Thalassemic Red Blood Cells Research Articles

Revisting red blood cell heterogeneity, membrane molecules and surface charge changes in normal, anaemic and thalassaemic red blood cells for blood microsample test development

Revisting red blood cell heterogeneity, membrane molecules and surface charge changes in normal, anaemic and thalassaemic red blood cells for blood microsample test development

Erythrocytic phosphatidylserine exposure and hemostatic alterations in β-thalassemia intermediate patients

IntroductionHypercoagulable state is one of the common findings in beta-thalassemia intermedia (β-TI), particularly in splenectomized patients, with infrequent blood transfusion. Abnormality of the red blood cells (RBC) membrane due to oxidative damage is suggestive of possible etiologies. Membrane lipid peroxidation increases the exposure of phosphatidylserine (PS) that plays a role in the activation of coagulation factors V and X, subsequently initiating thrombosis. Our aim of this study was to find the probable correlation of the alteration of the PS on the RBC outer membrane with the hypercoagulable state in the β-TI patients.Materials and methodsOur cross-sectional study was conducted on 39 splenectomized β-TI patients and 38 age-matched healthy controls. The mean age was 37 years. Analysis of the PS exposure on the RBCs was performed by fluorescein isothiocyanate (FITC) conjugated AV protein .Measurement of the coagulation factors X, V and antithrombin III (AT-III) was performed. We also checked the D-dimer levels .Analysis was performed by SPSS16.ResultsFluorescence of FITC-Annexin V labeling on patients RBCs were higher than healthy controls; (2.8 ± 2.2%) of the patients versus (0.4 ± 0.18%) in the control group and was statistically significant (P < 0.05). Mean levels of factor X and AT-III of the patients as compared with the control group decreased and showed significant difference (P < 0.05).ConclusionsCirculation of thalassemic RBCs, which abnormally possess PS on RBC membrane outer surface, suggests the possibility of the gradual consumption of the coagulation factors in the presence of a chronic coagulability state.

β-Thalassemia: New Therapeutic Modalities, Genetics, Complications, and Quality of Life

Beta-thalassemia is considered one of the most common genetic disorders which mass migration is introducing to countries worldwide and challenging them with its management. Advanced and improved medical care has allowed us to prolong the lives of thalassemia patients, simultaneously uncovering novel complications and outlining new challenges. We herein present in this special issue of Anemia some of the most cutting-edge research outcomes pertaining to the latest complications and the novel treatment strategies in iron-chelation therapy. We also study the effects of these interventions on the quality of life of patients. It is studies like these that serve as the basis of evidence-based medicine and guide clinicians through their process of decision making. One article of this special issue addresses the new method of using transactional Doppler ultrasonography for measuring intracranial blood flow velocity in patients with beta-thalassemia intermedia. This study shows higher blood flow velocity in these patients compared to the control group, which may point to a higher risk of ischemic events in the future. Another paper discusses that thalassemic DNA-containing red blood cells are under oxidative stress, which induces externalization of phosphatidylserine. This mechanism is involved in the removal of these cells from the circulation by the spleen, similar to that of the removal of senescent red blood cells. This special issue also includes a paper explaining the pathophysiology of bone modifications in beta-thalassemia. Imbalance in mineral turnover resulting in abnormal regulation of bone metabolism may be related to hormonal and genetic factors, iron overload, and iron chelation therapy. These factors and their contribution are addressed. Moreover, a review article that addresses the mechanism of tissue damage arising from iron overload in patients with β-thalassemia major is presented. It is the result of oxidative stress from free radical production, altered antioxidant enzymes, and its interaction with other essential trace element levels. The last paper presented is a study comparing the quality of life in patients with β-thalassemia major and myelodysplastic syndrome with iron-overload treated either with deferasirox or deferoxamine injections. The results show that deferasirox can improve health-related quality of life treatment satisfaction and adherence compared to subcutaneous deferoxamine injection. This issue is crucial and often neglected in the long-term treatment of patients with iron overload. Mehran Karimi Sezaneh Haghpanah Ali T. Taher Maria Domenica Cappellini

Transcription Factors and MicroRNAs Expression Profiles in Reticulocytes From Beta Thalassemia Intermedia and Sickle Cell Anemia Patients,

Abstract 3201During erythroid differentiation, the expression profile of erythroid-specific genes follows a precise timing that is controlled by a complex network of transcription factors (TFs) and microRNAs (miRNAs) that regulate the expression of a set of target genes. The aim of this study was to analyze the expression profile in sickle cell anemia (SCA) and β-thalassemia (BT) patients of the TFs GATA-1, TAL-1, NFE-2, LDB1, LMO2, EKLF and of the miRNAs 24, 144, 155, 210, 221, 222, 223 and 451, which all of have been previously described in normal erythropoiesis. Reticulocytes were separated from peripheral blood samples of subjects with normal hematological data (n≥8), from untransfused BT IVS-I-6 (T→C) homozygous patients (n=9) and from SCA patients (n=8), without HU therapy. Spherocytosis patient samples were included in the results analysis. Extraction of RNA and miRNA, transcription to cDNAs and qRT-PCR were performed to analyze TFs and miRNAs expressions. In BT patients, statistical analyzes showed that the expression levels of the TFs GATA-1, TAL-1 and NFE-2 were significantly higher (p<0.05) compared to those of normal subjects. GATA-1 is a central mediator of erythroid gene expression and interacts with multiple proteins, such as TAL-1 and NFE-2, and this regulatory network is essential for normal human erythroid maturation in vivo. Many studies have proposed an interaction between these transcriptional mechanisms and some miRNAs, such as 451. MiRNA 451 is coexpressed with miRNA144 in a bicistronic pri-mRNA transcript whose synthesis is directly activated by GATA-1. This study showed that the expression profile of these miRNAs was decreased in BT patients, compared to controls, in agreement with previous studies which demonstrating that miRNA 144/451 deficiency results in erythroid hyperplasia, ineffective erythropoiesis and anemia. In addition to these miRNAs, we also analyzed the expression profile of the miRNAs 24, 155, 210, 221, 222 and 223, which were significantly lower in BT patients (p<0.05). The low expression of these miRNAs could result from oxidative processes in thalassemic red blood cells, due to the imbalance in the synthesis of alpha and beta chains that leads to a decrease in hemoglobin production and promotes free iron accumulation. This event possibly contributes to membrane’s lipid peroxidation and may cause miRNA degradation by a toxic microenvironment. In SCA patients, LMO2 and EKLF presented lower expressions (p<0.05) compared to control subjects. The expression of miRNA 221 was significantly lower in SCA patients (p<0.05), while the miRNAs 223 and 451 were significantly overexpressed in this group, compared to control subjects. One biologically relevant miRNA 223 target appears to be LMO2, a critical component of a multiprotein DNA-binding complex required for erythropoiesis. The expression of miRNA 223 was described as antagonist to the expression LMO2, corroborating our results. This study showed that the miRNAs and TFs present significant changes in their expression levels in the reticulocytes of SCA and in BT patients. Taken together these findings suggest that the miRNA post-transcriptional mechanism of erythropoietic TFs regulation could provide insights into phenotypic variation of BT and SCA patients and may contribute to the understanding of molecular pathways of these erythrocyte disorders.Support by FAPESP, CNPq and INCTS Disclosures:No relevant conflicts of interest to declare.

Cytokine-Induced Apoptosis of Beta-Thalassemia/Hemoglobin E Erythroid Progenitor Cells via Nitric Oxide-Mediated Process in vitro

Background/Aim: β-Thalassemia/hemoglobin E (β-thal/HbE) is a common hereditary anemia in Thailand. Ineffective erythropoiesis due to apoptosis and decreased lifespan of circulating thalassemic red blood cells are the major causes of anemia. Changes to bone marrow microenvironment could contribute to apoptotic events. This study examined the effects of cytokines interleukin-1β, tumor necrosis factor-α and interferon-γ on apoptosis of β-thal/HbE erythroid progenitor cells in vitro, including nitric oxide-mediated apoptotic processes. Methods: Percent apoptosis of erythroid progenitor cells from 5 β-thal/HbE patients and 5 normal control subjects was examined using flow cytometry. In addition, the inducible nitric oxide synthase (iNOS) mRNA level and nitrite production were measured using quantitative PCR and the Griess method, respectively. Results: Upon cytokine treatment, a higher percent apoptosis was obtained with β-thal/HbE erythroid progenitor cells compared with control, and the maximum effect was observed using 20 ng/ml interferon-γ on day 14 of culture. There was an increase in iNOS mRNA level and a concomitant elevation of nitrite concentration in culture medium. Apoptosis and nitrite level were abrogated when β-thal/HbE and control cells were treated with S-methylisothiourea sulfate, an iNOS inhibitor. Conclusion: The marked sensitivity of erythroid progenitor cells from β-thal/HbE patients to cytokine-induced apoptosis via an NO-mediated process reflects a proapoptotic status of such thalassemic red blood cells.

Peroxiredoxin-2 (Prx2) Membrane Displacement is A New Additional Factor to the Severity of β-Thalassemic Mouse Red Cell Membrane Damage.

Abstract 2009Poster Board I-1031Peroxiredoxin 2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells (RBCs) and is involved in defence against oxidative stress. Although much is known regarding the structure and function of Prx2 in healthy RBCs, its role in protecting pathological RBCs remains largely unexplored. Here, we studied Prx2 dimerization and membrane translocation in mouse RBCs (wild-type mice, n=20) in response to in vitro oxidative stress by either phenylhydrazine-PHZ (50 μM) or diamide (2mM) or H2O2 (75-100 μM) and in vivo in β thalassemic (β thal) mouse RBCs (Hbbth/th and Hbbth3/+ mouse strains; n=16 from each group).In RBCs lysates we observed (i) slightly increased Prx2 dimerization in PHZ treated RBCs; (ii) complete Prx2 dimerization in presence of diamide; (iii) not apparent Prx2 dimerization in H2O2, suggesting a different sensitivity of Prx2 to the various oxidant agents. In RBCs membrane, Prx2 association was (i) reduced in PHZ treated RBCs; (ii) increased in diamide treated RBCs as both monomers and dimers and (iii) reduced to almost undetectable amount in H2O2 treated RBCs. In order to evaluate whether Prx2 native or from RBCs exposed to oxidative stress possess different degrees of affinity to native or oxidized membrane, we compared Prx2 membrane association in control, diamide, PHZ treated membranes with the cytoplasmic RBC fractions obtained from control, diamide and PHZ treated RBCs. We observed a remarkable loss of interactions only between Prx2 and the membranes obtained from PHZ treated red cells (n=3), suggesting that PHZ may inhibit Prx2 binding to the membrane through the masking of its docking sites. Using multiple techniques (immunofluorescence confocal microscopy, immunoblot and flow cytometric analysis), we showed that Prx2 was displaced from PHZ treated RBC membrane in a dose dependent manner. We then investigated the connection between Prx2 membrane displacement and hemicrome binding in time course experiments with PHZ treated RBCs. Prx2 was displaced from the membrane concomitantly to hemichrome membrane binding, indicating that PHZ blocks the Prx2 binding to the membrane through the masking of its docking sites by hemichromes.Since PHZ membrane oxidative damage mimics that of b thal RBCs we have examined Prx2 in RBCs from two mouse models of b thalassemia. We showed that Prx2 content was higher in b thal mouse RBCs than in controls and correlated with the clinical severity of b thalassemia, but the amount of Prx2 associated to the membrane was markedly reduced as observed in PHZ-treated RBCs. These data suggest that in b thal RBCs the masking of Prx2 membrane binding sites by hemichromes and the accumulation of its oxidized/dimerized state in the cytoplasm may affect Prx2 catalytic efficiency on b thal membrane proteins, therefore contributing to their lack of repair from oxidative insults. Thus, the abnormally low membrane association of Prx2 in b thal mouse RBCs represents a new additional factor amplifying the oxidative damage characterizing b thal RBCs and responsible for their reduced lifespan in the periferal circulation. Disclosures:No relevant conflicts of interest to declare.

Biophysical Characterization of β-Thalassemic Red Blood Cells

Thalassemia is the world's most common hereditary disease; therefore, more interest has been devoted for the development of the screening procedure of this disease. In beta-thalassemia major, the subject of the current study, impaired biosynthesis of beta-globin leads to accumulation of unpaired alpha-globin chain. The objective of the present study, was to examine many of the biophysical properties of beta-thalassemia major red blood cells (RBCs) and to study the possibility of use of any of them as a preliminary screening tool for beta-thalassemia. The percentage of normal hemolysis, osmotic fragility test, turbidity test, rheological properties, and dielectric properties, were studied in 20 regularly blood transfused thalassemia major patients who were under chelation therapy and their status were compared with those of 10 healthy subjects. There was an increase in the percentage of hemolysis for beta-thalassemia by 114.6% compared to the normal RBCs. The fragility curve for beta-thalassemia RBCs showed a shift toward lower NaCl concentration compared to the normal curve. The average osmotic fragility (H(50): the NaCl concentration producing 50% homolysis) for beta-thalassemia was found to be 3.21 +/- 0.67 g/l, whereas for normal RBCs it was 5.5 +/- 0.31 g/l. The turbidity curve of the beta-thalassemic RBCs showed a shift toward higher detergent concentration of the normal curve, with higher value for the average membrane solubilization (S(50)). The viscosity value of whole blood beta-thalassemia was found to be 3.916 +/- 0.56 cp whereas for normal blood was 2.516 +/- 0.36 cp. The relative permittivity, dielectric loss, and AC conductivity of RBCs decreased significantly compared to normal samples. This could be attributed to the loss of the insulating properties of the membrane and loss of its surface charge of thalassemic RBCs. As can be noticed, several factors showed clear difference between thalassemic and normal blood samples. Some of these parameters could be measured immediately after sample withdrawal and require short time to perform the measurements. This offers the advantages of being effective, low cost, and fast techniques, therefore, we suggest that these techniques could be applied for beta-thalassemia major screening purposes.

A flow cytometry approach for quantitative analysis of cellular phosphatidylserine distribution and shedding

Phospholipids are asymmetrically distributed across the membrane of all cells, including red blood cells (RBCs). Phosphatidylserine (PS) is mainly localized in the cytoplasmic membrane leaflet, but during RBC ageing it flip-flops to the external leaflet—a process that is increased in certain pathological conditions (e.g., β-thalassemia). PS externalization in RBCs mediates their phagocytosis by macrophages and removal from the circulation. PS is usually measured by flow cytometry and is reported as the percentage of cells with external PS. In the current study, we developed a novel two-step flow cytometry procedure to quantitatively measure not only the external PS but also the intracellular and shed PS. In this method, PS is first bound to fluorescent annexin V, and then the residual nonbound annexin is quantified by binding to PS exposed on apoptotic cells. Using this method, we measured 1.1 ± 0.2 and 0.12 ± 0.04 μmol inner and external PS, respectively, per 10 7 normal RBCs. Thalassemic RBCs demonstrated increased PS externalization (1.7-fold) and shedding (11-fold) that was accompanied by lower intracellular PS (31%). These results suggest that quantitative flow cytometry of PS could have a diagnostic value in evaluating the pathology of RBCs in hemolytic anemias associated with increased PS externalization and shortening of the RBC life span.

Aminoacids and Fatty Acids Levels in Patients with Thalassemia Major

Introduction: The red blood cell(RBC) membrane is a complex of lipids an proteins. When thalassemic RBCs are challenged with oxidant stress, incorporation of fatty acids in RBC phospholipids is affected. We investigated the aminoacids, carnitine and fatty acid levels of thalassemic patients.Material-Method: A total number of 40 thalassemia major patient aged between 7–30 years, who has been taken regular transfusion and deferasirox therapy are included. Serum lipid profil (trigliserit, cholesterol, HDL and LDL) and ferritin levels were measured. Serum levels of aminoacids (valine, leucine/isoluecine, methionine, phenylalanine, tyrosine, aspartat, glutamat, glysine, alanine, ornitine, sitrulline, carnitine and fatty acids (C2 asetil, C3 propionil, C4 butiril, C5:1 tigilil, C5 izovaleril, C4OH3Ohbutiril, C6 hekzanol, C5OH-3 hidroksiizovaleril, C8oktanol, C10 dekanol, C5DC glutaril, C12 dodekenoil, C14.1 metilglutaril, C1 miristoil, C14OH 3 hidoksimiristoil, C16 1-palmitoleil, C16 OH 3 hidroksipalmitoleil, C18 1 oleil, C18 stearoil, C18.1OH 3 hidrosioil) were measured by using tanden-mass spectrometry.Results: There was found high level of phenylalanine in 7 (17.5%) and fatty acid: c18 oleil 1 patients (2.5%) and low level of glisin in 4 (10%), metionin 1 patients(2.5%), carnitin 1 patients (2.5%) and as fatty acids; C16 palmitol in 11 (27.5%), C3 propiponil in 8 (20%) and C2 asetil 2 patients. (5%). Other amino acids and fatty acids were normal levels. There was significant relationship between fatty acids such as C16 palmitol, C3 propiponil and lipid profil.Conclusion: Normal lipid profil and fatty acids is changed in thalassemic cells which in turn leads to altered interactions.

Spectroscopical and mechanical characterization of normal and thalassemic red blood cells by Raman Tweezers

In this work, the effects of thalassemia, a blood disease quite diffuse in the Mediterranean sea region, have been investigated at single cell level using a Raman Tweezers system. By resonant excitation of hemoglobin Raman bands, we have examined the oxygenation capability of beta-thalassemic erythrocytes. A reduction of this fundamental erythrocyte function has been found. The measurements have been performed on a significant number of red blood cells; the relative statistical analysis is presented. Moreover, the response to photo-induced oxidative stress of diseased cells with respect to the normal ones has been analyzed. Finally, the deformability of thalassemic erythrocytes has been quantified by measuring the membrane shear modulus by using a double-trap system: the measurements have revealed an increase in membrane rigidity of more than 40%, giving evidence that the genetic defect associated to thalassemia, which manly relies on hemoglobin structure, also strongly affects the erythrocyte mechanical properties. Our results demonstrate that the developed set-up may have potential for the monitoring of blood diseases and their response to drug therapies.

Fermented papaya preparation as redox regulator in blood cells of β‐thalassemic mice and patients

Many aspects of the pathology in beta-hemoglobinopathies (beta-thalassemia and sickle cell anemia) are mediated by oxidative stress. Fermented papaya preparation (FPP) was tested for its antioxidant effects: the scavenging effect was determined spectrofluorometrically in a cell-free system using 2'-7'-dichlorofluorescin-diacetate (DCF). Both spontaneous and H(2)O(2)-induced DCF oxidations were decreased by FPP in a dose-dependent fashion. Using flow cytometry, it was shown that in vitro treatment of blood cells from beta-thalassemic patients with FPP increased the glutathione content of red blood cells (RBC), platelets and polymorphonuclear (PMN) leukocytes, and reduced their reactive oxygen species, membrane lipid peroxidation and externalization of phosphatidylserine. These effects result in (a) reduced thalassemic RBC sensitivity to hemolysis and phagocytosis by macrophages; (b) improved PMN ability to generate oxidative burst - an intracellular mechanism of bacteriolysis, and (c) reduced platelet tendency to undergo activation, as reflected by fewer platelets carrying external phosphatidylserine. Oral administration of FPP to beta-thalassemic mice (50 mg/mouse/day for 3 months) and to patients (3 g x 3 times/day for 3 months), reduced all the above mentioned parameters of oxidative stress (p < 0.001 in mice and p < 0.005 in patients). These results suggest that FPP, as a potent antioxidant, might alleviate symptoms associated with oxidative stress in severe forms of thalassemia.

Increased platelet adhesion under flow conditions is induced by both thalassemic platelets and red blood cells

Thromboembolic complications are not uncommon in thalassemia. Previous studies suggest increased platelet aggregation and a potential role of pathological changes in the red blood cell (RBC) lipid membrane, induced by oxidative stress. In the present study, platelet adhesion and the effect of thalassemic RBC on platelet adhesion under flow conditions were evaluated, using the Cone and Plate (let) Analyzer(CPA). Twenty-two beta-thalassemia patients and 22 blood type-matched healthy controls were studied. An increased platelet adhesion (% surface coverage, SC), was observed in patients as compared to controls (p < 0.05). When platelet count and haematocrit were normalized by autologous reconstitution, a significant increase in platelet aggregation (average size, AS) was observed (p < 0.05). Increased platelet adhesion (SC and AS), was demonstrated in six patients with a history of thrombosis as compared to 16 patients without any history of thrombosis (p < or = 0.007) and in 17 splenectomized patients as compared to five non-splenectomized patients (p = 0.003). In reconstitution studies, thalassemic RBC mixed with normal platelet-rich plasma significantly increased platelet adhesion compared to normal RBC (SC p < 0.03, AS p < 0.02). Thalassemic platelets reconstituted with normal RBC, had increased aggregation (AS, p < 0.004) in comparison with normal platelets. The results indicate that increased platelet adhesion in beta-thalassemia is induced by both platelets and RBC. Increased platelet adhesion correlated with clinical thrombotic events and thus may suggest a mechanism of thrombosis in thalassemic patients. The potential application of the CPA in identifying thalassemic patients with high risk for thrombosis should be studied prospectively in a larger cohort of patients.

N-acetylcysteine amide (AD4) attenuates oxidative stress in beta-thalassemia blood cells

Many aspects of the pathology in β-hemoglobinopathies (β-thalassemia and sickle cell anemia) are mediated by oxidative stress. In the present study we tested a novel thiol compound, N-acetylcysteine amide (AD4), the amide form of N-acetyl cysteine (NAC) for its antioxidant effects. Using flow-cytometry, we showed that in vitro treatment of blood cells from β-thalassemic patients with AD4 elevated the reduced glutathione (GSH) content of red blood cells (RBC), platelets and polymorphonuclear (PMN) leukocytes, and reduced their ROS. These effects resulted in a significant reduced sensitivity of thalassemic RBC to hemolysis and phagocytosis by macrophages. Intra-peritoneal injection of AD4 to β-thalassemic mice (150 mg/kg) reduced the parameters of oxidative stress (p<0.001). Our results show the superiority of AD4, compared to NAC, in reducing oxidative stress markers in thalassemic cells both in vitro and in vivo.

The In Vitro and In Vivo Antioxidant Effects of Erythropoietin in Thalassemia.

Erythropoietin (EPO), a hormone produced on hypoxia mainly in the kidneys, enhances red blood cell (RBC) production (erythropoiesis) by stimulating the proliferation of erythroid progenitors and precursors in the bone marrow. This effect is mediated by the homodimeric EPO receptor, a class 1 cytokine receptor. Recombinant human EPO is widely used for the treatment of anemia, e.g., in patients on dialysis, patients with myelodysplastic syndrome and oncology patients undergoing chemotherapy. Treatment with EPO was also tried experimentally in patients with thalassemia. In these patients, in spite the state of chronic anemia, the levels of EPO is usually low relative to the degree of anemia. Administration of EPO to thalassemic patients have been shown to increase erythropoiesis and in some cases to elevate fetal hemoglobin production. In addition, EPO has been suggested to have cardio- and neuro-protecting effects and to increase RBC survival in dialysis patients. We have previously shown that RBC and platelets derived from patients with beta-thalassemia and sickle cell disease are under oxidative stress; they have elevated potential to generate reactive oxygen species (ROS) and membrane lipid peroxides, and have lower content of reduced glutathione (GSH) than normal RBC. This oxidative stress resulted in high exposure of phosphatidylserine (PS) that is considered a major factor in shortening the life span of thalassemic RBC, and in the tendency of platelets to undergo activation and thus contributes to the high incidence of thromboembolic complications in thalassemia. In the present study, we investigated the in vitro and in vivo effects of EPO as an antioxidant on RBC and platelets from beta-thalassemic patients and mice. Using flow-cytometry methodology, we showed that in vitro treatment of blood cells from beta-thalassemic patients with 1–4 U/ml of EPO for 2 hours increased the GSH content of RBC (2.1-fold) and platelets (1.7-fold) and reduced their ROS (1.5-fold), membrane lipid peroxidation and externalization of PS. Intraperitoneal injection of EPO to heterozygotes (Hbbth3/+) beta-thalassemic mice (3,000U/kg) significantly reduced ROS and increased GSH in their RBC within 3 hours. The in vitro effects of EPO on oxidative stress resulted in reduced sensitivity of thalassemic RBC to undergo hemolysis and phagocytosis by macrophages, and reduced tendency of platelets to undergo activation, as reflected by fewer platelets carrying external PS. Our results suggest that in addition to its effect on erythropoiesis and fetal hemoglobin production, EPO might alleviate symptoms in thalassemia and other hemolytic anemias as a potent antioxidant.

Na, K ATPase ?3 subunit (CD298): association with ? subunit and expression on peripheral blood cells

Beta3 subunit is described as one of the Na, K ATPase subunits. Recently, we generated a monoclonal antibody (mAb), termed P-3E10. This mAb was shown to react with the Na, K ATPase beta3 subunit or CD298. By immunofluorescence analysis using mAb P-3E10, it was found that all peripheral blood leukocytes express Na, K ATPase beta3. The presence of beta3 subunit on leukocytes is not in a quantitative polymorphic manner. Upon phytohemagglutinin or phorbol myristate acetate activation, the expression level of the Na, K ATPase beta3 subunit on activated peripheral blood mononuclear cells was not altered in comparison with those of unstimulated cells. Red blood cells (RBCs) of healthy donors showed negative reactivity with mAb P-3E10. However, more than 80% of thalassemic RBCs showed positive reactivity. By immunoprecipitation, moreover, a protein band of 55-65 kDa was precipitated from normal RBC membrane using mAb P-3E10. These results evidenced that the beta3 subunit of Na, K ATPase is expressed on RBC membrane but the epitope recognized by mAb P-3E10 is hidden in normal RBCs. Furthermore, we showed the association of beta3 subunit and alpha subunit of Na, K ATPase. This information is important for further understanding of the functional roles of this molecule.

Dysregulation of Phosphatidylserine Scrambling by Oxidative Stress.

An abnormal population of red blood cells (RBC) that exposes phosphatidylserine (PS) has been found in patients with sickle cell anemia and thalassemia. Enhanced formation of PS-exposing RBC results in their premature demise as these cells are removed from the circulation, and contributes to disease complications in the case that these cells are insufficiently removed. We hypothesize that increased oxidative stress, a common indication in these RBC, contributes to increased PS exposure. To evaluate the underlying mechanism, we compared the PS scrambling rate in three different mouse strains that sustain oxidative stress to their RBC: sickle mice, thalassemic mice (C57/Hbb[th3]), and mice deficient in peroxiredoxin II (prdx −/−). Oxidative damage is prominent in sickle and thalassemic RBC because of the presence of damaged hemoglobin and free heme components, and in prdx −/− mice because they lack a key component of oxidative damage repair. We determined the PS scrambling rate resulting from loading RBC with 100 microM intracellular Ca2+, in presence of either NEM (sulfhydryl alkylation), PDA (sulfhydryl modification), vanadate (flippase/ATPase inhibitor), or oligomycin (scrambling stimulation). As previously reported, sulfhydryl modifications cause flippase inhibition, and also change the PS scrambling rate with NEM treatment stimulating scrambling but PDA treatment reducing it. Sickle cells showed a 10% reduced susceptibility to NEM modification and no susceptibility to PDA, indicating shielding of the scramblase sulfhydryl groups. Thalassemic and Prdx −/− RBC showed a 30% and 10% reduced response to NEM modification, respectively, but normal reaction with PDA. The prdx −/− RBC contain a subpopulation that is severely oxidized as detected by an increase in autofluorescence. This cell population had a 10–15% lower susceptibility to NEM and PDA compared to the remaining population. The maximum percentage of PS-exposing cells (with oligomycin) was 10–20% reduced in all transgenic strains compared to normal, and even further reduced in the highly oxidized prdx−/− cells, indicating that a subset of cells had lost its ability to expose PS altogether. Yet, the baseline scrambling rate with 100 microM Ca2+ was increased 3-fold in thalassemic RBC, 1.5-fold in sickle mice, and 3-fold in the highly oxidized prdx −/− cells compared to normal mouse RBC, confirming the hypothesis that increased oxidative damage results in a higher propensity to scramble. Vanadate treatment had a stimulating effect on mouse RBC scrambling resulting in similar PS exposure in all mouse strains. While flippase activity was reduced in sickle RBC, this was not the case in thalassemic and prdx −/− RBC. In all RBC, increased scrambling activity resembled alterations similar to those seen after NEM treatment. Our data show that a subpopulation of oxidatively damaged cells is more prone to PS exposure during Ca2+ influx due to sulfhydryl modification. In addition, some oxidatively damaged cells lose their ability for Ca-induced scrambling. Together, we conclude that scrambling is dysregulated in oxidatively challenged RBC.

Cytoadherence between endothelial cells and P. falciparum infected and noninfected normal and thalassemic red blood cells

Cytoadhesion of P. falciparum infected red blood cells (RBCs) to endothelial cells (ECs) is an important phenomenon that causes cerebral malaria in man. Reduced adhesion especially in thalassemia and hemoglobinopathies may be related to a protective mechanism against malaria in such people. The cytoadherence assay was performed using both conventional and floating conditions between ECs (ECV 304) and P. falciparum infected and noninfected RBCs from both normal and thalassemia subjects. In floating condition, RBC was fluorescently labeled with anti-glycophorin A antibody, whereas EC was identified by surface expression of PECAM-1, CD-36, ICAM-1, and E-selectin. The condition of floating EC was similar to the condition for subcultivation as they can adhere or bind to any surface. The phosphatidylserine (PS) exposure was also determined by using flow cytometer. The adhesion of noninfected heterozygous thalassemic RBCs (all genotypes) to ECs was significantly increased as compared with normal RBCs (P < 0.02). Interestingly, after P. falciparum infection, the number of normal RBCs bound to ECs was significantly increased as compared with noninfected RBCs (P < 0.01), whereas heterozygous thalassemic RBCs infected by P. falciparum showed no significant difference compared with noninfected RBCs. In addition, we found a similar level of PS exposure in normal and thalassemic infected RBCs, which was related to the cytoadherence phenomenon. The reduced adhesion between heterozygous thalassemic RBCs infected by P. falciparum to ECs provides an explanation for their protective mechanism against malaria, as increased adhesion is a high risk for cerebral malaria and nonbinding infected RBCs can be removed by the reticuloendothelial system and other mechanism(s) in vivo.

H 2O 2 injury in β thalassemic erythrocytes: Protective role of catalase and the prooxidant effects of GSH

Redox-mediated injury is an important pathway in the destruction of β thalassemic red blood cells (RBC). Because of the autoxidation of the unstable hemoglobin chains and subsequent release of globin free heme and iron, significant amounts of superoxide (O 2 −) and, more importantly, hydrogen peroxide (H 2O 2) are generated intracellularly. Hence, catabolism of H 2O 2 is crucial in preventing cellular injury. Removal of H 2O 2 is mediated via two primary pathways: GSH-dependent glutathione peroxidase or catalase. Importantly, both pathways are ultimately dependent on NADPH. In the absence of any exogenous oxidants, model thalassemic RBC demonstrated significantly decreased GSH levels ( P < 0.001 at 20 h). Perhaps of greater pathophysiologic importance, however, was the finding that the model thalassemic RBC exhibited significantly ( P < 0.001) decreased catalase activity. Following 20 h incubation at 37°C only 61.5 ± 2.9% of the initial catalase activity remained in the α-hemoglobin chain-loaded cells versus 104.6 ± 4.5 and 108.2 ± 3.2% in the control and control-resealed cells, respectively. The mechanism underlying the loss of both catalase activity and GSH appears to be the same in that both catabolic pathways require adequate NADPH levels. As shown in this study, model β thalassemic cells are unable to maintain a normal (∼1.0) NADPH/NADP total ratio and, after 20 h, the model β thalassemic cells have a significantly ( P < 0.001) lower ratio (∼0.5) which is quite similar to a G6PD-deficient RBC. In support of these findings, direct inactivation of catalase gives rise to significantly increased oxidant damage. In contrast, GSH depletion is not closely associated with oxidant sensitivity. Indeed, the consumption of GSH noted in the thalassemic RBC may be via a prooxidant pathway as augmentation of cellular GSH levels actually enhances α-hemoglobin chain-mediated injury.

Ultrastructural assessment of Plasmodium falciparum in age-fractionated thalassaemic erythrocytes

Culture of Plasmodium falciparum in age-fractionated thalassaemic red blood cells (RBC) has shown evidence of parasite damage on light microscopy in older cells during the third culture cycle (96-144 h). In this report, parasites growing in thalassaemic trait and normal RBC were examined ultrastructurally from 96 to 144 h. All parasite stages in old thalassaemic RBC showed evidence of damage worsening with culture duration. There were cytoplasmic alterations with ribosomal damage, and parasite cytoplasm became increasingly loose and grainy, with multiple fissures. Discontinuity of the nuclear membrane with an abnormal nucleolus was seen at l20 h. Cytosomes remained normal, but damage to the food vacuole and shrunken disintegrating parasites were observed at 144 h. These changes are compatible with cellular degeneration and developmental retardation and would account for the schizont maturation arrest and reduced reinvasion rates previously reported. Increased free radicals associated with thalassaemic erythrocytes would explain these changes, further supporting the role for oxidant stress in the protective mechanism.

Measuring Chromosome Breaks in Patients with Thalassemia

Iron-mediated oxidative stress plays an important role in the pathophysiology of thalassemia. Oxidative stress can cause lesions in DNA, including double-strand breaks. DNA damage, which is a cause of cancer (although not the only one), is recognized as deleterious. Unlike cancer, DNA damage can be assayed easily and relatively inexpensively in humans. In this study, a sensitive micronucleus assay was used to measure the frequency of chromosomal breaks in patients with alpha- and beta-thalassemia. The micronucleus test is based on the observation that a secondary nucleus (micronucleus) is formed around a chromosomal fragment, outside the main nucleus of a dividing cell. Micronuclei are readily apparent in red blood cells (RBCs), which otherwise lack DNA. We combined an immunomagnetic separation technique with single-laser flow cytometry to isolate and analyze reticulocytes in peripheral blood for the presence of micronuclei before these cells are removed by the spleen. Blood samples were obtained from patients with thalassemia and healthy volunteers. After immunomagnetic enrichment of CD71-positive reticulocytes, the cells were stained for micronuclei using the DNA dye 7-aminoactinomycin D (7-AAD) and evaluated by flow cytometry. Our findings indicate that higher levels of micronuclei frequencies are present in thalassemic RBCs.