CSTM 2011,Toronto, Canada

CBR News

Effect of Iron on Antigen Presenting Cells: Implications For Transfusion Dependent Hemoglobinopathies.

Ibrahim Mustafa, Duncheng Wang and Mark D. Scott

Canadian Blood Services and the Centre for Blood Research and the Department of Pathology and Laboratory Medicine at the University of British Columbia, Vancouver, BC, Canada

Background: The thalassemias and Sickle Cell Disease (SCD) are characterized by destabilized hemoglobin that can lead to a potentially life-threatening anemia. This anemia arises due to iron-driven destruction of the RBC. Removal of oxidatively damaged RBC in vivo occurs primarily via erythrophagocytosis by the mononuclear phagocytic system (MPS). This clearance mechanism may result in negative immune consequences such as the observed increased risk of bacterial infections in these patients. Methods: To determine the functional consequences of iron and iron chelators on the MPS, the effects of ferric iron (Fe3+; ferric ammonium citrate, FAC) on dendritic cells (DC) antigen presentation [tetanus toxoid (TT Ag)] and the proliferation of peripheral blood mononuclear cells (PBMC) were examined. The iron chelators tested included Desferal (DFO) and Deferiprone (L1). PBMC were labelled with the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) to measure cell proliferation. In addition, the effects of iron +/- iron chelators on the expression of CD83, CD80, CD86 and HLA-DR on mature DC were examined. Results: Importantly, iron significantly inhibited antigen presentation and PBMC proliferation. Treatment of DC cells with 200 µM FAC for 24 hours resulted in a ~70% reduction in PBMC proliferation in response to the TT Ag following 14 days culture. However, inclusion of iron chelators (e.g., 200 µM DFO or L1) restored near normal proliferation. Similarly, CD83 an important co-stimulatory molecule expressed on DC cells was also negatively affected by FAC in a dose (0-200 µM) dependent manner. Following 24 hours treatment with 200 µM FAC, a ~30% reduction in the mean fluorescence of CD83 was observed via flow cytometric assay. Treatment with DFO or L1 overcame the effects of iron on CD83 expression. There were no significant effects of iron on CD80 or CD86. Conclusions: As shown, iron has significant immunosuppressive effects on antigen presentation and lymphocyte proliferation. Iron chelators can effectively bind and remove free and complexed iron and reverse iron-mediated immunosuppression. These data suggest that iron chelation may provide a mechanism to diminish the risk of recurrent bacterial infections in patients with unstable hemoglobins or with iron-overload (hemochromatosis or secondary iron overload).

Note: This abstract was published on conference proceedings, of Canadian Society for Transfusion Medicine 2010 held at Vancouver

Immunological Inhibition Arising From Misplaced Iron: Implications for Thalassemia and Sickle Cell Disease

I. Mustafa, D. Wang and M.D. Scott

Canadian Blood Services and the Centre for Blood Research and the Department of Pathology and Laboratory Medicine at the University of British Columbia, Vancouver, BC, Canada

Background: The thalassemias and Sickle Cell Disease (SCD) arise from mutations to the globin subunits of adult hemoglobin (HbA) resulting in destabilized hemoglobin and, potentially, a life-threatening anemia due in part to iron-driven redox reactions. While transfusions corrects the anemia, secondary iron overload can occur. Thus, both the primary and secondary pathology of thalassemia and SCD arise from “misplaced” iron. Removal of oxidatively damaged RBC in vivo occurs primarily via erythrophagocytosis by the mononuclear phagocytic system (MPS). This clearance mechanism may result in negative immunoregulatory effects such as the observed increased risk bacterial infections in these patients.

Methods: To determine the functional consequences of iron on the MPS, the effects of ferric iron (Fe³⁺; ferric ammonium citrate, FAC), heme, purified HbA and oxidized RBC on antigen presentation/proliferation by PBMC and cultured dendritic like (DC) cells was examined. Antigens examined included tetanus toxoid (TT Ag), formalin-fixed Streptococcus mutans (SM Ag) and RhD peptide. PBMC proliferation was determined by ³H-thymidine incorporation or via flow cytometry using carboxyfluorescein diacetate, succinimidyl ester (CFSE) stained cells. To determine if iron-driven immunomodulation could be reversed, an iron shuttle chelation system using Desferal (DFO; shuttle chelator) and S-DFO (a high molecular weight DFO-starch conjugate) was examined.

Results: Importantly, all forms of iron, including oxidized RBC, significantly inhibited antigen presentation and PBMC proliferation. For example, 100 µM hemin resulted in a >98% reduction in proliferation in response to the TT or SM Ag. Similarly, phagocytosis of oxidized RBC virtually abolished the ability of antigen presenting cells within the PBMC to present antigen and abolished the response to the TT and SM antigens. DC cells were similarly affected by FAC (200 µM) exposure (7 days) with a ~78% reduction PBMC response to an immunodominant RhD peptide. Iron chelators could partially overcome the effects of the bioreactive iron. Of interest, prolonged treatment with S-DFO (unlike DFO) did not adversely affect purified hemoglobin.

Conclusions: As shown, iron has significant immunodepressive effects on immune function (antigen presentation and lymphocyte proliferation). Iron chelation can effectively bind and remove free and complexed iron /heme preventing both redox-driven damage and immuosuppression. These data suggest that a two component iron shuttle chelation system may effectively slow/prevent iron-driven damage within cells and may also protect immune competency.

Note: This was published Transfusion AABB Annual Meeting, New Orleans, LA USA 24-27 Oct 2009.

Fe3+ as a potent accelerator for hemoglobin oxidation in vitro and prooxidant effects of reduced glutathione (GSH).

Authors: Mustafa, I. and Scott, M.D. Department of Pathology and Laboratory Medicine and the Centre for Blood Research at the University of British Columbia and the Canadian Blood Services.

Abstract

Iron is an essential trace element for all living cells. Indeed, iron cores constitute the functional sites of many enzymes and proteins involved in generating energy, transporting oxygen, and DNA synthesis. Containing approximately 20 mM iron, normal erythrocytes are the most iron and oxygen rich somatic cell. While this important metal is vital, maintaining its biological balance in an organism is far more crucial than virtually any other trace element (with the possible exception of copper). Excess iron, due to its catalysis of one electron redox chemistry, plays a key role in the formation of toxic oxygen radicals. Indeed, this is readily observed in diseases such as thalassemia and sickle cell anemia. This potentially dangerous combination of oxygen and iron within the erythrocyte is kept in check by a number of endogenous mechanisms. These include the hemoglobin (Hb) tetramer itself, as well as a number of antioxidants such as superoxide dismutase (SOD), catalase, glutathione/glutathione peroxidase, and methemoglobin reductase.

Experiments were conducted in vitro using purified hemoglobin (HbA; a₂ ß₂) exposed to ferric (Fe³⁺) iron. Upon Fe³⁺ addition (0-250 µM ferric ammonium sulfate), spectrophotometric shifts in the absorption spectra (500 –700 nm) of purified hemoglobin (~10 µM HbA; pH 7.4) was determined. HAb concentration was determined by the cyanomethemoglobin (Drabkin’s) method. The inhibitory effects of glutathione (GSH), iron chelators (desferrioxamine, DFO; and Deferiprone, L1), SOD and catalase on Fe³⁺-driven oxidation was assessed. In the absence of Fe³⁺ no HbA oxidation was noted. Similarly, in contrast to erythrocyte hemolysates, simple addition of Fe³⁺ to purified HbA also had minimal oxidative effects. However, addition of the “anti-oxidant” GSH, resulted in a potent GSH (0-10 mM) and Fe³⁺ (0-250 µM) dose-dependent oxidation of the purified HbA to methemoglobin. Methemoglobin is characteristized by a peak (or shoulder) at 630 nm. This data demonstrates that a reducing agent is necessary for iron-driven oxidation. Notably the intact erythrocyte is rich in both GSH and ascorbate. This oxidation was further enhanced by the presence of hydrogen peroxide (H₂O₂), a normal byproduct of HbA auto-oxidation. Importantly, inclusion of the iron chelators (DFO or L1) inhibited Fe³⁺-mediated HbA oxidation in a dose-dependent manner. For example, >250 µM DFO, inhibited virtually all iron mediated HbA oxidation induced by the addition of 250 µM Fe³⁺ and 0. 4 mM GSH. Further experiments were carried out to see if Fe³⁺ mediated HbA oxidation could be inhibited by SOD or catalase. These studies demonstrated no protective effects, suggesting that Fe³⁺ - GSH degradation is not due to either O^-₂ or H₂O_2, or even hydroxy radicals, because SOD or catalase would have blocked hydroxyl radical formation. Consequently this data implicates thiol radicals as the agent of injury.

These findings have significant implications in the mechanisms of injury in disease such as the thalassemias and sickle cell anemia. Furthermore, these results suggest that chelation of bioreactive iron within the abnormal erythrocyte may be an effective therapeutic intervention. My research is currently focusing on a novel iron-shuttle chelation therapy regime utilizing low and high molecular weight iron chelators.

Iron Shuttle Chelation Therapy: A Novel Approach To Treating Hemoglobinopathies

I. Mustafa, N. Rossi, J. N. Kizhakkedathu and M.D. Scott
Canadian Blood Services and the Centre for Blood Research and the Department of Pathology and Laboratory Medicine at the University of British Columbia, Vancouver, BC, Canada

Background: Thalassemias arise from deficiency of the globin subunits of adult hemoglobin (HbA) and results in ineffective erythropoiesis and the rapid destruction of RBC in the periphery. These iron-driven events give rise to anemia. While transfusion therapy corrects the anemia, it give rise to secondary iron overload. Thus, both the primary and secondary pathology of thalassemia arise from “misplaced” iron. Our previous studies suggest that chelation of bioreactive iron within the thalassemic/sickle RBC may be an effective therapeutic intervention. Our research is focused on a novel iron-shuttle chelation therapy utilizing both low (shuttle) and high (docking) molecular weight iron chelators.
Methods: The effect of shuttle and docking chelators on ferric iron (Fe3+) – driven Hb and lipid oxidation was assessed singularly and in combination. Experiments were conducted in vitro using HbA exposed to Fe3+ (0-175µM). HbA oxidation was quantitated spectrophotometrically. Shuttle chelators included DFO, L1, HBED and ICL-670.The docking chelators consisted of S-DFO (a starch conjugate of DFO) and P-DFO (a novel poly(ethylene glycol)-acrylate based copolymer of DFO).Lipid peroxidation was measured by thiobabutaric acid reactive substances (TBARS) formation
Results: In the absence of Fe3+ no HbA oxidation was noted. Addition of 175µM Fe3+ resulted in rapid methemoglobin formation. Importantly, inclusion of any of our chelators inhibited HbA oxidation in a dose-dependent manner. For example,175 or 200 µM DFO , P-DFO or S-DFO respectively, inhibited >90% HbA oxidation induced by 175 µM Fe3+. Similarly, lipid peroxidation was inhibited in a chelator dose dependent manner: 400 µM DFO or P-DFO equivalents resulted in a 58 or 70% reduction TBARS formation (respectively).
Conclusions: As shown, both shuttle and docking chelators can effectively bind and remove free and complexed iron /heme from aqueous and lipid environments and prevent redox-driven damage. These data suggest that a two component iron shuttle chelation system may effectively slow/prevent iron-driven damage and improve both effective erythropoiesis and the viability of abnormal RBC within the periphery. Thus this iron shuttle system may have therapeutic importance in the treatment of hemoglobinopathies.

Note: This abstract was published on AABB Transfusion journal 2008 special edition for the anual conference in Montreal.

Saga of thalassemia - Maldivian perspectives

Ibrahim Mustafa
National Thalassemia Centre, Male' Maldives

Maldives is a country with a distinctive geography that portrays a model of a small island nation. The total landmass of the 1190 small islands of the country is less than one percent of its national territory. Population of the 200 inhabited islands varies considerably. The country's 2000 population census shows a total of 270,101 people living in the country.

Origins of the Maldivians are not very certain. However, it is believed that the history goes as far back as BC 1000. Early settlers were travelers on the Silk Route, and from the Indus Valley Civilization. The Maldivians are innately warm, friendly and generous by nature, and anyone can easily establish a casual conversation with the Maldivians.

It is believed that the genetically inherited heterogeneous disease “Thalassaemia” have been imported to Maldives through traders’ route, how ever there is no scientific evidence to prove this, or the gene is due to a mutation in the midst of Maldivian population. The earliest case of Thalassaemia diagnosed goes back to early 1970s. During 1970 – 1980 few cases registered in Government Hospital where there was no proper regimen for transfusion and proper management of this disease. During 1980 -1990 there was dramatic increase of Thalassaemia patients in the country, due to this special clinics for these patients were been made in the paediatric ward of the hospital. The impact of Thalassaemia and its social stigma has been recognized by the government and many experts from various countries have been brought in collaboration with WHO during 1980s. Ever since 1992 one of the leading NGO in the country ,Society for Health Education (SHE) working towards well being of Maldivian’s health has started one of its essential element, Thalassaemia Prevention Program, comprising ,health education , population screening and genetic counseling .During 1993 National Thalassaemia Program has been formulated and in December 1994 National Thalassaemia Centre has been inaugurated with 17 beded ward , blood banking facilities and a diagnostic laboratory services.

The enormous contribution of many people in both Government and NGOs across the country, thalassaemia awareness has increased dramatically and now the word thalassaemia is a house hold story amid all the alliance of communities. The combine effort of these institutions has screened thalassaemia and other hemoglobinopathies over 30 % of the entire population in the archipelago using modern diagnostic methodologies, including hemoglobin electrophoresis, automatic cell counting and quantification of hemoglobin. Some collaborative studies has been done with International Thalassaemia Research Institutions abroad, such as molecular defect of Beta Thalassaemia in the Maldives has been studies and their prevalence has been identified (74% being IVS 1 – 5 G – C ) ( Journal of Hemoglobin 1998, Fucheron et al ). This knowledge has contributed and paves the way to guiding principle for the prevention of thalassaemia for the sustainable development of health sector in the country.

Around 18% of the Maldivian population is found to be carriers for hereditary blood disorder, beta thalassaemia. This prevalence level leads to a birth of a thalassaemic child (thalassaemia major) for every 120 births. At the end of the year 2002, nearly 504 thalassaemia majors were registered at the National Thalassaemia Center. It has been estimated that for the eight-year period 1990-1998 more than 50% of the Thalassaemic children born in Maldives would have died in infancy or before their third birthday.

Due to the impact of emerging issues of thalssaemia and its impact to the community directly and indirectly, a six year National Thalassaemia Program has been formulated, many areas have been highlighted and it’s on the way for implementations. This includes, population screening, thalassaemia education, prenatal diagnosis (PND) and research. In near future PND can be done in Maldives with the establishment of molecular biology laboratory in the country. The couples at risk can be counseled and if they desire medical termination of pregnancy (MTP) can be recommended.

Screening for thalassaemia, as well as treatment of thalassaemics, is costly but at the moment it’s free in the Maldives. Thalassaemic children require continuous and regular care and treatment to stay alive. They require monthly transfusions and treatment with the drug Desferrioxamine, injected five times a week. The annual cost of treatment exceeds US$6,000. At present only, Bone Marrow Transplant (BMT) ensures permanent cure for Thalassaemics. But the cost of this treatment ranks between US$30,000 and US$50,000. Due to the low income of average people, this costly treatment BMT cannot be afforded by many families.

The perspective of Thalassaemia seems to be a successful story in Maldives, as it is integrating government and non governmental efforts to control thalassaemia in Maldives. A look at the expectations therefore indicates the need for a more comprehensive incorporated program to strengthen the existing agenda for the prevention of thalassaemia in Maldives.

Note: This article was written on 2002 and published on Thalassemia Awareness Week supplement at Baqai Medical University.

References:

1. Ministry of Planning National Development: Population and Housing Census of Maldives 2000, Population and Housing Tables (1st Edition) Novelty Press. Male’
2. Society for Health Education (2002),Thalassaemia Status in Maldives: a report from retrospective studies
3. National Thalassaemia Centre(2002) Laboratory data ,Male’ Maldives
4. UN Building, WHO Reference Library, Reports on Thalassaemia in Maldives
5. Health Master Plan, Sustainable Development of Health 1996
6. National Thalassaemia Program 2001 – 2006, Ministry of Health, Maldives.
7. Daniel W. Byrne(1988) Publishing Your Medical Research Paper(1stEdition)Williams