Oxidative Stress of RBC in a Murine Model of Beta-Thalassemia Can Be Reversed by Treatment with Antioxidants.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3643-3643
Author(s):  
Eitan Fibach ◽  
Johnny Amer ◽  
Eliezer Rachmilewitz ◽  
Ella Guy ◽  
Stefano Rivella
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Mouse Model ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Beta Thalassemia ◽  
Light Scatter ◽  
Intracellular Ros ◽  
Mercury Orange ◽  
Dichlorofluorescin Diacetate

Abstract Oxidative stress is a prominent contributor to the premature destruction of RBC as well as anemia in thalassemia and sickle cell anemia. The oxidative status within RBC is maintained by the balance between oxidative systems, such as Reactive Oxygen Species (ROS), and antioxidative systems, such as reduced glutathione (GSH). Using flow cytometric methods, we previously showed that RBC obtained from patients with thalassemia (Amer et al. Eur J Haematol70:84,2003; Cytometry60:73,2004) or sickle cell anemia (Amer et al. Blood, 104:972a,2004) exhibit oxidative stress. In the present study, we assessed the extent of RBC oxidative stress and the effects of antioxidant administration, using the thalassemic mouse model Th3/+. This model closely mimics the phenotype observed in patients affected by beta-thalassemia intermedia, such as low hemoglobin levels (7 to 9 gr/dL), splenomegaly and iron overload. In addition, the mature RBC have a shorter life-span and are characterized by anisocytosis, poikilocytosis and hypochromatism. RBC were derived from normal and thalassemic mice before and 4 hours after i.p. injection of the antioxidants N-acetyl cysteine (NAC), vitamin C (Vit. C) or tocotrinol - a mixture of vitamin E derivatives, at a dose of 150 mg/kg. Intracellular ROS was determined in dichlorofluorescin diacetate-stained RBC following stimulation with 2 mM H2O2; GSH content was assessed in RBC stained with mercury orange. Cells were analyzed by flow cytometry: RBC were gated according to size (forward light scatter) and granularity (side light scatter), their fluorescence was measured and the Mean Fluorescence Channel (MFC) was calculated. Fig. 1 shows the average MFC of ROS and GSH of normal and thalassemic mice treated or not treated with anti-oxidants (N=6 in each group). The results show a significantly higher (2.6-fold) production of ROS and lower (three-fold) levels of GSH in RBC from the thalassemic mice versus those in RBC from normal mice. Administration of antioxidants decreased the ROS of normal and thalassemic RBC by 1.4-fold and 2.6-fold, respectively, whereas GSH levels were significantly increased both in the normal (2.7 fold) and in the thalassemic (9.4-fold) RBC. The results show that the RBC of thalassemic mice are under oxidative stress that could be ameliorated by a short antioxidant treatment. Hence, this mouse model recapitulates the oxidative stress found in thalassemic patients and can serve as a model for studying the effects of antioxidant therapy. The flow cytometry methodology used is helpful in following up the results of the treatment and in evaluating its efficacy in reducing oxidative stress. Figure Figure

Platelets and PMN as Well as RBC of Patients with Sickle Cell Anemia Exhibit Oxidative Stress Which Can Be Corrected by Antioxidants.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3575-3575
Author(s):  
Johnny Amer ◽  
Hussam Ghoti ◽  
Eliezer Rachmilewitz ◽  
Koren Ariel ◽  
Eitan Fibach
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Sickle Cell ◽  
Clinical Manifestations ◽  
Oxidative Status ◽  
Polymorphonuclear Neutrophils ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Beta Thalassemia ◽  
Mercury Orange

Abstract Sickle cell disease (SCD) is caused by an abnormal hemoglobin (HbS), which results mainly in sickling and hemolysis of RBC. However, the platelets and the polymorphonuclear neutrophils (PMN) are also involved in the pathophysiology of the disease. Similar to the findings in thalassemia, some patients develop thromboembolic phenomena with hypercoagulable state, which is due in part to platelet activation. In addition, a growing body of evidence suggests that WBC, particularly PMN, are abnormal; their number is elevated during painful crises and the severity of the disease increases with their number. Several aspects of the changes in the three blood lineages are thought to result from oxidative stress, which represents the imbalance between enhanced generation of reactive oxygen species (ROS) and a low cellular content of antioxidants such as reduced glutathione (GSH), the major intracellular scavenger of ROS. In SCD, oxidative stress results primarily from the premature precipitation of the unstable HbS and the accumulation of excess iron, a catalyst in the formation of ROS. Accumulation of ROS may result in hemolysis of RBC, activation of platelets and a respiratory burst of PMN. We developed flow cytometry techniques for measuring oxidative-state markers, ROS generation and GSH content, simultaneously in RBC, platelets and PMN. Peripheral blood samples obtained from 15 normal donors, 10 patients with SCD and 5 SCD/beta-thalassemia patients (two with the IVS2-1 mutation and 3 with the IVS1-6 mutation) were studied. Out of the 15 SCD patients, 5 were splenectomized, and all were treated with folic acid, except one who was treated with hydroxyurea. The blood was mixed with 3% gelatin, and following 30 min. incubation the upper layer, containing RBC, platelets and WBC, was collected. ROS and GSH were measured by FACS analysis following staining with 2′, 7′-dichlorofuoresceine (DCF) and mercury orange, respectively. Cells were gated on the basis of size and granularity to include platelets, RBC or PMN, exclusively. The cells in each gate were analyzed for green (DCF) or orange (mercury orange) fluorescence and the Mean Fluorescence Channel (MFC) was calculated. The results showed that ROS production increased by 10 to 30-fold in RBC, platelets and PMN from SCD patients compared with that of their normal counterparts. Concomitantly, the GSH content decreased by 20–50% in the SCD cells. It was possible to modulate the oxidative status of cells from both normal donors and SCD patients: Exposure of the cells to oxidants such as hydrogen peroxide (2 mM), hemin (0.1 mM) or iron (ferric ammonium citrate - 0.1 mM) increased the oxidative status in all cell types, while antioxidants such as N-acetyl cysteine, vitamin C (both at 1 mM) and vitamin E (0.2 mM) significantly decreased the oxidative stress. Our results indicate that similar to the findings in thalassemia, in SCD, RBC and platelets, as well as PMN, are in a state of oxidative stress, which could in part account for the clinical manifestations. Addition of antioxidants, which reduced the ROS and enhanced the GSH content of the cells, could protect against oxidative damage. The flow cytometry techniques we developed may prove useful for studying the effects of various antioxidants and for monitoring the patient’s oxidative status during therapy thereby providing an objective, quantitative evaluation of their efficacy.

In Vitro and In Vivo Antioxidant Effect of Fermented Papaya Preparation (FPP) on Blood Cells of Beta-Thalassaemia Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1766-1766
Author(s):  
Eitan Fibach ◽  
Johnny Amer ◽  
Ada Goldfarb ◽  
Eliezer Rachmilewitz
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Peripheral Blood ◽  
Thalassemia Major ◽  
Oxidative Status ◽  
Beta Thalassemia ◽  
Thalassemia Intermedia

Abstract In sickle cell anemia (SCD) and thalassemia, although the basic lesions are mutations in the globin genes, the pathophysiology involves oxidative stress-mediated cell damage in the bone marrow (ineffective erythropoiesis due to apoptosis of early erythroid precursors) and in the peripheral blood (chronic hemolysis of mature RBC). In addition, some patients develop thromboembolic complications and recurrent bacterial infections, the etiology of which is related at least in part, to documented oxidative stress in platelets and neutrophils (PMN), respectively. To study the presence and the role of oxidative stress in thalassemia and SCD, we adapted flow cytometry techniques for measuring the generation of Reactive Oxygen Species (ROS), the content of reduced glutathione (GSH), membrane lipid peroxidation and externalization of phosphatidylserine (PS) moieties in RBC, platelets and PMN. Cells derived from the peripheral blood of patients with beta-thalassemia major, intermedia or SCD showed increased oxidative status (increased ROS, lipid peroxidation and PS externalization, and decreased GSH) compared with their normal counterparts. Incubating fresh blood samples from patients with thalassemia major and thalassemia intermedia with 10 mg/ml FPP for 16 hours at 37oC reduced the oxidative status of RBC as well as platelets and PMN. Experiments carried out in normal and thalassemic mice (Th3/+, a mouse model of human beta-thalassemia intermedia demonstrated that mice treated for one week with 10 mg/ml FPP (dissolved in the drinking water) had reduced oxidative stress compared to control mice. The in-vivo effect of FPP was tested on 9 patients with beta-thalassemia (6 - major and 3 - intermedia) treated with 3 gr FPP per os three times a day for 12–15 weeks. Following the treatment, the ROS in RBC, platelets and PMN decreased and the GSH increased in all patients (see table). Six of these patients responded by a modest increase in RBC, reticulocytes and hemoglobin levels. These results suggest that FPP may have an important clinical efficacy as an antioxidant in thalassemia and sickle cell anemia. The in vivo effect of FPP treatment of beta-thalassemia patients Baseline After treatment n Mean ± SE Mean ± SE P-value* * Paired samples t-test RBC 9 324.07 ± 29.19 209.55 ± 23.65 0.001 ROS Platelets 9 223.73 ± 26.49 109.11 ± 8.71 0.001 PMN 9 222.72 ± 46.42 117.61 ± 8.98 0.045 RBC 9 55.37 ± 5.37 94.88 ± 3.71 0.001 GSH Platelets 9 59.41 ± 4.98 97.55 ± 5.26 <0.0001 PMN 9 58.29 ± 5.35 90.06 ± 5.87 0.005

scholarly journals Inheritance of the Bantu/Benin haplotype causes less severe hemolytic and oxidative stress in sickle cell anemia patients treated with hydroxycarbamide

2016 ◽  
Vol 61 (7) ◽  
pp. 605-611 ◽  
Author(s):  
Jéssika V Okumura ◽  
Danilo G H Silva ◽  
Lidiane S Torres ◽  
Edis Belini-Junior ◽  
Willian M Barberino ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
And Oxidative Stress

Prevalence of Red Blood Cell Alloimmunization Among Beta-Thalassemia and Sickle Cell Anemia Patients: a Study from Saudi Arabia

2021 ◽  
Vol 67 (10/2021) ◽  
Author(s):  
Raed Felimban ◽  
Ahmed Alsharyufi ◽  
Jasem Aljehani ◽  
Ahmed Sahlool ◽  
Hamead Aljabri ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Blood Cell ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Red Blood Cell ◽  
Beta Thalassemia

scholarly journals The -158 site 5' to the G gamma gene and G gamma expression

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1463-1465
Author(s):  
D Labie ◽  
O Dunda-Belkhodja ◽  
F Rouabhi ◽  
J Pagnier ◽  
A Ragusa ◽  
...  
Keyword(s):  
Linkage Disequilibrium ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Beta Thalassemia ◽  
Surrounding Area ◽  
Area 5 ◽  
Highly Correlated

To test the hypothesis advanced by Gilman and Huisman that the -158 site 5′ to the G gamma gene determines the G gamma expression after the first 4 months of life, we have examined DNA from sickle cell anemia (SS) patients from Africa and beta-thalassemic homozygotes from Algeria. We find that the Xmnl site is strongly linked to the Senegal haplotype among SS patients, to haplotype IX (most probably identical to the Senegal haplotype), and to haplotype III among the Algerian thalassemics. Thalassemics with haplotypes I/I and V/V have no Xmnl site and low G gamma expression. In contrast, beta-thalassemia- associated haplotype II (also characterized by high G gamma expression) fails to exhibit the Xmnl site. We conclude that, although highly correlated, the -158 C----T substitution does not perfectly predict the presence of high G gamma expression. These findings also exclude the possibility that the Xmnl site is solely involved in the determination of high G gamma expression and suggest that either several different site substitutions in the area 5′ to the gamma gene might have the same effect or that, alternatively, the Xmnl site and its surrounding area is not involved in G gamma expression and may be only in linkage disequilibrium with a controlling sequence elsewhere.

scholarly journals The influence of hydroxyurea on oxidative stress in sickle cell anemia

2012 ◽  
Vol 34 (6) ◽  
pp. 421-425
Author(s):  
Lidiane de Souza Torres<sup></sup> ◽  
Danilo Grünig Humberto da Silva<sup></sup> ◽  
Edis Belini Junior<sup></sup> ◽  
Eduardo Alves de Almeida<sup></sup> ◽  
Clarisse Lopes de Castro Lobo<sup></sup> ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sickle Cell ◽  
Sickle Cell Anemia

scholarly journals Sickle Cell Anemia Mediates Carotid Artery Expansive Remodeling That Can Be Prevented by Inhibition of JNK (c-Jun N-Terminal Kinase)

2020 ◽  
Vol 40 (5) ◽  
pp. 1220-1230 ◽  
Author(s):  
Hannah Song ◽  
Philip M. Keegan ◽  
Suhaas Anbazhakan ◽  
Christian P. Rivera ◽  
Yundi Feng ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Mouse Model ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Structural Damage ◽  
Proteolytic Enzymes ◽  
Arterial Compliance ◽  
Cathepsin K ◽  
Large Artery ◽  
Elastic Lamina

Objective: Sickle cell anemia (SCA) causes chronic inflammation and multiorgan damage. Less understood are the arterial complications, most evident by increased strokes among children. Proteolytic mechanisms, biomechanical consequences, and pharmaceutical inhibitory strategies were studied in a mouse model to provide a platform for mechanistic and intervention studies of large artery damage due to sickle cell disease. Approach and Results: Townes humanized transgenic mouse model of SCA was used to test the hypothesis that elastic lamina and structural damage in carotid arteries increased with age and was accelerated in mice homozygous for SCA (sickle cell anemia homozygous genotype [SS]) due to inflammatory signaling pathways activating proteolytic enzymes. Elastic lamina fragmentation observed by 1 month in SS mice compared with heterozygous littermate controls (sickle cell trait heterozygous genotype [AS]). Positive immunostaining for cathepsin K, a powerful collagenase and elastase, confirmed accelerated proteolytic activity in SS carotids. Larger cross-sectional areas were quantified by magnetic resonance angiography and increased arterial compliance in SS carotids were also measured. Inhibiting JNK (c-jun N-terminal kinase) signaling with SP600125 significantly reduced cathepsin K expression, elastin fragmentation, and carotid artery perimeters in SS mice. By 5 months of age, continued medial thinning and collagen degradation was mitigated by treatment of SS mice with JNK inhibitor. Conclusions: Arterial remodeling due to SCA is mediated by JNK signaling, cathepsin proteolytic upregulation, and degradation of elastin and collagen. Demonstration in Townes mice establishes their utility for mechanistic studies of arterial vasculopathy, related complications, and therapeutic interventions for large artery damage due to SCA.

scholarly journals 5-Azacytidine increases gamma-globin synthesis and reduces the proportion of dense cells in patients with sickle cell anemia

Blood ◽  
1983 ◽  
Vol 62 (2) ◽  
pp. 370-380 ◽  
Author(s):  
TJ Ley ◽  
J DeSimone ◽  
CT Noguchi ◽  
PH Turner ◽  
AN Schechter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Beta Thalassemia ◽  
Globin Genes ◽  
Gamma Globin ◽  
Globin Synthesis ◽  
Marrow Cells

Abstract We previously demonstrated that 5-azacytidine can selectively increase gamma-globin synthesis in a patient with beta +-thalassemia, prompting us to treat two patients with sickle cell anemia and two additional patients with beta + thalassemia. 5-Azacytidine (2 mg/kg/day) was continuously infused for 7 days with no apparent clinical toxicity. The gamma/beta-globin biosynthetic ratio increased fourfold to sixfold in the bone marrow cells of each patient after treatment and remained elevated for 7–14 additional days. Hypomethylation of DNA near the gamma-globin genes in bone marrow cells was demonstrated 2 days after beginning the 5-azacytidine infusion. The peripheral blood fetal hemoglobin (HbF) level increased from 6.0% to 13.7% in one patient with sickle cell anemia and from 1.6% to 8.9% in the second. Stractan gradient analyses of peripheral blood from patients with sickle cell anemia revealed a marked decrease in the percentage of dense cells (cells that contain increased amounts of HbS polymer when deoxygenated) following treatment. These observations provide an impetus to investigate the effects of repeated courses of 5-azacytidine in a small group of severely ill patients to determine whether this drug may have a role in the treatment of patients with sickle cell anemia and beta- thalassemia.

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