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Introduction:Patients with transfusion dependent thalassemia (TDT) have a genetic anemia that causes incomplete erythropoiesis and iron overload. Plasma zinc deficiency is also seen in roughly 25% of patients with TDT. Iron overload is thought to be related to a number of secondary complications in TDT including cardiomyopathy and diabetes. However, in TDT the effects of altered Zn status are not as well characterized and the effects of low copper (Cu) are even less well known. One possible cause of these complications is oxidative damage to tissues. This oxidative stress can be caused by labile plasma iron (LPI), a component of the non-transferrin bound iron pool, which is often seen in individuals suffering from iron overload. LPI is both redox-active and chelatable and is the likely culprit distributing iron to extra-hepatic tissues. Through reactive Fenton chemistry, LPI, can also cause lipid peroxidation releasing malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which are damage-associated molecular patterns and markers of oxidative tissue damage that activate the immune system to induce inflammation. Incomplete erythropoiesis as well as transfusional iron overload are responsible for an increase in the amount of poorly handled, redox active LPI in TDT. Thus, we hypothesized that changes in the levels of key iron trafficking proteins (such as soluble transferrin receptor (sTfR) or hemopexin) would affect oxidative stress levels in TDT. We also hypothesized that metal dyshomeostasis, such as a functional Zn or Cu deficiency would affect oxidative stress. Aims: The purpose of this pilot project is to 1) Determine the state of circulating levels of oxidative stress markers and iron trafficking proteins in TDT patients and 2) Explore the relationship between the markers and proteins measured in (1)and the Zn and Cu status of TDT patients. Methods:39 subjects with informed consent were enrolled (29 patients with TDT and 10 controls). Liver iron concentration (LIC) was measured by a superconducting quantum interference device (SQUID™). LPI was measured using dihydrorhodamine 123. Both MDA and MDA + 4-HNE were measured using N-methyl-2-phenylindole. The iron trafficking proteins sTfR, transferrin, haptoglobin and hemopexin were measured by immunoassay isolation followed by multiplex multiple reaction monitoring mass spectrometry. Zn and Cu were assessed by inductively coupled plasma atomic emission spectroscopy. Fructosamine was measured by quantitative spectrophotometry. Results: Patients with TDT had elevated LIC levels of 2681 ± 2424 ug iron/g wet weight. Plasma levels of the iron trafficking proteins, transferrin, hemopexin and haptoglobin were all decreased in TDT patients (P Conclusion: Metal dyshomeostasis involving Zn and Cu may be important contributors to oxidative stress and iron injury in TDT. We confirm previous findings in TDT of elevated levels of LPI as well as the oxidative stress markers MDA and 4-HNE. We expand previous findings of reduced transferrin levels in TDT to show a similar reduction in both haptoglobin and hemopexin. StfR levels were elevated in TDT patients, possibly due to a strong erythropoietic drive. Both reduced haptoglobin and hemopexin as well as decreased Zn and Cu levels and increased Zn excretion appear to be present in TDT. These preliminary findings suggest that low levels of Zn, Cu, haptoglobin and hemopexin may be related to increased oxidative stress and LPI in TDT, which could be important contributors to secondary complications of TDT. Disclosures Walter: Apopharma: Research Funding.

Introduction: β-thalassemia major requires a lifetime of transfusion and chelation therapy and despite improved chelation therapies, patients endure organ iron overload and increased hemolysis leading to organ injury and release of damage associated bio-markers. The structure of these markers are recognized by pattern recognition receptors (PRR) of the innate immune system as damage associated molecular patterns (DAMPs), which trigger inflammation and increased reactive oxygen species (ROS). The specificity of this recognition depends on PRRs, such as toll-like receptor 4 (TLR4), to provide the first line of defense against pathogens and DAMPS to initiate inflammation. Therefore, chronic organ iron loading is implicated in the chronic inflammation and morbidity of β-thalassemia major; however, the mechanism of how monocytes respond to iron loading in β-thalassemia remains largely unknown. This study investigated how chronic iron and heme overload affected innate immune cell morphology and expression of TLR4 and ROS by modeling THP-1 cellscompared to β-thalassemia monocytes. Methods: Cultured THP-1 monocytic cells were exposed to increasing concentrations of ferric citrate (0-250 μM) or heme (0-50 μM) for 24 hours or to chronic exposures of 40 μM ferric citrate for up to 3 weeks. Anti-human TLR4 phycoerythrin conjugated antibodies (TLR4-PE) were used to quantify TLR4 expression and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure ROS. Flow cytometry was done using a BD FACSCalibur. Peripheral-blood samples were obtained from 13 consenting β-thalassemia patients as part of the Novartis sponsored study investigating the effects of combined chelation therapy (CICL670AUS42T). Samples were also obtained from ethnically and age matched healthy controls. Peripheral-blood monocytes and THP-1 cells were gated on the parameters of forward and side scatter to stratify cells based on size and granularity, separating cells into two populations: larger more granular (LG) and smaller less granular (SG) cells. Results are reported as the MFI ratios of LG versus SG cells for TLR4-PE or DCFH-DA as a functional measure of the difference in TLR4 or ROS expression between these populations. Results: In vitro, LG THP-1 cells expressed 4-fold more TLR4 and 10-fold more ROS than SG cells (p < 0.001). Treatment of THP-1 cells with increasing concentrations of iron or heme for 1 day or 3 weeks, increased the ROS and TLR4 heterogeneity between LG and SG monocytes. The LG versus SG ratio of ROS production significantly correlated to the concentration of ferric citrate or heme added to the culture media (r = 0.64, p = 0.019 and r = 0.58 and p = 0.048, respectively). The mean ratio for iron but not heme treated THP-1 cells was significantly greater than for untreated cells (p = 0.008 and p = 0.406, respectively). Mean LG versus SG ratios of TLR4-PE MFI were 5.20 ± 1.56 for ferric citrate, 4.11 ± 0.90 for heme, and 3.78 ± 1.41 for untreated cells. We observed a similar TLR4 morphological heterogenic pattern in peripheral-blood monocytes from iron overloaded β-thalassemia patients. β-thalassemia LG monocytes had significantly greater TLR4 MFI than SG monocytes (LG mean = 11.91 ± 1.17, SG mean = 6.56 ± 1.02, p < 0.001). Long-term treatment of THP-1 monocytic cells for 3 weeks with 40 μM ferric citrate also resulted in the same phenomena regarding TLR4. No difference was observed in ROS production for control LG cells and SG cells; however, THP-1 cells treated for 3 weeks with 40uM ferric citrate revealed populations of LG cells that expressed 3 fold more ROS than SG cells. Conclusion: THP-1 monocytic cells and β-thalassemic peripheral-blood monocytes exhibit a morphological heterogeneity where LG cells express more TLR4 and in vitro, produce more ROS than SG cells. As TLR4 is one of the receptors for the initiation of inflammation, LG cells are likely greater contributors to the chronic inflammation experienced by β-thalassemia patients. Thus, we hypothesize that iron overload may give rise to monocytes that are larger and more granular and express more TLR4 and ROS. Disclosures Walter: Novartis: Research Funding. Porter:Shire: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy.

Introduction The thalassemias are inherited anemias sometimes characterized by severe transfusion dependence that can lead to extra-hepatic cardiac iron overload, causing cardiomyopathy. Despite improved chelation therapies, patients with transfusion-dependent thalassemia still endure cardiomyopathy and chronic inflammation. The innate immune system provides the first line of defense against infection and specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) interacts with CD14 on innate immune cells transducing the signal for bacterial lipopolysaccharide. Another cell surface protein that is not a PRR, but aids phagocytosis and is important in granulocytes function and chemotaxis is the adhesive polysaccharide antigen, CD15. The role that excess iron plays in determining expression level of these innate immune proteins is unknown. Thus, the goal in these studies is to investigate the relationship of cardiac iron overload and its chelation to innate immune cell expression of TLR4 and CD15 in patients with transfusion-dependent thalassemia. Patients and Methods Eighteen patients with transfusion dependent thalassemia (11 – 29 years old) (participating in the Novartis sponsored CICL670AUS24T) were enrolled in a substudy investigating innate immunology (Novartis sponsored CICL670AUS42T). Patients were investigated at baseline, then after 6 months and one year of combined chelation therapy with deferasirox and deferoxamine. Fasting blood samples were obtained after a 72 hr washout with no chelators. Fourteen healthy controls (10 - 35 yrs old) were also enrolled. Changes in LIC (ferritometer), cardiac function (MRI) and myocardial iron (MRI T2*) were monitored. Peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from blood samples using density gradients. Monocytes and granulocytes were further purified using antibody-linked magnetic microbeads. Highly enriched populations of CD14+ monocytes and CD15+ granulocytes were verified by flow cytometry. The expression level of CD15 and TLR4 was determined. Results Previously we found that transfusion-dependent thalassemia patients had 37% higher TLR4+ neutrophils than control patients and a smaller percentage of CD15+ neutrophils. We have also observed a decrease in TLR4 expression during the course of combined chelation therapy on neutrophils but not monocytes, indicating that TLR4 is differentially modulated on neutrophils compared to monocytes. Now we find that these flow cytometry parameters show significant relationships to markers of iron burden. The percentage of TLR4+ monocytes was related to liver iron concentration (r=- 0.49, p = 0.039), ferritin concentration (r=-0.47, p = 0.049), serum iron level (r=0.61, p = 0.008), and total iron binding capacity (TIBC; r=0.51, p = 0.021), while the percentage of CD15 positive neutrophils predicted myocardial iron, as measured by MRI T2* (r= 0.69, p Conclusions This study found evidence that the innate immune system may be modulating iron trafficking not only to the liver but to the heart as well. The negative correlation between LIC and TLR4 expression suggests that severe iron overload may lead to heptocellular damage causing monocyte dysfunction, the pathology of which could be due to altered TLR4 expression. This relationship may also be driving the positive correlation observed between TLR4 expression and TIBC. CD15 seems to play an important role in cardiac health as it is positively correlated to LVEF and MRI T2*. This relationship is further validated by our previous finding that transfusion-dependent thalassemia patients had a smaller percentage of CD15+ neutrophils, which we now show improved during one year of combination chelation therapy. Taken together, this suggests that chelation therapy may enhance cardiac health by increasing the percentage of CD15+ neutrophils. Disclosures: Walter: Novartis: Research Funding. Porter:Novartis: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria; Celgene: Consultancy. Vichinsky:Novartis: Honoraria, Research Funding.

Abstract 1025 Introduction: Thalassemia major patients endure chronic RBC transfusions, high levels of tissue iron, iron chelation and organ injury. Patients with thalassemia also have reduced immune function and are at risk for infection. Infection is in fact the second most common cause of death in thalassemia. The innate immune system provides the first line of defense against infection and it specificity depends on pattern recognition receptors (PRRs) specific to microbial pathogens. One class of PRR called the toll-like receptors (TLRs) interact with CD14 on innate immune cells transducing the signal for bacterial Lipopolysaccharide (LPS), resulting in cytokine production. The role iron plays in thalassemia in determining expression level of PRRs is unknown. Thus, the goal in these studies is to investigate the relationship of iron overload and its chelation to innate immune cell expression of PRRs in thalassemia. Patients and Methods: Eighteen transfusion dependent thalassemia patients (11 – 29 yrs old) participating in the combination trial of deferasirox and deferoxamine (Novartis sponsored CICL670AUS24T) were enrolled in a substudy investigating innate immunology (Novartis sponsored CICL670AUS42T). Fasting blood samples were obtained i) at baseline after a 72 hr. washout of chelator, and ii) at 6 and 12 months on study. Fourteen healthy controls (10 – 35 yrs old) were also enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and then from these cells monocytes and granulocytes were purified using antibody-linked magnetic microbeads (Miltenyi Biotec Inc). Highly enriched populations of CD14+ monocytes and CD15+ granulocytes were verified by flow cytometry. The expression level of CD14 and CD15 in PBMCs and TLR4 in purified cells were determined and reported as the median fluorescent intensity (MFI). Liver iron concentration (LIC) was determined by biomagnetic susceptibility (“SQUID”, Ferritometer®) in patients with thalassemia; healthy controls were shown to have normal ferritin. Results: In PBMCs from thalassemia patients at baseline, the expression of monocyte CD14 and TLR4 were significantly increased 22% and 6.5% respectively compared to healthy controls (p < 0.05). Granulocytes from patients with thalassemia at baseline were also found to have a 50% higher expression of TLR4 compared to controls. Markers of iron burden, such as LIC and ferritin also significantly correlated with the expression of monocyte TLR4. In longitudinal analysis markers of iron burden, the expression of TLR4 on monocytes and granulocytes all significantly decreased in the follow-up period in thalassemia patients receiving intensive combination chelator therapy (p Conclusions: These studies support the hypothesis that iron burden influences the innate immune response in thalassemia as demonstrated by the increased monocyte expression of both CD14 and TLR4 at baseline, both of which likely contribute to the commonly observed susceptibility to infection. After intensive chelation, the levels of CD14 and TLR4 decreased, indicating that decreased iron overload with chelation may improve innate immune responsiveness. These changes in CD14 and TLR4 may be able to restore proper innate immune function in thalassemia patients. Disclosures: Walter: Novartis: Research Funding. Harmatz:Novartis: Research Funding; FerroKin BioSciences: Research Funding. Porter:Novartis: Consultancy, Research Funding. Vichinsky:ARUP Research Lab: Research Funding; ApoPharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Sickle cell disease (SCD) is a hemoglobinopathy characterized by micro-vascular hypoxia-reperfusion, inflammation and leukocytosis. Studies in SCD have shown that leukocytosis is a strong predictor of stroke and disease severity. It is known that leukocytosis and inflammation contribute to increased leukocyte-endothelial adhesion and vasoocclusive events. Leukocytosis or increased leukocyte number is determined by the balance between cell death programs (apoptosis) and proliferation. In this study, we examine markers of apoptosis and proliferation in SCD as compared to thalassemia (a hemoglobinopathy that is vasculitis negative) and a control group. Methods: Markers of leukocyte turnover, inflammation and free iron (NTBI, non-transferrin bound iron) were compared in 11 patients with SCD (7M, 13 ± 4 yrs), 18 with thalassemia (7M, 24 ± 9 yrs) and 10 disease-free controls (5M, 27 ± 12 yrs). All SCD and thalassemia patients were healthy and event free in the previous 4 months. Blood was obtained fasting and prior to RBC transfusion; and plasma, serum and cells were separated by centrifugation. The pro-apoptotic markers nucleosome protein (DNA laddering) and Bax (initiator of mitochondrial permeability) and inflammatory marker, high-sensitivity C Reactive Protein (hsCRP) were determined by ELISA. NTBI (DNA damage inducing, pro-apoptotic) was measured by HPLC. Plasma levels of cytokines (anti-apoptotic, proliferation stimulating) were determined by using a multiplex bead-based immunoassay. Plasma lactate dehydrogenase (LDH), a marker of hemolysis, was also measured because hemolysis products have been shown to inhibit apoptosis. Results: Leukocyte number and absolute neutrophil count were 1.8 and 2.2 fold higher in SCD compared to thalassemia (P

Objective: Chronic red blood cell transfusion therapy is life-saving for patients with β-thalassemia (THL) and sickle cell disease (SCD), but often results in severe iron overload. Clinical observations suggest that organ dysfunction (heart, liver, endocrine dysfunction, bone disease) resulting from iron overload is seen more often in patients with THL than SCD. This study examines the possible correlation between increased organ injury in these patients and oxidative stress, iron metabolism, inflammation and plasma vitamin E. Methods: Markers of oxidant stress were compared in 18 subjects with THL (7M, 24 ± 9 yrs) and 11 with SCD (7M, 13 ± 4 yrs) with 10 disease-free controls (5M, 27 ± 12 yrs). All THL and SCD patients admitted to the study were healthy and had not had a recent medical event including vascular crises for SCD within the last 4 months. Blood was drawn from fasted subjects, prior to blood transfusion, and plasma, serum and cells were separated by centrifugation. Plasma levels of malondialdehyde (MDA), a marker of lipid peroxidation, were determined by GC-MS. Non-transferrin bound iron (NTBI), protein carbonyls and tocopherol content were measured by HPLC. C Reactive Protein (CRP) was determined by ELISA. Liver iron content was analyzed by ICP-mass spectrometry from disease patients. Results: Table 1 Values are means ± SD (subject n). Within a row, underlined values are significantly different from control and starred values are significantly different between THL and SCD (P