Hussam Ghoti

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.

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.

Evidence for tissue iron overload in long-term hemodialysis patients and the impact of withdrawing parenteral iron

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.

Changes in parameters of oxidative stress and free iron biomarkers during treatment with deferasirox in iron-overloaded patients with myelodysplastic syndromes

Approximately 60–80% of patients with myelodysplastic syndromes (MDS) present with symptomatic anemia and, of these, 80–90% will require red blood cell (RBC) transfusions as supportive therapy.[1][1],[2][2] Excess transfusional iron causes accumulation of labile plasma iron (LPI), the

Addition of fresh frozen plasma as a source of complement to rituximab in advanced chronic lymphocytic leukaemia.

However, during the fi nal year of treatment the disease progressed, with severe malaise and marked B symptoms (eg, night sweats and weight loss) associated with massive lymphadenopathy and splenomegaly. Concentration of peripheral blood lymphocytes increased to 450×10 9 /L and serum lactate dehydrogenase reached 2400 IU/L. This time the disease was resistant to regimens that contained fl udarabine. Alemtuzumab was not yet approved for use in Israel at that time. The disease was also resistant to treatment with six cycles of rituximab combined with dose-modifi ed cyclo phos phamide, doxorubicin, vincristine, and prednisone-like chemotherapy (CHOP-R). Modest response was noted with the fi rst four CHOP-R cycles, whereas two additional cycles yielded no lasting response. After the last cycle, complete blood count showed haemoglobin (Hb) concentration as 110 g/L, white blood cells (WBC) 180×10 9

Urinary hepcidin excretion in patients with myelodysplastic syndrome and myelofibrosis

Many patients with myelodysplastic syndrome (MDS) develop severe anaemia that requires multiple blood transfusions with consequent iron overload. The absorption of iron from the intestine, iron release from hepatic stores and the recycling of iron by macrophages is negatively regulated by hepcidin, the principal iron-regulatory hormone (Nicolas et al, 2002; Ganz & Nemeth, 2006). Hepcidin regulates iron absorption and distribution by binding to ferroportin, the iron exporter of these cells, and inducing its internalization and degradation, (Nemeth et al, 2004). Hepcidin is increased in response to increased body iron levels, transferrin saturation (Ganz & Nemeth, 2006) or inflammation, and is decreased in response to hypoxia, (Nicolas et al, 2002) erythropoietic activity (Adamsky et al, 2004) and oxidative stress (Choi et al, 2007). Patients with congenital haemolytic anaemia, such as thalassaemia, develop severe iron overload both due to multiple blood transfusions and to increased iron absorption. Hepcidin levels are inappropriately decreased in thalassaemia intermedia, whereas in thalassaemia major, hepcidin levels were found to be elevated, presumably due to transfusions which decrease erythropoietic drive (Origa et al, 2007). The present study investigated whether urinary hepcidin levels are also low in patients with various forms of MDS, similar to the findings in thalassaemia intermedia. Approval was obtained from the University of California, Los Angeles (UCLA) and Wolfson Medical Centre institutional review boards for the study. Urine samples were collected during visits to the outpatient department of the Wolfson Medical Centre. The patients lacked any clinical signs of inflammation. Urine samples were obtained from four patients with refractory anaemia (RA), four with refractory anaemia with ringed sideroblasts (RARS), three with refractory cytopenia with multilineage dysplasia (RCMD), two with refractory anemia with excess of blasts I (RAEB I), four with refractory anaemia with excess of blasts II (RAEB II) and four with idiopathic myelofibrosis (MF), with an age range from 54 to 87 years (Table I). Nine patients had received less than 10

The role of endocytic pathways in cellular uptake of plasma non-transferrin iron

Background In transfusional siderosis, the iron binding capacity of plasma transferrin is often surpassed, with concomitant generation of non-transferrin-bound iron. Although implicated in tissue siderosis, non-transferrin-bound iron modes of cell ingress remain undefined, largely because of its variable composition and association with macromolecules. Using fluorescent tracing of labile iron in endosomal vesicles and cytosol, we examined the hypothesis that non-transferrin-bound iron fractions detected in iron overloaded patients enter cells via bulk endocytosis. Design and Methods Fluorescence microscopy and flow cytometry served as analytical tools for tracing non-transferrin-bound iron entry into endosomes with the redox-reactive macromolecular probe Oxyburst-Green and into the cytosol with cell-laden calcein green and calcein blue. Non-transferrin-bound iron-containing media were from sera of polytransfused thalassemia major patients and model iron substances detected in thalassemia major sera; cell models were cultured macrophages, and cardiac myoblasts and myocytes. Results Exposure of cells to ferric citrate together with albumin, or to non-transferrin-bound iron-containing sera from thalassemia major patients caused an increase in labile iron content of endosomes and cytosol in macrophages and cardiac cells. This increase was more striking in macrophages, but in both cell types was largely reduced by co-exposure to non-transferrin-bound iron-containing media with non-penetrating iron chelators or apo-transferrin, or by treatment with inhibitors of endocytosis. Endosomal iron accumulation traced with calcein-green was proportional to input non-transferrin-bound iron levels (r2=0.61) and also preventable by pre-chelation. Conclusions Our studies indicate that macromolecule-associated non-transferrin-bound iron can initially gain access into various cells via endocytic pathways, followed by iron translocation to the cytosol. Endocytic uptake of plasma non-transferrin-bound iron is a possible mechanism that can contribute to iron loading of cell types engaged in bulk/adsorptive endocytosis, highlighting the importance of its prevention by iron chelation.

Increased serum hepcidin levels during treatment with deferasirox in iron-overloaded patients with myelodysplastic syndrome.

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.

Non-transferrin bound iron in Thalassemia: differential detection of redox active forms in children and older patients.

Non‐transferrin bound iron (NTBI) is commonly detected in patients with systemic iron overload whose serum iron‐binding capacity has been surpassed. It has been perceived as an indicator of iron overload, impending organ damage and a chelation target in poly‐transfused thalassemia patients. However, NTBI is a heterogeneous entity comprising various iron complexes, including a significant redox‐active and readily chelatable fraction, which we have designated as “labile plasma iron” (LPI). We found that LPI levels can be affected by plasma components such as citrate, uric acid, and albumin. However, the inclusion of a mild metal mobilizing agent in the LPI assay (designated here as “eLPI”), at concentrations that do not affect transferrin‐bound iron, largely overcomes such effects and provides a measure of the full NTBI content. We analyzed three distinct groups of poly‐transfused, iron overloaded thalassemia patients: non‐chelated children (3–13 yrs, Gaza, Palestine), chelated adolescents‐young adults (13–28 yrs, Israel), and chelated adults (27–61 yrs, Israel) for LPI and eLPI. The eLPI levels in all three groups were roughly commensurate (r2 = 0.61–0.75) with deferrioxamine‐detectable NTBI, i.e., DCI. In older chelated patients, eLPI levels approximated those of LPI, but in poly‐transfused unchelated children eLPI was notably higher than LPI, a difference attributed to plasma properties affected by labile iron due to lack of chelation, possibly reflecting age‐dependent attrition of plasma components. We propose that the two formats of NTBI measurement presented here are complementary and used together could provide more comprehensive information on the forms of NTBI in patients and their response to chelation. Am. J. Hematol., 2011.

Decreased hemolysis following administration of antioxidant-fermented papaya preparation (FPP) to a patient with PNH.

Dear Editor, Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disease (HSC), characterized by intravascular hemolysis [1] due to inactivating mutation of the X-linked PIG-A gene in an HSC; the gene product is essential for the synthesis of glycosylphosphatidylinositol (GPI) anchor molecules [2]. Intravascular hemolysis is a major cause of anemia in PNH. Two surface proteins, CD55 and CD59, which regulate complement activation on the cell surface are GPI-linked [3], and their deficiency explains the hypersusceptibility of PNH red blood cells (RBC) to complementmediated lysis, intravascular hemolysis, and release of free hemoglobin (Hb). Hb has a vasculotoxic potential, directly impairing endothelial function and generating inflammatory and oxidative stress [4]. Cell-free Hb disintegrates into heme and globin, and the iron from heme catalyzes the formation of reactive oxygen species (ROS) [5] causing damage to various components of the cell. Flow cytometry analyses of RBC, platelets, and polymorphonuclear cells from patients with PNH disclosed a significant increase in ROS, while reduced glutathione was decreased. Oxidative stress was more profound in cells derived from the pathological clone with a CD55–CD59 phenotype. Increased membrane lipid peroxidation was also documented in PNH RBC [6]. Consequently, there is evidence for increased oxidative stress in PNH, which might play a significant role in the pathophysiology of the disease. Indeed, in vitro treatment of PNH-RBC with antioxidants decreased their hemolysis [6]. Therefore, there is a rationale for treatment with antioxidants in order to reduce the oxidative stress and improve its clinical manifestations in PNH patients. One antioxidant is fermented papaya preparation (FPP), a natural health food product obtained by yeast biofermentation of carica papaya [7], which decreases oxidative stress both in vitro and in vivo [8].