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The major hemoglobin (Hb) during fetal life is fetal Hb (Hb F). It is mostly replaced by adult Hbs before birth and during the first year of life. In adults, where Hb F comprises <2.0% of the total Hb, it is not homogenously distributed among the red blood cells (RBCs) but is concentrated in a few RBCs, termed F-cells. Interestingly, for reasons that are unclear, Hb F increases in the maternal circulation during pregnancy. This increased Hb F could have two potential origins that are not mutually exclusive: A) maternal origin, due to inducing environment of Hb F in the maternal erythroid precursors; B) fetal origin, due to fetal cells crossing the placenta and entering the maternal circulation. The question we present herein is whether the observed increased Hb F in the maternal circulation during pregnancy is, at least partially, derived from the fetal origin. Peripheral blood was obtained from normal neonates (1-3 days old), adult men and pregnant and non pregnant women. The RBCs were stained for Hb F and carbonic anhydrase (CA) using a fetal cell count kit and analyzed by flow cytometry. Fetal and adult F-cells were distinguished by their expression of Hb F and CA. Fetal F-cells were Hb F ++ /CA - , while adult F-cells were Hb F + /CA + . Comparing pregnant and non pregnant women samples (n = 10), we found six samples of pregnant women with 0.2-1.7% fetal cells, but none in the non pregnant group. These results support the possibility that at least part of the increase in Hb F during pregnancy is due to fetal cells entering the maternal circulation.

Background: Fetal-maternal hemorrhage (FMH) occurs when fetal red blood cells (RBC) pass into the maternal circulation as a result of obstetric- or trauma-related complications to pregnancy. Their detection in the maternal blood is commonly used as a diagnostic test. There is, however, a serious and general limitation to this test that is sometimes ignored. Fetal RBC carrying the father's antigens (most crucially, the ABO blood antigens) may be incompatible with the mother's plasma. They are expected to be eliminated by the maternal natural antibodies, thus, negative results may be false., Objectives: By simulating fetal-maternal ABO incompatibility, we studied the fate of fetal RBC in vitro., Methods: Adult blood samples (n = 6) of O-blood group (type) were mixed with 1-5% cord blood or neonatal blood of A- or O-type, representing incompatible and compatible fetal RBC, respectively. The survival of fetal RBC was quantified after an overnight incubation. The supernatant was assayed for fetal hemoglobin (HbF) using the spectrophotometric alkaline-resistance benzidine assay, while the pellet was assayed for HbF/carbonic anhydrase (CA) expression in RBC by flow cytometry. The HbFhigh/CAlow phenotype characterizes fetal RBC., Results: Both assays demonstrated disappearance of the fetal RBC due to lysis upon incubation in incompatible blood., Conclusions: A similar situation may also occur in vivo. Thus, under these conditions, negative results in the FMH test may be false, and lead to misdiagnosis., (© 2018 S. Karger AG, Basel.)

Thalassemia (thal) is an autosomal recessive, hereditary, chronic hemolytic anemia due to a partial or complete deficiency in the synthesis of α-globin chains (α-thal) or β-globin chains (β-thal) that compose the major adult hemoglobin (α 2 β 2). It is caused by one or more mutations in the corresponding genes. The unpaired globin chains are unstable; they precipitate intracellularly, resulting in hemolysis, premature destruction of red blood cell [RBC] precursors in the bone marrow, and a short life-span of mature RBCs in the circulation. The state of anemia is treated by frequent RBC transfusions. This therapy results in the accumulation of iron (iron overload), a condition that is exacerbated by the breakdown products of hemoglobin (heme and iron) and the increased iron uptake for the chronic accelerated, but ineffective, RBC production. Iron catalyzes the generation of reactive oxygen species, which in excess are toxic, causing damage to vital organs such as the heart and liver and the endocrine system. Herein, we review recent findings regarding the pathophysiology underlying the major symptoms of β-thal and potential therapeutic modalities for the amelioration of its complications, as well as new modalities that may provide a cure for the disease., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

While most common symptom of impairment of iron homeostasis is iron deficiency anemia, some hematological disorders are associated with iron overload (IO). These disorders are related mainly to chronic severe hemolytic anemia, where red blood cells (RBC) or their precursors are destroyed prematurely (hemolyzed), leading to anemia that cannot be compensated by increased production of new RBC. In such cases, IO is mainly due to repeated RBC transfusions and/or increased uptake of iron in the gastrointestinal tract. Normally, iron is present in the plasma and in the cells bound to compounds that render it redox inactive. Iron overload leaves a fraction of the iron free (labile iron pool) and redox active, leading to the generation of excess free radicals such as the reactive oxygen species. This condition upsets the cellular redox balance between oxidants and antioxidants, leading to oxidative stress. The free radicals bind to various cellular components, thereby becoming toxic to vital organs. Oxidative stress may also affect blood cells, such as RBC, platelets and neutrophils, exacerbating the anemia, and causing recurrent infections and thrombotic events, respectively. The toxic effect of IO can be decreased by treating the patients with iron chelators that enter cells, bind free iron and remove it from the body through the urine and feces. Iron toxicity may be also ameliorated by treatment with anti-oxidants that scavenge free radicals and/or correct their damage. The use of iron chelators is widely accepted when started in young patients with severe chronic anemia, but is still debatable as a therapeutic modality for older patients suffering from IO due to myelodysplastic syndromes. It should be noted that in addition to preventing iron toxicity, some compounds with iron chelator activity may also benefit other aspects of hematological disorders. These aspects include stimulation of platelet production, inhibition of leukemic cell proliferation and induction of their differentiation. Compounds with such multiple activities may prove beneficial for at least some patients with leukemia and myelodysplastic syndromes., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

The formation of new blood vessels plays a crucial for the development and progression of pathophysiological changes associated with a variety of disorders, including carcinogenesis. Angiogenesis inhibitors (anti-angiogenics) are an important part of treatment for some types of cancer. Some natural products isolated from marine invertebrates have revealed antiangiogenic activities, which are diverse in structure and mechanisms of action. Many preclinical studies have generated new models for further modification and optimization of anti-angiogenic substances, and new information for mechanistic studies and new anti-cancer drug candidates for clinical practice. Moreover, in the last decade it has become apparent that galectins are important regulators of tumor angiogenesis, as well as microRNA. MicroRNAs have been validated to modulate endothelial cell migration or endothelial tube organization. In the present review we summarize the current knowledge regarding the role of marine-derived natural products, galectins and microRNAs in tumor angiogenesis., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Iron is an essential element for key cellular metabolic processes. However, transfusional iron overload (IOL) may result in significant cellular toxicity. IOL occurs in transfusion dependent hematologic malignancies (HM), may lead to pathological clinical outcomes, and IOL reduction may improve outcomes. In hematopoietic stem cell transplantation (SCT) for HM, IOL may have clinical importance; endpoints examined regarding an impact of IOL and IOL reduction include transplant-related mortality, organ function, infection, relapse risk, and survival. Here we review the clinical consequences of IOL and effects of IOL reduction before, during and following SCT for HM. IOL pathophysiology is discussed as well as available tests for IOL quantification including transfusion history, serum ferritin level, transferrin saturation, hepcidin, labile plasma iron and other parameters of iron-catalyzed oxygen free radicals, and organ IOL by imaging. Data-based recommendations for IOL measurement, monitoring and reduction before, during and following SCT for HM are made., (Copyright © 2017 Elsevier B.V. All rights reserved.)

β-Thalassemia (β-thal) is a very common disease in the Palestinian population of the Gaza Strip. We studied their mutation frequency and clinical features. Thirteen different mutations were identified. The most common mutation was IVS-I-1 (G>A) (HBB: c.92+1G>A), which was prevalent in 31.5% of the thalassemia alleles studied. The IVS-I-110 (G>A) (HBB: c.93-21G>A) mutation was found in 25.0% of the alleles. Homozygotes for the IVS-I-1 mutation had higher mean hemoglobin (Hb) levels, required less blood transfusions, and lower transferrin saturation than the homozygotes for the IVS-I-110 mutation. This milder phenotype was, most likely, the result of the persistent production of Hb F; it was 9-fold higher in absolute terms (g/dL) and 7.7-fold higher in relative terms (percentage of total Hb). About half of our IVS-I-1 patients carried the XmnI polymorphism, which is known to be associated with elevated Hb F levels.

The JAK2V617F mutation that results in a hyper-activation of the JAK2 kinase in the erythropoietin pathway is a molecular marker for myeloproliferative neoplasms. Using allele-specific Real-Time PCR, we detected the mutation in the blood of 17.3% (17/98) of normal donors; the mutant allele burden was, however, very low (<0.01% compared to >1% in polycythemia vera). It was much higher in differentiated blood cells in the peripheral blood than in undifferentiated CD34 + cells. Erythropoietin-stimulated differentiation of normal CD34 + cells in liquid culture increased the mutation frequency by 3.34-fold. When progenitors from 9 normal donors were grown in erythropoietin-stimulated semi-solid cultures, the mutation was found in 8.69% of the colonies, but only in <3% of the JAK2 alleles in each positive colony, suggesting that the mutation occurred only in a few cells per colony. In mouse erythroleukemia cells carrying human JAK2 DNA, wild-type or JAK2V617F, the frequencies of mutations from JAK2 wild-type to JAK2V617F and vice versa increased following erythroid differentiation. These results suggest that the mutation occurs and accumulates during differentiation. We hypothesize that genetic stability, which relies on DNA repair, is efficient in normal hematopoietic stem cells but is downgraded in differentiating cells, rendering them susceptible to mutations, including JAK2V617F., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Anemia is a major cause of morbidity and mortality worldwide resulting from a wide variety of pathological conditions. In severe cases it is treated by blood transfusions or injection of erythroid stimulating agents, e.g., erythropoietin (Epo), which can be associated with serious adverse effects. Therefore, there is a need to develop new treatment modalities. We recently reported that treatment of erythroleukemic cells with the novel the bi-functional prodrugs of 5-aminolevulinic acid (ALA) and butyric acid (BA), AN233 and AN908, enhanced hemoglobin (Hb) synthesis to a substantially higher level than did ALA and BA individually or their mixture. Herein, we describe that these prodrugs when given orally to mice induced histone deacetylase inhibition in the kidneys, bone marrow and spleen, thus, indicating good penetrability to the tissues. In mice where anemia was chemically induced, treatment with the prodrugs increased the Hb, the number of red blood cells (RBCs) and the percentage of reticulocytes to normal levels. The prodrugs had no adverse effects even after repeated treatment at 100-200mg/kg for 50days. The lack of increased levels of Epo in the blood of mice that were treated with the prodrugs suggests that AN233 and AN908 affected the Hb and RBC levels in an Epo-independent manner. Taken together with our previous studies, we propose that the prodrugs increase globin expression by BA inhibition of histone deacetylase and elevation heme synthesis by ALA. These results support an Epo-independent approach for treating anemia with these prodrugs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The complement (C') system and redox status play important roles in the physiological functioning of the body, such as the defense system, but they are also involved in various pathological conditions, including hemolytic anemia. Herein, we review the interaction between the C' and the redox systems in C'-mediated hemolytic anemias, paroxysmal nocturnal hemoglobinuria (PNH) and autoimmune hemolytic anemia, including acute hemolytic transfusion reaction. Blood cells in these diseases have been shown to have increased oxidative status, which was further elevated by interaction with activated C'. The results suggest that oxidative stress, in conjunction with activated C', may cause the underlying symptoms of these diseases, such as intra- and extravascular hemolysis and thrombotic complications. Antioxidants ameliorate oxidative stress by preventing generation of free radicals, by scavenging and preventing their accumulation, and by correcting their cellular damage. Antioxidants have been shown to reduce the oxidative stress and inhibit hemolysis as well as platelet activation mediated by activated C'. This raises the possibility that treatment with antioxidants might be considered as a potential therapeutic modality for C'-mediated hemolytic anemias. Currently, eculizumab, a humanized monoclonal antibody that specifically targets the C' protein C5, is the main treatment modality for PNH. However, because antioxidants are well tolerated and relatively inexpensive, they might be considered as potential adjuvants or an alternative therapeutic modality for PNH and other C'-mediated hemolytic anemias., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Exposure to ionizing radiation causes cellular damage, which can lead to premature cell death or accumulation of somatic mutations, resulting in malignancy. The damage is mediated in part by free radicals, particularly reactive oxygen species. Fermented papaya preparation (FPP), a product of yeast fermentation of Carica papaya Linn, has been shown to act as an antioxidant. In this study, we investigated the potential of FPP to prevent radiation-induced damage. FPP (0-100 μg/ml) was added to cultured human foreskin fibroblasts and myeloid leukemia (HL-60) cells either before or after irradiation (0-18 Gy). After 1-3 days, the cells were assayed for: intracellular labile iron, measured by staining with calcein; reactive oxygen species generation, measured with dichlorofluorescein diacetate; apoptosis, determined by phosphatidylserine exposure; membrane damage, determined by propidium iodide uptake; and cell survival, determined by a cell proliferation assay. DNA damage was estimated by measuring 8-oxoguanine, a parameter of DNA oxidation, using a fluorescent-specific probe and by the comet assay. These parameters were also assayed in bone marrow cells of mice treated with FPP (by adding it to the drinking water) either before or after irradiation. Somatic mutation accumulation was determined in their peripheral red blood cells, and their survival was monitored. FPP significantly reduced the measured radiation-induced cytotoxic parameters. These findings suggest that FPP might serve as a radioprotector, and its effect on DNA damage and mutagenicity might reduce the long-term effects of radiation, such as primary and secondary malignancy.

Several investigations have demonstrated a mild clinical status in patients with β-globin disorders and congenital high persistence of foetal haemoglobin. This can be mimicked by a pharmacological increase of foetal γ-globin genes expression and foetal haemoglobin production. Our goal was to apply a multistep assay including few screening methods (benzidine staining, RT-PCR and HPLC analyses) and erythroid cellular model systems (the K562 cell line and erythroid precursors collected from peripheral blood) to select erythroid differentiation agents with foetal haemoglobin inducing potential. With this methodology, we have identified a butyric acid derivative, namely the 4174 cyclopropanecarboxylic acid compound, able to induce erythroid differentiation without antiproliferative effect in K562 cells and increase of γ-globin gene expression in erythroid precursor cells. The results are relevant for pharmacological treatments of haemoglobinopathies, including β-thalassaemia and sickle cell anaemia., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Patients with β-thalassemia major and mainly intermedia have an increased risk for developing venous and arterial thrombosis which may be related to circulating pathological red blood cells (RBC) and continuous platelet activation. In the present study we used a modified thalassemic mice model in conjunction with a "real-time" carotid thrombus formation procedure to investigate thrombotic complications of thalassemia. Heterozygous Th3/+ mice, which lack one copy of their β-major and β-minor globin genes, exhibit anomalies in RBC size and shape, chronic anemia and splenomegaly which recapitulate the phenotype of human β-thalassemia intermedia. Flow cytometry measurements showed higher reactive oxygen species generation, indicating oxidative stress, in platelets and RBC of the thalassemic mice compared with wild type mice concomitant with an increase in reduced glutathione content which may represent a compensatory response to oxidative stress, and exposed phosphatidylserine which indicates platelet activation. To elucidate the effect of thalassemia on the development of arterial thrombosis, we studied photochemical-induced real-time thrombus formation in the carotid artery of these mice. The results indicated a significantly shorter "time to occlusion" in the thalassemic mice compared to wild type mice, which was prolonged following in vivo aspirin treatment. We suggest that this mouse model may contribute to our understanding of platelet activation and the hypercoagulable state in thalassemia and lay foundations to screening of anti-platelet drugs as well as anti-oxidants as possible therapeutics for prevention of thrombosis in thalassemia patients., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This review presents the indications and contraindications (pros and cons) for the potential use of erythropoietin (Epo) as a treatment in β-thalassemia and sickle cell anemia (SCA). Its high cost and route of administration (by injection) are obvious obstacles, especially in underdeveloped countries, where thalassemia is prevalent. We believe that from the data summarized in this review, the time has come to define, by studying in vitro and in vivo models, as well as by controlled clinical trials, the rationale for treating patients with various forms of thalassemia and SCA with Epo alone or in combination with other medications., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Fetal hemoglobin (HbF), which is largely replaced after birth by the adult Hb, is concentrated in a few "F cells." Their number significantly increases in certain physiologic and clinical situations, including in β-thalassemia (β-thal). Their quantification is used to detect fetal-maternal hemorrhage (FMH), where fetal cells enter the maternal circulation. We were confronted with a pregnant woman with β-thal who was suspected to have FMH. To establish the usefulness of a flow cytometric procedure to differentiate between fetal cells and the maternal F cells, we screened adult β-thal patients., Study Design and Methods: Blood samples were simultaneously stained with fluorescent antibodies to HbF and to carbonic anhydrase (CA) isotype II, which is specific to adult red blood cells (RBCs)., Results: A heterogeneous distribution of RBCs with respect to HbF and CA expression was observed: adult non-F cells (CA+HbF-) and F cells (CA+HbF+/HbF++) as well as F cells with characteristics of fetal cells (CA-HbF++)., Conclusions: The presence of CA-HbF++ RBCs in nonpregnant women, and even men, with thal indicates that the CA/HbF method is inappropriate for detection of FMH. The coexistence of F cells carrying fetal or adult markers suggests that they originate from two types of stem cell, adult and fetal, lineages. Normally, the fetal lineage is insignificant, but in β-thal, as HbF-containing RBCs have a selective advantage, the "fetal" lineage gains significance., (© 2012 American Association of Blood Banks.)

Phosphatidylserine (PS), which is normally localized in the cytoplasmic leaflet of the membrane, flip-flops to the external leaflet during aging of, or trauma to, cells. A fraction of this PS undergoes shedding into the extracellular milieu. PS externalization and shedding change during maturation of erythroid cells and affect the functioning, senescence and elimination of mature RBCs. Several lines of evidence suggest dependence of PS shedding on intracellular Ca concentration as well as on interaction between plasma membrane phospholipids and microtubules (MTs), the key components of the cytoskeleton. We investigated the effect of Ca flux and MT assembly on the distribution of PS across, and shedding from, the membranes of erythroid precursors. Cultured human and murine erythroid precursors were treated with the Ca ionophore A23187, the MT assembly enhancer paclitaxel (Taxol) or the inhibitor colchicine. PS externalization and shedding were measured by flow cytometry and the cholesterol/phospholipids in RBC membranes and supernatants, by ¹H-NMR. We found that treatment with Taxol or colchicine resulted in a marked increase in PS externalization, while shedding was increased by colchicine but inhibited by Taxol. These results indicate that PS externalization is mediated by Ca flux, and PS shedding by both Ca flux and MT assembly. The cholesterol/phospholipid ratio in the membrane is modified by PS shedding; we now show that it was increased by colchicine and A23187, while taxol had no effect. In summary, the results indicate that the Ca flux and MT depolymerization of erythroid precursors mediate their PS externalization and shedding, which in turn changes their membrane composition.

The pathophysiology of oxidative hemolytic anemia is closely associated with hemoglobin (Hb) stability; however, the mechanism of how Hb maintains its stability under oxidative stress conditions of red blood cells (RBCs) carrying high levels of oxygen is unknown. Here, we investigated the potential role of peroxiredoxin II (Prx II) in preventing Hb aggregation induced by reactive oxygen species (ROS) using Prx II knockout mice and RBCs of patients with hemolytic anemia. Upon oxidative stress, ROS and Heinz body formation were significantly increased in Prx II knockout RBCs compared to wild-type (WT), which ultimately accelerated the accumulation of hemosiderin and heme-oxygenase 1 in the Prx II knock-out livers. In addition, ROS-dependent Hb aggregation was significantly increased in Prx II knockout RBCs. Interestingly, Prx II interacted with Hb in mouse RBCs, and their interaction, in particular, was severely impaired in RBCs of patients with thalassemia (THAL) and sickle cell anemia (SCA). Hb was bound to the decameric structure of Prx II, by which Hb was protected from oxidative stress. These findings suggest that Prx II plays an important role in preventing hemolytic anemia from oxidative stress by binding to Hb as a decameric structure to stabilize it., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background/aims: Erythropoiesis in long-term hemodialyzed (LTH) patients is supported by erythropoietin (rHuEpo) and intravenous (IV) iron. This treatment may end up in iron overload (IO) in major organs. We studied such patients for the parameters of IO in the serum and in major organs., Methods: Patients were treated with rHuEpo (6-8 x 10(3) units × 1-3/wk) and IV 100 mg ferric saccharate., Results: Of 115 patients, 21 had serum ferritin (SF) > 1000 ng/mL. This group was further analyzed. Their SF and transferrin saturation (TSAT) were 2688 ± 1489 ng/mL and 54.2 ± 32.7%, respectively (vs. 125-360 ng/mL and 20-50% in normal controls). Serum hepcidin was 60.1 ± 29.5 nm (vs. 10.61 ± 6.44 nm in controls) (P < 0.001). Nineteen patients had increased malonyldialdehyde, a product of lipid peroxidation, indicating oxidative stress. T2* MRI disclosed in 19 of 21 patients moderate to severe IO in the liver and spleen, in three of eight patients in the pancreas, but in no patient in the heart. After stopping IV iron for a mean of 12 months, while continuing rHuEpo, the mean SF decreased in 11 patients to 1682 ng/mL and the mean TSAT decreased to 28%, whereas hemoglobin did not change indicating that tissue iron was utilized., Conclusion: High SF correlates with IO in the liver and spleen, but not in the heart., (© 2012 John Wiley & Sons A/S.)

Thalassemia and sickle-cell anemia (SCA) present a major public health problem in countries where the number of carriers and affected individuals is high. As a result of the abnormalities in hemoglobin production, cells of thalassemia and SCA patients exhibit oxidative stress, which ultimately is responsible for the chronic anemia observed. Therefore, identification of compounds exhibiting both antioxidant and hemoglobin-inducing activities is highly needed. Our results demonstrate resveratrol to be such a compound. This was shown both in the human K562 cell line, as well as in erythroid precursors derived from normal donors and β-thalassemia patients. Resveratrol was shown to exhibit antioxidant activity and to stimulate the expression of the γ-globin genes and the accumulation of fetal hemoglobin (HbF). To the best of our knowledge, this is the first report pointing to such a double effect of resveratrol. Since this natural product is already marketed as an antioxidant, future investigations should concentrate on demonstrating its potential to augment HbF production in experimental animal models (e.g., thalassemia and SCA mice) as well as in patients. We believe that the potential of clinical use of resveratrol as an antioxidant and HbF stimulator may offer a simple and inexpensive treatment to patients.

Strategies that increase homing to the bone marrow and engraftment efficacy of ex vivo expended CD34(+) cells are expected to enhance their clinical utility. Here we report that nicotinamide (NAM), a form of vitamin B-3, delayed differentiation and increased engraftment efficacy of cord blood-derived human CD34(+) cells cultured with cytokines. In the presence of NAM, the fraction of CD34(+)CD38(-) cells increased and the fraction of differentiated cells (CD14(+), CD11b(+), and CD11c(+)) decreased. CD34(+) cells cultured with NAM displayed increased migration toward stromal cell derived factor-1 and homed to the bone marrow with higher efficacy, thus contributing to their increased engraftment efficacy, which was maintained in competitive transplants with noncultured competitor cells. NAM is a known potent inhibitor of several classes of ribosylase enzymes that require NAD for their activity, as well as sirtuin (SIRT1), class III NAD(+)-dependent-histone-deacetylase. We demonstrated that EX-527, a specific inhibitor of SIRT1 catalytic activity, inhibited differentiation of CD34(+) cells similar to NAM, while specific inhibitors of NAD-ribosylase enzymes did not inhibit differentiation, suggesting that the NAM effect is SIRT1-specific. Our findings suggest a critical function of SIRT1 in the regulation of hematopoietic stem cell activity and imply the clinical utility of NAM for ex vivo expansion of functional CD34(+) cells., (Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Iron-overload is a major clinical problem in various diseases. Under this condition, serum iron which surpasses the binding capacity of transferrin is present as non-transferrin bound iron and cellular unbound Labile Iron Pool (LIP) is increased. LIP participates in the generation of free radicals, including reactive oxygen species (ROS). Increased ROS, with concomitant decrease in anti-oxidants, results in oxidative stress and toxicity to the liver, heart and other tissues, causing serious morbidity and eventually mortality. Therapeutic iron chelation reduces the LIP and thereby ameliorates oxidative stress-mediated toxicity. Many food-derived antioxidants have the capacities to scavenge ROS and chelate iron. We have reported that fermented papaya preparation (FPP) has ROS scavenging effect on blood cells in vitro or in vivo (in thalassemic patients and experimental animals). We now investigated FPP's iron chelating effect - its ability to prevent (and revert) LIP accumulation. Liver- and heart-derived cells, and RBCs were exposed to non-transferrin bound iron in the form of ferrous ammonium sulfate and the effect of FPP on their LIP content and ROS generation was measured by flow-cytometry. The results indicate that FPP reduces LIP and ROS, and suggests that its antioxidant mechanism is related, at least in part, to iron chelation.

Diamond-Blackfan anemia (DBA) is congenital pure red-cell anemia due to a differentiation block in erythroid precursors. The disease is commonly caused by mutations in genes for ribosomal proteins. Despite the identification of disease causal genes, the disease pathogenesis is not completely elucidated. The ribosomal abnormalities are assumed to inhibit globin translation which may lead to excess free heme, stimulating a generation of free radicals and thereby damaging the precursors. We studied the effect of hemin (heme chloride) on cultured human erythroid precursors and found that contrary to aforementioned hypothesis, although hemin moderately stimulated free radicals, it did not cause apoptosis or necrosis. In erythroid precursors derived from DBA patients, hemin significantly stimulated growth and hemoglobinization. Thus, heme toxicity is unlikely to play a role in the pathophysiology of most DBA cases. Moreover, its beneficial effect in culture suggests a therapeutic potential.

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-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 β-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 β-globin through a novel mechanism that links the rate of transcription of the transgenic β-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 β-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 β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-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.

We studied the nature of enucleated RBCs containing DNA remnants, Howell-Jolly (HJ) RBCs and reticulocytes (retics), that are characteristically present in the circulation of thalassemic patients, especially after splenectomy. Using flow cytometry methodology, we measured oxidative status parameters of these cells in patients with β-thalassemia. In each patient studied, these cells had higher content of reactive oxygen species and exposed phosphatidylserine compared with their DNA-free counterparts. These results suggest that oxidative stress in thalassemic developing erythroid precursors might, through DNA-breakage, generate HJ-retics and HJ-RBCs and that oxidative stress-induced externalization of phosphatidylserine is involved in the removal of these cells from the circulation by the spleen, a mechanism similar to that of the removal of senescent RBCs.

Maturation and aging of erythroid cells are accompanied by extensive remodeling of the membrane and a marked decrease in cell size, processes that are mediated by externalization and shedding of phosphatidylserine (PS). In the present study, we investigated the redistribution of PS in the plasma membrane of erythroid precursors during their maturation and of mature RBCs during senescence, and the involvement of changes in calcium (Ca)-flux in these processes. Maturation was studied by analyzing normal human bone marrow cells as well as cultured human normal erythroid precursors induced by erythropoietin and murine erythroleukemia cells induced by hexamethylene-bisacetamide. Senescence was studied in normal human peripheral RBCs following density fractionation. PS and Ca were determined by flow cytometry using annexin-V and Flu-3, respectively. The outer, inner and shed PS were quantified by a novel two-step binding inhibitory assay. The results indicate a bi-phasic modulation of intracellular Ca and PS externalization/shedding; both of which decreased during maturation and increased during aging. The role of intracellular Ca in PS externalization/shedding was demonstrated by modulating intracellular Ca: Ca was decreased by incubating the cells with an ion chelator (EDTA) or with decreasing concentrations of Ca, whereas treatment with the ionophore A23187 elevated intracellular Ca. The results showed that low Ca resulted in decreased outer and shed PS, whereas high Ca had the opposite effect. The results suggest that PS externalization and shedding are mediated by increased cellular Ca-flux, and that they play an important role in erythroid maturation and RBC senescence., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the present study, we questioned the role of oxidative stress in hereditary spherocytosis (HS), where red blood cells (RBC) have a shortened survival due to primary deficiency in membrane proteins. Using flow cytometry techniques, we showed that RBC derived from 17 HS patients of seven families generate more reactive oxygen species, membrane lipid peroxides, and less reduced glutathione than normal RBC. Following in vitro incubation of HS-RBC from seven patients with a fermentation bioproduct of Carica papaya (fermented papaya preparation (FPP)) with known antioxidative properties, oxidative stress markers were significantly reduced. Similar results were obtained following treatment with FPP for 3 months of 10 adult HS patients, as well as decreased tendency to undergo hemolysis. The hemoglobin levels increased by >1 g/dl, mean corpuscular hemoglobin concentration decreased by >1 g/dl, and the reticulocyte count decreased by 0.93%. Concomitantly, lactic dehydrogenase decreased by 17% and indirect bilirubin by 50%. A significant decrease in malonyldialdehyde was also detected. These data indicate that oxidative stress plays an important role in the pathophysiology of HS which can be ameliorated by an antioxidant such as FPP. Additional clinical trials with FPP and other antioxidants are warranted.

Hepcidin is a major regulator of iron metabolism. We evaluated changes in serum hepcidin during 3 months of therapy with the iron-chelator deferasirox in patients with low-risk myelodysplastic syndrome and iron overload. Serum hepcidin was found to be high in these patients, correlated with their iron and oxidative status, and further increased by treatment with deferasirox. These findings support the concept that the hepcidin level represents a balance between the stimulating effect of iron overload and the inhibitory effects of erythropoietic activity and oxidative stress. These preliminary findings favour the rationale for iron chelation therapy in such patients., (© 2011 Blackwell Publishing Ltd.)

Phosphatidylserine (PS), which is normally localized in the cytoplasmic leaflet of the membrane, undergoes externalization during aging or trauma of red blood cells (RBCS: ). A fraction of this PS is shed into the extracellular milieu. Both PS externalization and shedding are modulated by the oxidative state of the cells. In the present study we investigated the effect of calcium (Ca) flux on oxidative stress-induced membrane distribution of PS and its shedding and on the membrane composition and functions. Normal human RBCs were treated with the oxidant t-butyl hydroperoxide, and thalassemic RBCs, which are under oxidative stress, were treated with the antioxidant vitamin C or N-acetylcystein. The intracellular Ca content was modulated by the Ca ionophore A23187 and by varying the Ca concentration in the medium. Ca flux was measured by Fluo-3, PS externalization and shedding were measured by quantitative flow cytometry and membrane composition was measured by (1)H-NMR analysis of the cholesterol and phospholipids. The results indicated that increasing the inward Ca flux induced PS externalization and shedding, which in turn increased the membrane cholesterol/phospholipid ratio and thereby increased the RBC osmotic resistance. In addition, these processes modulated the susceptibility of RBCs to undergo phagocytosis by macrophages; while PS externalization increased phagocytosis, the shed PS prevented it. These results indicate that PS redistribution and shedding from RBCs, which are mediated by increased calcium, have profound effects on the membrane composition and properties and, thus, may control the fate of RBCs under physiological and pathological conditions.

Production of RBCs is triggered by the action of erythropoietin (Epo) through its binding to surface receptors (Epo-R) on erythroid precursors in the bone marrow. The intensity and the duration of the Epo signal are regulated by several factors, including the balance between the activities of kinesase and phosphatases. The Epo signal determines the proliferation and maturation of the precursors into hemoglobin (Hb)-containing RBCs. The activity of various protein tyrosine phosphatases, including those involved in the Epo pathway, can be inhibited by sodium orthovanadate (Na(3)VO(4), vanadate). Adding vanadate to cultured erythroid precursors of normal donors and patients with β-thalassemia enhanced cell proliferation and arrested maturation. This was associated with an increased production of fetal hemoglobin (HbF). Increased HbF in patients with β-hemoglobinopathies (β-thalassemia and sickle cell disease) ameliorates the clinical symptoms of the disease. These results raise the possibility that specific and nontoxic inhibitors of phosphatases may be considered as a therapeutic modality for elevating HbF in patients with β-hemoglobinopathies as well as for intensifying the Epo response in other forms of anemia.

Most of the iron in the plasma is bound to transferrin (Tf) and is taken up by cells through their surface Tf receptors (TfRs). Under pathological conditions of iron-overload, the plasma iron which is in excess of the binding capacity of Tf is present as non-Tf-bound iron. We probed the uptake of non-Tf iron and its consequences on the oxidative status of peripheral RBC and reticulocytes as well as developing erythroid precursors grown in vitro. The cells were exposed to ferrous ammonium sulfate under Tf-supplemented and Tf-free conditions. Using flow cytometry techniques, we found that both the TfR-deficient mature RBC and their TfR-containing precursors at all stages of maturation can take up non-Tf iron that accumulates as redox-active labile iron and generates reactive oxygen species. Such a mechanism may account for ineffective erythropoiesis of developing precursors in the bone marrow and for the shortening of the lifespan of mature RBCs in the circulation.

On the basis of all the presented data, one can conclude that oxidative stress plays a major role in the pathophysiology of thalassemia and other congenital and acquired hemolytic anemias. Free extracellular (labile plasma iron, LPI) and intracellular (labile iron pool, LIP) iron species that have been identified in thalassemic blood cells are responsible for generation of oxidative stress by catalyzing formation of oxygen radicals over the antioxidant capacity of the cell. Consequently, there is a rationale for iron chelation to eliminate the free-iron species, which in this respect, act like antioxidants. In addition, antioxidants such as vitamin E and polyphenols are also capable of ameliorating increased oxidative stress parameters and, given together with iron chelators, may provide a substantial improvement in the pathophysiology of hemolytic anemias and particularly in thalassemia.

Lentiviral-mediated beta-globin gene transfer successfully treated beta-thalassemic mice. Based on this result, clinical trials were initiated. To date, however, no study has investigated the efficacy of gene therapy in relation to the nature of the different beta-globin mutations found in patients. Most mutations can be classified as beta(0) or beta(+), based on the amount of beta-globin protein produced. Therefore, we propose that a screening in vitro is necessary to verify the efficacy of gene transfer prior to treatment of individual patients. We used a two-phase liquid culture system to expand and differentiate erythroid progenitor cells (ErPCs) transduced with lentiviral vectors. We propose the use of this system to test the efficiency of lentiviral vectors carrying the human beta-globin gene, to correct the phenotype of ErPCs from patients preparing for gene therapy. This new approach might have profound implications for designing gene therapy and for understanding the genotype/phenotype variability observed in Cooley's anemia patients.

In the present study we analysed the mechanism of intracellular routing of iron acquired by erythroid cells via receptor-mediated endocytosis of Tf-Fe [Tf (transferrin)-iron]. Using real-time fluorimetry and flow cytometry, in conjunction with targeted fluorescent metal sensors, we monitored concurrently the cytosolic and mitochondrial changes in labile iron evoked by endocytosed Tf-Fe. In K562 human erythroleukaemia cells, most of the Tf-Fe was found to be delivered to the cytosolic labile iron pool by a saturable mechanism [60-120 nM Km (app)] that was quantitatively dependent on: Tf receptor levels, endosomal acidification/reduction for dislodging iron from Tf and ensuing translocation of labile iron into the cytosolic compartment. The parallel ingress of iron to mitochondria was also saturable, but with a relatively lower Km (app) (26-42 nM) and a lower maximal ingress per cell than into the cytosol. The ingress of iron into the mitochondrial labile iron pool was blocked by cytosol-targeted iron chelators, implying that a substantial fraction of Tf-Fe delivered to these organelles passes through the cytosol in non-occluded forms that remain accessible to high-affinity ligands. The present paper is the first report describing intracellular iron routing measured in intact cells in real-time and in quantitative terms, opening the road for also exploring the process in mixed-cell populations of erythroid origin.

Oxidative stress mediates damage to various cells and is thought to be involved in various pathologies, including hereditary and acquired hemolytic anemias. It is induced by a multitude of physiological and environmental factors, including extracorporeal manipulation of blood. As a result, hemodialysis induces oxidative damage to red blood cells, thereby increasing their susceptibility to hemolysis and shortening their life span. We studied the effect of apheresis on the oxidative status of blood components. Using flow cytometric measurements, we showed that red blood cells, lymphocytes, monocytes, and polymorphonuclear cells undergo oxidative stress induced by the procedure. Their reactive oxygen species and externalization of phosphatidylserine increased, while their levels of reduced glutathione decreased. This oxidative stress, which may be caused by a direct interaction with the membranous system, may lead to cellular abnormalities with clinical consequences such as hemolysis and platelet hyperactivation.

Background/aims: Iron accumulation in vital organs such as heart and liver is a major pathology in beta-thalassaemia. It may also affect mature RBCs and developing erythroid precursors. The cellular damage is mainly caused by the labile iron pool (LIP) and is mediated by reactive oxygen species (ROS). We have previously shown that thalassaemic RBCs and their precursors have more LIP and ROS than their normal counterparts. We now report the effect of clinically relevant iron chelators on these parameters., Methods: RBCs, reticulocytes and cultured erythroid precursors derived from patients with beta-thalassaemia were studied for LIP and oxidative stress parameters by flow-cytometry., Results: In vitro treatment with deferiprone, deferasirox and deferoxamine reduced the cytosolic LIP in RBCs and reticulocytes, and both the cytosolic and mitochondrial LIP in cultured erythroid precursors. This was associated with reduced oxidative stress (ROS and external phosphatidylserine). While the effect of deferiprone and deferasirox was fast (within 10 min), deferoxamine affected these parameters after 24 h, suggesting a slower rate of entry., Conclusion: The chelators studied reduce the LIP and the oxidative status of thalassaemic RBC and their precursors. Whether these effects directly improve ineffective erythropoiesis and RBC survival remains to be shown., (Copyright (c) 2009 S. Karger AG, Basel.)

Because of its stimulating effect on RBC production, erythropoietin (Epo) is used to treat anemia, for example, in patients on dialysis or on chemotherapy. In β-thalassemia, where Epo levels are low relative to the degree of anemia, Epo treatment improves the anemia state. Since RBC and platelets of these patients are under oxidative stress, which may be involved in anemia and thromboembolic complications, we investigated Epo as an antioxidant. Using flow-cytometry technology, we found that in vitro treatment with Epo of blood cells from these patients increased their glutathione content and reduced their reactive oxygen species, membrane lipid peroxides, and external phosphatidylserine. This resulted in reduced susceptibility of RBC to undergo hemolysis and phagocytosis. Injection of Epo into heterozygous (Hbb(th3/+)) β-thalassemic mice reduced the oxidative markers within 3 hours. Our results suggest that, in addition to stimulating RBC and fetal hemoglobin production, Epo might alleviate symptoms of hemolytic anemias as an antioxidant.

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., beta-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 micromol 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.

Purpose: Combat soldiers have a higher prevalence of anemia than age- and gender-matched civilians. This may be caused by hemodilution, which is typical among athletes, or by reduced body iron stores. The aim of this study was to investigate the incidence of iron-deficiency anemia in recruits to the Israel Defense Force after 6 months of training., Methods: Blood was collected from recruits before training. After 6 months of follow-up, 153 paired blood samples were collected from the initial cohort. Total blood count and serum iron, transferrin, and ferritin were measured at both time points. Soluble transferrin receptor (sTfR) was measured in 119 of the paired samples and the sTfR/log ferritin ratio was calculated., Results: At recruitment, mean hemoglobin concentration was 14.7 +/- .9 g/dl. Iron-transferrin saturation was 34.1% +/- 13.6%, and mean ferritin concentration was 53.6 +/- 33.2 ng/ml. Anemia prevalence (Hb <14 g/dl) was 17.6%, and 14.9% of participants were iron-deficient (ferritin <22 mg/dl). At 6 months, 50.3% of the cohort was anemic, and 27.3% demonstrated iron-store depletion. Paired analysis showed an average reduction of .83 g/dl in hemoglobin (p < .001), and of 9.8 mg/dl in ferritin (p < .001). sTfR increased from 1.9 to 2.1 mg/dl (p < .003) among recruits who became anemic. Half of the recruits experienced mild anemia after 6 months of training. Iron store depletion was observed among 24.5% of the cohort after training, as opposed to 15% at recruitment. Overall, these changes were not accompanied by a significant increase in sTfR, but among the subset of anemic subjects, there was a slight increase in index value., Conclusions: In half of the cases, new-onset anemia was attributable to iron deficiency, and in the remainder, to hemodilution.

Hemoglobin (Hb) disorders are common, potentially lethal monogenic diseases, posing a global health challenge. With worldwide migration and intermixing of carriers, demanding flexible health planning and patient care, hemoglobinopathies may serve as a paradigm for the use of electronic infrastructure tools in the collection of data, the dissemination of knowledge, the harmonization of treatment, and the coordination of research and preventive programs. ITHANET, a network covering thalassemias and other hemoglobinopathies, comprises 26 organizations from 16 countries, including non-European countries of origin for these diseases (Egypt, Israel, Lebanon, Tunisia and Turkey). Using electronic infrastructure tools, ITHANET aims to strengthen cross-border communication and data transfer, cooperative research and treatment of thalassemia, and to improve support and information of those affected by hemoglobinopathies. Moreover, the consortium has established the ITHANET Portal, a novel web-based instrument for the dissemination of information on hemoglobinopathies to researchers, clinicians and patients. The ITHANET Portal is a growing public resource, providing forums for discussion and research coordination, and giving access to courses and databases organized by ITHANET partners. Already a popular repository for diagnostic protocols and news related to hemoglobinopathies, the ITHANET Portal also provides a searchable, extendable database of thalassemia mutations and associated background information. The experience of ITHANET is exemplary for a consortium bringing together disparate organizations from heterogeneous partner countries to face a common health challenge. The ITHANET Portal as a web-based tool born out of this experience amends some of the problems encountered and facilitates education and international exchange of data and expertise for hemoglobinopathies.

Derivatives of distamycin A modified at the C-terminal amidine moiety and tethered to bis-epoxyethyl moieties at the N-terminal position were tested for their ability to induce erythroid differentiation in the human erythroleukemic cell line K562. None of the compounds without bis-epoxyethyl moiety were active. A comparison of the biological activity of diepoxy compounds containing different non-basic amidine-modified moieties, showed low activity of amidoxime, carbamoyl and N-methyl carbamoyl derivatives as differentiation agents. In contrast, a cyanamidine derivative, compound 3, was able to induce erythroid differentiation of K562 cells. In addition, the cyanamidine derivative 3 was able to induce HbF accumulation following treatment of cultures of erythroid precursor cells isolated from the peripheral blood of normal subjects.

The oxidative status of cells is determined by the balance between pro-oxidants and antioxidants. Pro-oxidants, referred to as reactive oxygen species (ROS), are classified into radicals and nonradicals. The radicals are highly reactive due to their tendency to accept or donate an electron and attain stability. When cells experience oxidative stress, ROS, which are generated in excess, may oxidize proteins, lipids and DNA - leading to cell death and organ damage. Oxidative stress is believed to aggravate the symptoms of many diseases, including hemolytic anemias. Oxidative stress was found in the beta-hemoglobinopathies (sickle cell anemia and thalassemia), glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis, congenital dyserythropoietic anaemias and Paroxysmal Nocturnal Hemoglobinuria. Although oxidative stress is not the primary etiology of these diseases, oxidative damage to their erythroid cells plays a crucial role in hemolysis due to ineffective erythropoiesis in the bone marrow and short survival of red blood cells (RBC) in the circulation. Moreover, platelets and polymorphonuclear (PMN) white cells are also exposed to oxidative stress. As a result some patients develop thromboembolic phenomena and recurrent bacterial infections in addition to the chronic anemia. In this review we describe the role of oxidative stress and the potential therapeutic potential of anti-oxidants in various hemolytic anemias.

Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the relationship between oxidative stress experienced by RBCs and their phospholipid content and shedding. Using 1H-NMR, we demonstrated a higher lactate/pyruvate ratio, an indicator of oxidative stress, in normal RBCs treated with oxidants (t-butylhydroxyperoxide and H2O2) as well as in beta-thalassemic RBCs. Using 31P-NMR, we found 30% more phosphatidylcholine (PC), and unexpectedly, 35% less phosphatidylserine (PS) in the thalassemic RBCs. PS was decreased by treatment with oxidants and increased by anti-oxidants (vitamin C and N-acetyl cysteine); PC showed the opposite behavior. Thalassemic RBCs incubated in phosphate buffered saline produced more PS in the supernatant than normal RBCs. Anti-oxidants reduced the PS in the supernatant while oxidants increased it. Plasma of thalassemic patients contained 2.6-fold and 1.8-fold more PS and PC, respectively, than normal plasma. These results indicate that the decreased PS in RBCs resulted from increased shedding. The nature of the shed PS was studied by purifying and analyzing membranous microparticles from the plasma and RBC supernatants. More PS was found in microparticles purified from thalassemic plasma and RBC supernatants (5.6- and 4.8-fold, respectively) than in their normal counterparts. However, the bulk (80-90%) of the shed PS was not associated with microparticles. The significance of PS shedding for RBC survival needs further clarification.

Although most cellular iron is firmly bound (e.g. in haemoglobin), some, the labile iron pool (LIP), is bound to low-affinity ligands. The LIP is regarded as the crossroads of cellular iron traffic. Using multi-parameter flow-cytometry of cells treated with the metal-sensitive sensor calcein and the cell-permeable chelator deferiprone, we studied LIP in various human erythroid cell populations in peripheral blood, bone marrow and culture. Erythroid maturation was found to be associated with a decrease in the LIP. In the peripheral blood, nucleated erythrocytes (normoblasts) had 5.8-fold and 8.8-fold greater LIP than reticulocytes and erythrocytes respectively. Early reticulocytes had 2.5-fold more LIP than late reticulocytes. In the bone-marrow and in culture, LIP decreased by c. 30-fold as early erythroid precursors matured to late precursors. Adding holo-transferrin to iron-depleted cultures elevated LIP by 3.9-fold. We also show that in beta-thalassaemia, a disease associated with iron-overload, erythrocytes and reticulocytes in the blood and erythroid precursors in culture have a significantly greater LIP than their normal counterparts. In conclusion, the LIP in erythroid cells is altered under physiological (maturation) and pathological (thalassaemia) conditions. The methodology presented might be useful for evaluating the LIP in various diseases and for studying the efficacy of iron-chelators.

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., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Objective: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder associated with intravascular hemolysis and thrombosis. Hemolysis is caused by the hypersensitivity of PNH-red blood cells (RBC) to complement-mediated lysis due to deficiency in the surface glycosyl phosphatidylinositol-anchored antigens, CD55 and CD59. Thrombosis may be related to the platelet tendency to undergo hyperactivation. We previously suggested that hemolysis and thrombosis in other hemolytic anemias are related to oxidative stress. In the present study, we assessed the oxidative status of blood cells in PNH and tested the potential protective effects of antioxidants., Materials and Methods: Blood samples were obtained from 11 PNH patients and 11 normal control donors. Flow cytometry was used to measure oxidative stress markers in conjunction with the PNH immunophenotype., Results: Results indicated that abnormal, CD55/CD59-negative, RBC, neutrophils, and platelets are under oxidative stress. Their intracellular reactive oxygen species, membrane lipid peroxides, and external phosphatidylserine were higher and their reduced glutathione was lower than CD55/CD59-positive cells of the same patient or cells of normal controls. PNH-RBC were hypersensitive to an oxidative insult (e.g., hydrogen peroxide) and their oxidative status increased following interaction with complement, prior to hemolysis. Antioxidants reduced this hemolysis as well as activation of PNH platelets., Conclusion: We propose that oxidative stress mediates the symptoms of PNH and suggest that antioxidants might be considered as a therapeutic modality.

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin-containing red blood cells. The transferrin-transferrin receptor pathway is the main route for erythroid iron uptake. Using a two-phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin-free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor-mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co-cultured with macrophages under transferrin-free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin-dependent, receptor-mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin-free conditions by delivering essential iron in the form of metabolizable ferritin.

Many aspects of the pathology in beta-hemoglobinopathies (beta-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 beta-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 beta-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.

Developing erythroid cells are dependent on transferrin (Tf) to acquire iron in amounts sufficient for hemoglobin production. Previously, we showed that although these cells cannot grow in culture in the absence of Tf, ferritin (Ft) can substitute Tf to some extent and support the development of hemoglobin-containing cells. In the current study, we investigated the ability of various iron sources to replace Tf in cultures of normal human erythroid precursors. The results showed that whereas Ft and hemin supported erythroid cell proliferation and hemoglobinization in Tf-free cultures to some extent, ferric amonium citrate and iron complexed with several chelators had little or no effect. Although salicylaldehyde-isonicotinoyl-hydrazone, which is a tridentate lipid-soluble chelator, complexed with iron increased both cytosolic and mitochondrial labile iron pools, it failed to support heme synthesis and did not decrease the surface Tf receptors, suggesting that its iron is not recognized by the cells. Moreover, this iron-chelator complex did not support erythroid precursor proliferation and hemoglobinization. Thus, although under normal conditions, Tf is the major route of iron uptake, Ft and hemin, but not iron-chelator complexes, may serve as alternative iron sources under Tf-poor conditions.