Fetal hemoglobin rescues ineffective erythropoiesis in sickle cell disease.

While ineffective erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in the bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for ineffective erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased ineffective erythropoiesis.

Fyn Kinase Is Involved in EPO Receptor Signaling and Is Required to Harmonize the Response to Oxidation.

DISCLOSURES: No relevant conflicts of interest to declare.

Identification of binding domains on red blood cell glycophorins for Babesia divergens.

BACKGROUND: Invasion of red blood cells (RBCs) is one of the critical points in the lifecycle of Babesia. The parasite does not invade other host cells. Earlier work has shown that GPA and GPB function as putative receptors during parasite invasion. The primary focus of this study was the delineation of parasite-binding domains on GPA and GPB. STUDY DESIGN AND METHODS: The assay of choice to validate molecules that participate in invasion is an inhibition of invasion assay, in which changes in parasitemia are assessed relative to a wild-type assay (no inhibitors). Inhibition of invasion can be achieved by modification of different components of the assay or by the addition of competitors of the molecules that participate in invasion. In this study purified antibody fragments to various domains on GPA and GPB were tested for magnitude of inhibition of parasite invasion. Effects on invasion were monitored by assessment of Giemsa-stained smears every 24 hours. RESULTS: Among 10 selected antibodies directed at various epitopes on GPA and GPB, antibodies directed against GPA(M) epitopes had the most severe effect (up to 35%) on inhibition of invasion, followed by antibodies directed against GPB(S) epitope (up to 24%). CONCLUSION: This study confirms the role of RBC glycophorins A and B in Babesia divergens invasion and shows that the GPA(M) and GPB(S) epitopes are likely to play an important role in the entry process.

Single particle electron microscopy analysis of the bovine anion exchanger 1 reveals a flexible linker connecting the cytoplasmic and membrane domains.

Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO2 transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.

Therapeutic hemoglobin levels after gene transfer in ß-thalassemia mice and in hematopoietic cells of ß-thalassemia and sickle cells disease patients.

Preclinical and clinical studies demonstrate the feasibility of treating ß-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human ß-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients.We generated lentiviral vectors carrying the human ß-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human ß-globin through a novel mechanism that links the rate of transcription of the transgenic ß-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34(+) cells isolated from patients affected by ß-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S).Our results suggest two major findings. First, we discovered that for the purpose of expressing the ß-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from ß-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

New Views of Sickle Cell Disease Pathophysiology and Treatment.

This review addresses several areas of concern in the care of patients with sickle cell disease. In Sections I and II, the fundamental pathogenetic mechanisms of sickle cell disease and their clinical consequences are discussed. Dr. Narla presents the evidence for abnormal cell adhesiveness by SS cells and Dr. Rosse examines the role of the increased whole blood viscosity. In Section III, Dr. Petz reviews common and uncommon alloimmune consequences of transfusion in sickle cell disease and discusses the diagnosis and management of sickle cell patients with hyperhemolysis after transfusion. In Section IV, Dr. Steinberg gives an update on the use of hydroxyurea in the treatment of sickle cell disease, including the SC and S-beta thalassemia variants.