Early identification of infants with sickle cell disease (SCD) by newborn screening, now universal in all 50 states in the US, has improved survival, mainly by preventing overwhelming sepsis with the early use of prophylactic penicillin. Routine transcranial Doppler screening with the institution of chronic transfusion decreases the risk of stroke from 10% to 1% in paediatric SCD patients. Hydroxyurea decreases the number and frequency of painful crises, acute chest syndromes and number of blood transfusions in children with SCD. Genetic research continues to be driven toward the prevention and ultimate cure of SCD before adulthood. This review focuses on clinical manifestations and therapeutic strategies for paediatric SCD as well as the evolving topic of gene-focused prevention and therapy.
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.
erythropoiesis; serum-free media; holotransferrin; haemoglobin; enucleation; iron
Treatment-associated fetal hemoglobin (HbF) expression patterns in children with sickle cell disease (SCD) have not been fully described. The objective of this study was to compare HbF expression profiles (HbF and F-cells) in the peripheral blood of pediatric SCD patients receiving hydroxyurea (HU), chronic transfusions (Tx) or no chronic therapy (Ctrl).
Peripheral blood samples were collected from SCD patients between 1 month and 21 years of age and immunostained with anti-HbF and anti-HbA antibodies. Erythrocytes containing HbF (F-cells) were enumerated with this dual staining method. HbF was measured using chromatography (HPLC).
Blood from 44 Ctrl patients ≤4 years of age was compared with that from older children (50 Ctrl, 17 HU, 17 Tx). Among the older children, the percentage of both HbF and F-cells in the Tx group was significantly decreased compared to the control (HbF 5.4 ± 4.2% vs. 11.0 ± 7.2%, P = 0.003; F-cells 30.2 ± 16.3% vs. 43.8 ± 20.4%, P = 0.0071). While the distribution of F-cells was significantly increased in the HU group (56.3 ± 17.1% vs. 43.8 ± 20.4%, P = 0.016), the increase in HbF was less robust (14.7 ± 6.4% vs. 11.0 ± 7.2%, P = 0.051). Positive correlations of HbF and F-cell distributions were noted in all groups (P < 0.0001 for all groups). In serial samples from individual patients, relatively static patterns of HbF and F-cell distribution were noted.
Pediatric SCD patients possess distinct patterns of HbF switching and silencing in peripheral blood erythrocytes. Thereafter, erythrocyte HbF expression level and distribution are maintained with both patient- and treatment-specific patterns that may be useful for predicting the need or response to HbF-modulating therapy.
F-cells; fetal hemoglobin; sickle cell disease
Low serum hepcidin levels provide a physiologic response to iron demand in patients with iron deficiency (ID). Based on a discovery of suppressed hepcidin expression by a cytokine named growth differentiation factor 15 (GDF15), it was hypothesized that GDF15 may suppress hepcidin expression in humans with ID due to blood loss.
STUDY DESIGN AND METHODS
To test this hypothesis, GDF15 and hepcidin levels were measured in peripheral blood from subjects with iron-deficient erythropoiesis before and after iron supplementation.
Iron variables and hepcidin levels were significantly suppressed in iron-deficient blood donors compared to healthy volunteers. However, ID was not associated with elevated serum levels of GDF15. Instead, iron-deficient subjects’ GDF15 levels were slightly lower than those measured in the control group of subjects (307 ± 90 and 386 ± 104 pg/mL, respectively). Additionally, GDF15 levels were not significantly altered by iron repletion.
ID due to blood loss is not associated with a significant change in serum levels of GDF15.
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.
Iron deficiency anemia arises when the balance of iron intake, iron stores, and the body’s loss of iron are insufficient to fully support production of erythrocytes. Iron deficiency anemia rarely causes death, but the impact on human health is significant. In the developed world, this disease is easily identified and treated, but frequently overlooked by physicians. In contrast, it is a health problem that affects major portions of the population in underdeveloped countries. Overall, the prevention and successful treatment for iron deficiency anemia remains woefully insufficient worldwide, especially among underprivileged women and children. Here, clinical and laboratory features of the disease are discussed, and then focus is placed on relevant economic, environmental, infectious, and genetic factors that converge among global populations.
Purpose of review
Growth differentiation factor 15 (GDF15) was identified as a hepcidin-suppression factor that is expressed at high levels in patients with ineffective erythropoiesis. This review addresses the regulation, expression and potential functions of GDF15 in the context of erythroid biology.
GDF15 expression during late erythroid differentiation was discovered as part of an erythroblast transcriptome project. Since GDF15 expression is associated with cellular stress or apoptosis, further investigation of the cytokine was focused upon its involvement in ineffective erythropoiesis. Remarkably high serum levels were detected in patients with thalassemia syndromes, congenital dyserythropoiesis and some acquired sideroblastic anemias. Similarly high-level GDF15 expression is not a feature of normal erythropoiesis, or erythroid recovery after bone marrow transplantation. Since GDF15 is a TGF-β superfamily member, it was investigated as an effector of ineffective erythropoiesis that suppresses hepcidin expression despite iron overloading.
In contrast to the low-levels of GDF15 expressed during normal erythropoiesis, ineffective erythropoiesis causes high-level expression of GDF15. In patients with thalassemia and related anemias, GDF15 expression may contribute to iron overloading or other features of the disease phenotype.
GDF15; ineffective erythropoiesis; iron regulation
Erythropoiesis describes the hematopoietic process of cell proliferation and differentiation that results in the production of mature circulating erythrocytes. Adult humans produce 200 billion erythrocytes daily, and approximately 1 billion iron molecules are incorporated into the hemoglobin contained within each erythrocyte. Thus, iron usage for the hemoglobin production is a primary regulator of plasma iron supply and demand. In many anemias, additional sources of iron from diet and tissue stores are needed to meet the erythroid demand. Among a subset of anemias that arise from ineffective erythropoiesis, iron absorption and accumulation in the tissues increases to levels that are in excess of erythropoiesis demand even in the absence of transfusion. The mechanisms responsible for iron overloading due to ineffective erythropoiesis are not fully understood. Based upon data that is currently available, it is proposed in this review that loading and overloading of iron can be regulated by distinct or combined mechanisms associated with erythropoiesis. The concept of erythroid regulation of iron is broadened to include both physiological and pathological hepcidin suppression in cases of ineffective erythropoiesis.
Iron deficiency is one of the most common disorders affecting mankind, and iron deficiency anemia continues to represent a major public health problem worldwide. It is especially common among women of childbearing age due to pregnancy and menstrual blood loss. Additional patient groups include those with other sources of blood loss, malnutrition or gut malabsorption. Iron deficiency anemia remains quite prevalent despite the widespread ability to diagnose the disease and availability of medicinal iron preparations. Therefore, new approaches are needed to effectively manage these patient populations. In this review, the diagnosis and treatment of iron deficiency anemia are discussed with emphasis placed upon consideration of patient specific features. It is proposed that all patients participate in their own care by helping their physician to identify a tolerable daily iron dose, formulation, and schedule. Dosing cycles are recommended for iron replacement based upon the tolerated daily dose and the total iron deficit. Each cycle consists of 5000mg of oral elemental iron ingested over at least one month with appropriate follow-up. This approach should assist physicians and their patients with the implementation of individualized treatment strategies for patients with iron deficiency anemia.
iron; anemia; hepcidin; dosing cycles
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.
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.
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.
These data suggest that during early infancy, increased reticulocytosis among asymptomatic SCA subjects is associated with increased severity of disease in childhood.
Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14–21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia.
MicroRNAs are ~22nt-long small non-coding RNAs that negatively regulate protein expression through mRNA degradation or translational repression in eukaryotic cells. Based upon their importance in regulating development and terminal differentiation in model systems, erythrocyte microRNA profiles were examined at birth and in adults to determine if changes in their abundance coincide with the developmental phenomenon of hemoglobin switching.
Expression profiling of microRNA was performed using total RNA from four adult peripheral blood samples compared to four cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNAs were hybridized to custom spotted arrays containing 474 human microRNA species (miRBase release 9.1). Total RNA from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines provided a hybridization reference for all samples to generate microRNA abundance profile for each sample.
Among 206 detected miRNAs, 79% of the microRNAs were present at equivalent levels in both cord and adult cells. By comparison, 37 microRNAs were up-regulated and 4 microRNAs were down-regulated in adult erythroid cells (fold change > 2; p < 0.01). Among the up-regulated subset, the let-7 miRNA family consistently demonstrated increased abundance in the adult samples by array-based analyses that were confirmed by quantitative PCR (4.5 to 18.4 fold increases in 6 of 8 let-7 miRNA). Profiling studies of messenger RNA (mRNA) in these cells additionally demonstrated down-regulation of ten let-7 target genes in the adult cells.
These data suggest that a consistent pattern of up-regulation among let-7 miRNA in circulating erythroid cells occurs in association with hemoglobin switching during the fetal-to-adult developmental transition in humans.
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 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 structurally-related ribofuranose-substituted adenosine analogues act through an unknown mechanism to inhibit HbF expression in fetal and adult human erythroblasts.
Human erythropoiesis; cytokines; HbF inhibition; adenosine derivatives; SQ22536; hemoglobinopathies
Hembase (http://hembase.niddk.nih.gov) is an integrated browser and genome portal designed for web-based examination of the human erythroid transcriptome. To date, Hembase contains 15 752 entries from erythroblast Expressed Sequenced Tags (ESTs) and 380 referenced genes relevant for erythropoiesis. The database is organized to provide a cytogenetic band position, a unique name as well as a concise annotation for each entry. Search queries may be performed by name, keyword or cytogenetic location. Search results are linked to primary sequence data and three major human genome browsers for access to information considered current at the time of each search. Hembase provides interested scientists and clinical hematologists with a genome-based approach toward the study of erythroid biology.
Hydroxyurea treatment of patients with sickle-cell disease increases fetal hemoglobin (HbF), which reduces hemoglobin S polymerization and clinical complications. Despite its use in the treatment of myeloproliferative diseases for over 30 years, its mechanism of action remains uncertain. Recent studies have demonstrated that hydroxyurea generates the nitric oxide (NO) radical in vivo, and we therefore hypothesized that NO-donor properties might determine the hemoglobin phenotype. We treated both K562 erythroleukemic cells and human erythroid progenitor cells with S-nitrosocysteine (CysNO), an NO donor, and found similar dose- and time-dependent induction of γ-globin mRNA and HbF protein as we observed with hydroxyurea. Both hydroxyurea and CysNO increased cGMP levels, and the guanylyl cyclase inhibitors ODQ, NS 2028, and LY 83,538 abolished both the hydroxyurea- and CysNO-induced γ-globin expression. These data provide strong evidence for an NO-derived mechanism for HbF induction by hydroxyurea and suggest possibilities for therapies based on NO-releasing or -potentiating agents.