Johnny Amer

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.

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).

Amelioration of hepatic fibrosis by NK cell activation

Background and aims Interactions between hepatic stellate cells (HSCs) and immune cell subsets have emerged as important determinants of liver fibrosis progression and regression. Natural killer (NK) cells have an antifibrotic activity through killing of activated HSCs. In liver injury NK cell expression of activating/inhibitory killer immunoglobulin-related receptors (aKIR/iKIR) and their ratio are significantly increased, while class I major histocompatibilty (MHC) expression by activated HSCs is decreased. The aim of this study was to amplify the antifibrotic activity of NK cells and ameliorate hepatic fibrosis by iKIR silencing. Methods Human lymphocytes from patients with hepatitis C virus (HCV) infection were transfected with specific iKIR small interfering RNAs (siRNAs) or non-silencing control siRNAs, then co-cultured with a human HSC line and assessed for fibrogenic activity. To induce hepatic fibrosis, carbon tetrachloride was administrated to BALBc SCID-Beige male mice (lacking B/T/NK cells) for 4 weeks. Splenocytes from naive SCID donors (lacking B/T cells but with preserved NK cells) were transfected in vitro with either iKIR siRNA or non-silencing control siRNA, and then were transferred to the fibrotic SCID-Beige recipients. Results Transfection with iKIR or positive control siRNAs (mice and human) decreased mRNA expression of iKIR and mitogen-acivated protein kinase 1 (MAPK1). Consequently, total NK cells and NK cell degranulation were increased (p=0.01), consistent with NK cell stimulation. Compared with healthy lymphocytes, when HCV lymphocytes were transfected with non-silencing control siRNA and co-cultured with HSCs there was increased α-smooth muscle actin (αSMA) expression, reflecting HSC activation. Expression of αSMA in co-cultures was attenuated when HCV lymphocytes were transfected with iKIR siRNA. In SCID-Beige recipients, hepatic fibrosis and serum alanine aminotransferase (ALT) levels were significantly attenuated as a result of receiving iKIR siRNA. Conclusions iKIR knockdown stimulates NK cells and promotes their antifibrogenic activity in mice and human co-cultures. These findings have implications for possible immune therapeutic strategies in patients with advanced liver disease.

Oxidative stress in red blood cells, platelets and polymorphonuclear leukocytes from patients with myelodysplastic syndrome

Low‐risk myelodysplastic syndrome (MDS) is characterized by cytopenia, mainly anemia, because of ineffective hematopoiesis. Some of the patients with ineffective erythropoiesis, with or without ring sideroblasts in their bone marrow, develop severe anemia requiring frequent blood transfusions and consequently develop iron overload. Excess free iron in cells catalyses the generation of reactive oxygen species (ROS) that cause cell and tissue damage. Using flow cytometry techniques, we compared the oxidative status of red blood cells (RBC), platelets and neutrophils in 14 MDS patients with those of normal donors. The results show that ROS were higher while reduced glutathione (GSH) was lower in their RBC and platelets compared with normal cells. In neutrophils, no difference was found in ROS, while the GSH levels were lower. A correlation (r = 0.6) was found between serum ferritin levels of the patients and the ROS in their RBC and platelets. The oxidative stress was ameliorated by a short incubation with the iron‐chelators, the deferrioxamine and deferiprone or with antioxidants such as N‐acetylcysteine, suggesting that MDS patients might benefit from treatment with iron‐chelators and antioxidants.

Chronic oxidative stress reduces the respiratory burst response of neutrophils from beta-thalassaemia patients

Beta‐thalassaemia patients are susceptible to infections by mechanisms that are not fully understood. Polymorphonuclear neutrophils (PMN) destroy microbes by producing a burst of reactive oxygen species (ROS) (respiratory burst) in response to bacterial components, as well as to phorbol‐myristate‐acetate (PMA). In the present study, we compared ROS generation by normal and β‐thalassaemia PMN and assessed their response to PMA. Blood cells were subjected to gelatin separation, staining with dichlorofluorescin‐diacetate and flow cytometry. At basal level, the fluorescence (mean fluorescence channel) of normal and thalassaemia PMN were 12·7 ± 4·5 and 95·6 ± 19·8 respectively; it changed to 283·4 ± 72·5 and 39·5 ± 14·3, respectively, upon PMA stimulation, indicating that thalassaemia PMN have a higher basal ROS but a reduced response to PMA. When normal PMN were treated with the oxidants hydrogen peroxide and butyl‐hydroxyperoxide, as well as iron and haemin, which are elevated in thalassaemia, their basal ROS increased 5–22‐fold, but the PMA response was abolished. Treating thalassaemic PMN with antioxidants (N‐acetyl‐l‐cysteine or vitamins C and E) reduced their basal ROS but enhanced their PMA response. Our findings indicate that chronically stressed PMN, e.g. in thalassaemia, have reduced capacity to elicit a respiratory burst, which may compromise their antibacterial capacity, and imply prophylactic treatment with antioxidants for recurrent infections.

Oxidative stress causes membrane phospholipid rearrangement and shedding from RBC membranes--an NMR study.

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.

Fermented Papaya Preparation as Redox Regulator in Blood Cells of β-Thalassemic Mice and Patients

Many aspects of the pathology in β‐hemoglobinopathies (β‐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 H2O2‐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 β‐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 β‐thalassemic mice (50 mg/mouse/day for 3 months) and to patients (3 g × 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

Oxidative status of red blood cells, neutrophils, and platelets in paroxysmal nocturnal hemoglobinuria

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.

Amelioration of oxidative stress in red blood cells from patients with β-thalassemia major and intermedia and E-β-thalassemia following administration of a fermented papaya preparation.

In β‐hemoglobinopathies, such as β‐thalassemia (thal) and sickle cell anemia, the primary defects are mutations in the β‐globin gene. However, many aspects of the pathophysiology are mediated by oxidative stress. Fermented papaya preparation (FPP), a natural health food product obtained by biofermentation of carica papaya, has been shown to limit oxidative stress both in vitro and in vivo. We studied the effect of FPP on two groups of β‐thal patients: β‐thal, major and intermedia, (in Israel) and E‐β‐thal (in Singapore). The results indicated that in both groups FPP treatment increased the content of reduced glutathione (GSH) in red blood cells (RBC), and decreased their reactive oxygen species (ROS) generation, membrane lipid peroxidation, and externalization of phosphatidylserine (PS), indicating amelioration of their oxidative status, without a significant change in the hematological parameters. Since the turnover of the erythron is relatively slow, it is possible that longer duration of treatment, probably with the addition of an iron chelator, is required in order to achieve the latter goals. Copyright