of patients lacking splenic function, but their precise genomic origins are vague. Here we report studies of the genetic content of HJB using a molecular cytogenetic method known as Comparative Genomic Hybridization (CGH). Peripheral blood samples from six splenectomized human donors were studied. All donors had undergone splenectomies prior to this study for diagnostic and therapeutic indications related to underlying neoplastic and autoimmune disease. Ultrasound testing was used to rule out the presence of accessory splenic tissue, and peripheral blood smears confirmed the presence of HJB. Nucleated cells were removed by density gradients and leukocyte filtration for use as patient-matched controls. DNA was isolated from the leukocytes and HJB-containing erythrocytes. The presence of high molecular weight HJB DNA was confirmed in each sample using gel electrophoresis. As expected, it was not possible to obtain sufficient quantities of DNA from the control erythrocytes of healthy individuals with normal splenic function. CGH was performed using equivalent amounts of reference genomic DNA co-hybridized with HJB DNA or matched donor leukocyte DNA. Agilent oligonucleotide CGH arrays were hybridized and scanned according to the manufacturer’s instructions. The control hybridizations (genomic DNA isolated from donor leukocytes) demonstrated standard patterns consistent with the donor’s karyotype. In contrast, the HJB hybridization signals demonstrated an unusual pattern of centromeric-to-telomeric enrichment on each chromatid of each chromosome. The HJB hybridization signals also increased as a function of distance from the centromere according to the chromatid size. The enhanced signal relative to centromeric distance varied considerably between chromosomes, but was remarkably consistent between donors. These data suggest Howell-Jolly Bodies contain a non-random distribution of centromere-depleted chromatin generated after DNA double-strand breakage during erythroid differentiation.

Iron overload causes considerable morbidity associated with thalassemia due to inappropriate suppression of the iron regulator, hepcidin. One possible explanation for this phenomenon is that protein(s) that are normally secreted into the marrow microenvironment by erythroblasts become endocrine signals in patients with thalassemia due to the myeloproliferative nature of the disease. To test this hypothesis, progenitor and precursor cell transcriptional profiles were generated with Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays using primary erythroblasts cultured from 15 healthy human donors. For this study, informatic analyses were focused upon 54 members of the TGF–B/BMP superfamily. Among the subset of genes identified by this screen, evidence for high-level expression of a gene named growth differentiation factor 15 (GDF15) was discovered in the precursor cell profiles. Quantitative PCR, Western, and ELISA analyses confirmed expression and secretion of GDF15 during erythroblast maturation. GDF15 is an apoptosis-associated protein expressed primarily by the placenta. To determine whether GDF15 serves as a regulator of hepcidin expression, hepcidin expression assays were performed using a human hepatoma cell line (HuH-7). BMP2 and BMP4 were studied for comparison. Addition of BMP2 and BMP4 (range 10,000–500,000 pg/ml) resulted in dosed increases in hepcidin mRNA (5–30 fold). At the concentrations of GDF15 normally found in human blood (500 pg/ml), a 2-fold increase in the expression of hepcidin was measured compared to matched cultures containing no supplemental GDF15. However, GDF15 dosed to levels above 5,000 pg/ml resulted in a significant reduction in hepcidin expression. Next, plasma levels of GDF15 were measured in the peripheral blood of 162 donors (donor groups included healthy controls, sickle-cell syndromes, thalassemia syndromes, and other causes of anemia) to determine whether plasma GDF15 levels are dysregulated in thalassemia. Plasma samples from 21 hereditary hemochromatosis donors provided evidence that significantly elevated GDF15 expression is not associated with iron overload in the absence of erythroid pathology. Compared with mean plasma GDF15 levels of 536±222 pg/ml among the control samples, only patients with thalassemia intermedia, thalassemia major, and HbE-beta thalassemia showed significantly elevated plasma levels of GDF15 (9 donors; mean GDF15 24,600 pg/ml; range 8,980–75,100 pg/ml; p370 ug/L) were noted in each of those patients regardless of their transfusion or chelation history. These novel findings suggest that GDF15 is secreted from human erythroblasts, released into the circulation at extremely high levels in thalassemia patients, and contributes to iron overloading in those patients by suppressing hepcidin expression.

It was recently demonstrated that erythropoietin combined with stem cell factor and transforming growth factor beta (EST; EPO+SCF+TGF-B) signals a pancellular reversal of gamma-globin gene silencing among ex vivo cultures of CD34+ cells when compared to cultures supplemented with erythropoietin (EPO) alone (Bhanu et al., Blood.2005; 105(1):387–93). This primary human erythroblast culture model is now being utilized to study the molecular basis of globin gene regulation. In this study, we sought to determine whether EST-signaled effects are restricted to the gamma-globin gene(s). For this purpose, quantitative PCR (QPCR) assays were developed for the alpha-globin (zeta, mu, alpha, and theta) and beta-globin (epsilon, gamma, delta, and beta) gene clusters. EPO versus EST matched cultures from a total of 15 donors (3 single donors and two pools, 6 donors each) were investigated using culture day 7 proerythroblasts and day 14 hemoglobinized precursors. The QPCR products were quantified by comparison with plasmid-generated standard curves (20,000,000 to 200 copies). The patterns of mu-, alpha- and theta-globin gene expression were unchanged under all culture conditions. The delta- and beta-globin transcripts showed a significant reduction in levels in response to EST. In contrast, zeta-, epsilon- and gamma-globins all demonstrated significant increases in response to EST. The increases were consistently detected in the individual and pooled samples. The most dramatic increase in the zeta-, epsilon- and gamma-globins occurred at the proerythroblast stage of differentiation. On day 7, zeta-globin levels showed 190-fold increase (EPO: 6.9E+01±5.5E+01;EST: 1.3E+04±7.7E+03; p=0.01), epsilon-globin levels, a 27-fold increase (EPO: 1.0E+03±5.8E+02; EST: 2.7E+04±1.1E+04; p=0.001), and gamma-globin levels, a 13-fold increase (EPO: 9.1E+05±4.1E+05; EST: 1.2E+07±1.8E+06; p=0.001). Consistent with the 3–4 log difference between the levels of zeta- and epsilon-, versus gamma-globin copy numbers and the low sensitivity of HPLC compared with QPCR, no embryonic proteins were detected by reverse-phase HPLC (day 14 samples studied; 9 donors). However, G-gamma and A-gamma globin peaks confirmed EST-signaled activation of both gamma-globins genes (G-gamma/A-gamma= 1.0 in EPO versus 1.9 in EST; (total gamma)/(total gamma + beta-globin) = 3.7±2.4% in EPO versus 42.4 ±5.8% in EST; p=1.6E-08). These novel data suggest that cytokines can reverse the coordinated developmental silencing of human embryonic and fetal genes in both globin loci.

Hemoglobin switching patterns during human ontogeny are highly correlated with transcription of the alpha and beta globin genes on chromosomes 16 and 11, but the molecular and genetic mechanisms responsible for the switching phenomenon are not yet defined. For a better understanding of post-natal globin gene switching and silencing, the expression profiles from purified reticulocyte mRNA (28 separate clinical samples; 14 cord, 14 adult) were studied using Affymetrix HG-U133 arrays (44,229 probe sets). To validate the transcriptome expression levels derived from the microarrays and to discriminate the differences between cord blood and adult blood reticulocytes, we performed quatitative real-time PCR. Among 21 tested control genes, the ratios of adult-to-cord blood (AB/CB) array hybridization intensities versus PCR copy numbers demonstrated a correlation coefficient of 0.978. Hence, the AB/CB array hybridization intensities provide a reliable description of erythroid transcriptome patterns associated with post-natal hemoglobin switching. Stringent screening criteria were then used to identify genes that may be involved in fetal-to-adult hemoglobin switching. All 44,229 probe sets were filtered to identify genes that demonstrated greater than a five fold difference in AB and CB mean signal intensities as well as consistent differences among the 14 samples from each group. In addition, the screening criteria required high-level signals in either AB or CB (>1000 FU), and t-test p-values between AB and CB

It is well known that the globin genes are organized within the human genome as two gene clusters on chromosomes 16 and 11 (α cluster: ζ2-ζ1-α2-α1-𝛉 and β cluster: e-Gγ-Aγ-δ-β). Switching in the expression patterns of those genes during human ontogeny generally follows a pattern determined by their genomic arrangement, but the genetic mechanism is not defined. In an effort to better understand the γ-to-β globin switching phenomenon, gene expression patterns from reticulocyte RNA were studied. cDNA were generated from the reticulocytes of 28 separate donors (14 cord blood; 14 adult blood) and analyzed using Affymetrix HG-U133 arrays. Quantitative PCR was used to confirm the array expression patterns. Among those cDNA identified as having expression patterns that were relatively decreased among adult reticulocytes, we were surprised by expression associated with a cluster of expressed sequence tags (EST) located within the α-globin locus in the region between the ζ1 and α2 genes. In the context of the 44,229 arrayed probes, high-level expression from that EST cluster was detected (ranked < 25th). Informatic analysis revealed alignment with the second and third exons previously described for the Ψα2 globin gene, but no significant similarity with the first exon of Ψα2. Based upon these comparisons, we used adult human reticulocyte RNA to amplify a cDNA clone for further study (GenBank: [AY698022][1]; named μ-globin). Sequencing of that clone revealed an insert that encodes a predicted 423 nt. open reading frame that is one amino acid residue shorter than the α2-globin gene. While the mature μ-globin mRNA results from splicing of two introns, it lacks a canonical Kozak sequence. BLASTP and CLUSTALW analyses revealed the highest level of homology with the avian αD-globin protein with a sequence identity of 55% (78/141 amino acids). In addition, the predicted heme- and globin-binding amino acids of μ-globin and avian αD-globin are largely conserved. Experimentally, we analyzed the expression level of the μ-globin transcripts using quantitative real-time PCR. Non-erythroid tissues including whole brain, white blood cells, and the Jurkat cell line demonstrated only background levels. The expression level of μ-globin (copies per ng cDNA) was 3.04x10(4) ± 1.68x10(3) in fetal liver, 1.71x10(5) ± 9.51x10(4) in cord reticulocytes, 1.15x10(4) ± 1.19x10(3) in bone marrow, and 2.17x10(4) ± 6.84x10(3) in adult reticulocytes. Based upon the consistent decrease in μ-globin expression between fetal and adult erythroid tissues, we measured the expression of γ-globin in those tissues for comparison. The expression level of γ-globin (copies per ng cDNA) was 2.49x10(6) ± 1.48x10(5) in fetal liver, 1.49x10(8) ± 7.88x10(7) in cord reticulocytes, 3.84x10(3) ± 3.83x10(2) in bone marrow, and 5.70x10(5) ± 5.80x10(5) in adult reticulocytes. To determine if μ-globin expression is regulated during erythropoiesis, we assayed primary human erythroid progenitor cells cultured in erythropoietin. The expression pattern of μ-globin was similar to the other globin genes increasing from background levels on day 0 to maximum levels in committed erythroblasts (3.56x10(5) ± 1.99x10(4) copies per ng cDNA). These results suggest that the human genome encodes a previously unrecognized α-globin that most closely resembles avian αD-globin. Expression of that gene is decreased during the fetal-to-adult transition of human ontogeny and regulated during erythropoiesis. [1]: /lookup/external-ref?link_type=GEN&access_num=AY698022&atom=%2Fbloodjournal%2F104%2F11%2F502.atom

Adenosine signal transduction in human cells is achieved through four distinct G-protein-coupled receptors named A1, A2A, A2B and A3. Adenosine signaling events affect several physiological processes including growth regulation and cellular responses to hypoxia. Despite extensive studies of native and pharmacologically-targeted adenosine signal transduction, the potential for adenosine to regulate erythropoiesis remains largely unexplored. Here we systematically studied adenosine receptor expression, activation, and inhibition among CD34+ human erythroid progenitor cells cultured in the presence of erythropoietin. This 14 day culture system has been studied extensively in the context of erythroblast commitment and terminal differentiation (BLOOD 99(8):3005–13). Using real-time RT-PCR, we demonstrated that the A2A, A2B and A3 adenosine receptors are expressed in highly regulated patterns. The expression of A2A and A2B were highest early in the culture period and later declined as the cells underwent terminal differentiation. Receptor A3 gene expression gradually increased during the terminal differentiation. Expression of the A1 receptor gene was not detected above background levels at any stage of differentiation (compared with the positive control). Based upon those receptor expression patterns, several adenosine receptor signaling agonists and antagonists were screened for possible effects on erythroblast growth and differentiation. Among those screened molecules, the selective A3 adenosine receptor agonist, CI-IB-MECA, had effects on the proliferation and differentiation of the cells without overt toxicity in the dose range of 10–100uM. CI-IB-MECA significantly inhibited the proliferation of the cells with resulting cell counts being only 10% of those in matched controls after 14 days. Cell cycle analyses demonstrated that the growth inhibitory effects of CI-IB-MECA were largely due to a significant reduction in the percentage of S-phase proerythroblasts (S-phase: 16 ± 5% in CI-IB-MECA versus 51 ± 2% in matched controls; p80%) in the matched controls. Consistent with the morphological examination, HPLC analyses revealed barely detectable levels of hemoglobin in the presence of CI-IB-MECA after 14 days. These finding suggest that selective activation of adenosine A3 receptors permits erythroid commitment, but profoundly inhibits the proliferation of those committed cells and retards their terminal differentiation. As a novel regulator of erythropoiesis, adenosine A3 receptor signaling should be explored in patients with growth-related erythroid diseases.