Eitan Fibach

The Role of Oxidative Stress in Hemolytic Anemia

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.

Red blood cells, platelets and polymorphonuclear neutrophils of patients with sickle cell disease exhibit oxidative stress that can be ameliorated by antioxidants

Sickle cell disease (SCD) is basically a red blood cell (RBC) disorder characterised by sickling and haemolysis, but platelets and polymorphonuclear neutrophils (PMN) are also involved. Oxidative damage may play a role in the pathogenesis of SCD. Using flow cytometry, we measured oxidative‐state markers simultaneously in RBC, platelets and PMN obtained from 25 normal donors, nine homozygous (SS) patients and six SS/beta‐thalassaemia patients. Reactive oxygen species (ROS) and reduced glutathione (GSH) were measured following staining of blood samples with fluorescence probes and gating on specific subpopulations based on size and granularity. Ten‐ to 30‐fold higher ROS production and 20–50% lower GSH content were found in RBC, platelets and PMN from SCD patients versus those of their normal counterparts. This could in part account for the clinical manifestations, such as haemolysis, a hypercoagulable state, recurrent bacterial infections and vaso‐occlusive incidences, in SCD. We further showed that exposure of SCD samples to antioxidants, such as N‐acetyl‐cysteine, vitamin C and vitamin E, decreased their oxidative stress. These results suggest that antioxidant treatment of patients with SCD could reduce oxidative damage to RBC, PMN and platelets, thereby alleviating symptoms associated with their pathology. The flow cytometry techniques presented herein could assist in monitoring the efficacy of such treatment.

Flow cytometric measurement of reactive oxygen species production by normal and thalassaemic red blood cells.

Abstract: Reactive oxygen species (ROS) contribute to the pathogenesis of several hereditary disorders of red blood cells (RBCs), including thalassaemia. We report here on a modified flow cytometric method for measuring ROS in normal and thalassaemic RBCs. RBCs were incubated with 0.4 mM 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA), then washed and further incubated either with or without 2 mM H2O2. Flow cytometric analysis showed that RBC fluorescence increased with time; it increased faster and reached higher intensity (by 10–30‐fold) in H2O2‐stimulated RBCs as compared to unstimulated RBCs. In both cases, the antioxidant N‐acetyl‐l‐cysteine reduced fluorescence, confirming previous reports that DCFH fluorescence is mediated by ROS. While the fluorescence of unstimulated RBCs increased with time, probably because of exposure to atmospheric oxygen, in H2O2‐stimulated RBCs fluorescence decreased after 30 min. The latter effect is most likely related to H2O2 decomposition by catalase as both sodium azide, an antimetabolite that inhibits catalase and low temperature increased the fluorescence of stimulated RBCs. Washing had a similar effect, suggesting that maintenance of the oxidised DCF requires a constant supply of ROS. We next studied RBCs of β‐thalassaemic patients. The results demonstrated a significantly higher ROS generation by stimulated and unstimulated thalassaemic RBCs compared to their normal counterparts. These results suggest that flow cytometry can be useful for measuring the ROS status of RBCs in various diseases and for studying chemical agents as antioxidants.

Developmental regulation of DNA replication timing at the human β globin locus

The human β globin locus replicates late in most cell types, but becomes early replicating in erythroid cells. Using FISH to map DNA replication timing around the endogenous β globin locus and by applying a genetic approach in transgenic mice, we have demonstrated that both the late and early replication states are controlled by regulatory elements within the locus control region. These results also show that the pattern of replication timing is set up by mechanisms that work independently of gene transcription.

Flow cytometric analysis of the oxidative status of normal and thalassemic red blood cells

The oxidative status of cells has been shown to modulate various cell functions and be involved in physiological and pathological conditions, including hereditary chronic anemias, such as thalassemia. It is maintained by the balance between oxidants, such as reactive oxygen species (ROS), and antioxidants, such as reduced glutathione (GSH).

Cellular copper content modulates differentiation and self‐renewal in cultures of cord blood‐derived CD34+ cells

Summary.  Several clinical observations have suggested that copper (Cu) plays a role in regulating haematopoietic progenitor cell (HPC) development. To further study this role we used an ex vivo system. Cord blood‐derived CD34+ cells were cultured in liquid medium supplemented with Kit‐ ligand, FLt3, interleukin 6 (IL‐6), thrombopoietin and IL‐3. Under these conditions, Cu content, measured by atomic absorption, was 7 ng/107 cells. Modulation of intracellular Cu was achieved by supplementing the cultures with the Cu chelator tetraethylenepentamine, which reduced cellular Cu (4 ng/107 cells), or ceruloplasmin or Cu sulphate that elevated cellular Cu (18 and 14 ng/107 cells respectively). The results indicated that low Cu content delayed differentiation, as measured by the surface antigens CD34, CD14 and CD15, colony‐forming unit (CFU) frequency and cell morphology, while high Cu accelerated differentiation compared with Cu unmanipulated cultures. As a result, expansion of total cells, CFU and CD34+ cells in low Cu was extended (12–16 weeks), and in high Cu was shortened (2–4 weeks), compared with control cultures (6–8 weeks). These effects required modulation of intracellular Cu only during the first 1–3 weeks of the culture; the long‐term effects persisted thereafter, suggesting that the decision process for either self‐renewal or differentiation is taken early during the culture. This novel method of controlling cell proliferation and differentiation by copper and copper chelators might be utilized for ex vivo manipulation of HPC for various clinical applications.

Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine

BACKGROUND We have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+CD38-, CD34+CD38-Lin-) in human umbilical cord blood (CB)-derived CD34+ cell cultures. This study extends our previous findings that copper chelation can modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells. METHODS In the present study we established a clinically applicative protocol for large-scale ex vivo expansion of CB-derived progenitors. Briefly, CD133+ cells, purified from CB using Miltenyi Biotecs (Bergisch Gladbach, Germany) CliniMACS separation device and the anti-CD133 reagent, were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). This protocol was evaluated using frozen units derived from accredited cord blood banks. RESULTS Following 3 weeks of expansion under large-scale culture conditions that were suitable for clinical manufacturing, the median output value of CD34+ cells increase by 89-fold, CD34+CD38- increase by 30-fold and CFU cells (CFUc) by 172-fold over the input value. Transplantation into sublethally irradiated non-obese diabetic (NOD/SCID) mice indicated that the engraftment potential of the ex vivo expanded CD133+ cells was significantly superior to that of unexpanded cells: 60+/-5.5% vs. 21+/-3.5% CD45+ cells, P=0.001, and 11+/-1.8% vs. 4+/-0.68% CD45+CD34+ cells, P=0.012, n=32, respectively. DISCUSSION Based on these large-scale experiments, the chelator-based ex vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing CB transplantation for hematological malignancies.

Medicinal Chemistry of Fetal Hemoglobin Inducers for Treatment of β-Thalassemia

In this review we summarize the achievements of medicinal chemistry in the field of pharmacological approaches to the therapy of beta-thalassemia using molecules able to stimulate the production of fetal hemoglobin (HbF). We first describe the molecular basis of the pathology and the biochemical rational of using HbF inducers for therapy; we then outlined the in vitro and in vivo experimental systems suitable for screening of such potential drugs, and finally we describe the different classes of compounds with emphasis on their advantages and disadvantages in the treatment. The results of these reviewed studies indicate that: (a) HbF inducers can be grouped in several classes based on their chemical structure and mechanism of action; (b) clinical trials with some of these inducers demonstrate that they are effective in ameliorating the symptoms of beta-thalassemia; (c) a good correlation was found between HbF stimulation in vivo and in vitro indicating that in vitro testing might be predictive of the in vivo response; (d) combined use of different inducers might maximize the effect, both in vitro and in vivo. However, (e) the response to HbF inducers, evaluated in vitro and in vivo, is variable, and some patients might be refractory to HbF induction by certain inducers; in addition, (f) several considerations call for caution, including the fact that most of the inducers exhibit in vitro cytotoxicity, predicting side effects in vivo following prolonged treatments.

Two-phase liquid culture system models normal human adult erythropoiesis at the molecular level.

Abstract: We have studied the patterns of expression of various genes during maturation of normal human adult erythroid precursors cultured in a two‐phase liquid culture method. In the first phase, peripheral blood mononuclear cells are cultured for one week in the presence of a combination of growth factors, but not erythropoietin (Epo). In Phase II, Epo is included in the medium. Cell samples were taken throughout phase II, and expression of globins, transcription factors, and cytokine receptors was assayed by RT‐PCR and quantified by phosphor imaging. We have divided phase II into stages: early (days 0–5), intermediate (days 6–10) and late (days 11–15) and measured maximum expression of each gene. During early phase II, γ‐globin, Sp1, and GATA‐2 mRNAs were expressed at their highest levels. As the cells matured during the intermediate period, GATA‐2 levels remained high, and then declined, while the transcription factors GATA‐1, EKLF, NF‐E2, and the Epo receptor (EpoR) reached maximum expression. In late phase II, β‐globin increased and reached its maximum level of expression. This erythroid culture system appears to recapitulate normal adult erythropoiesis at the molecular level, and thus may be a suitable model to examine the molecular basis of severe congenital or acquired disorders oferythropoiesis.

Accumulation of γ‐globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine

Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5‐azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo‐fetal globin genes, such as the ζ‐, ε‐ and γ‐globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating γ‐globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal β‐globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of γ‐globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine‐mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of γ‐globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.