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A high tricuspid regurgitant jet velocity (TRV) signifies a risk for or established pulmonary hypertension (PH), which is a serious complication in thalassemia patients. The underlying pathophysiology in thalassemia subgroups and potential biomarkers for early detection and monitoring are not well defined, in particular as they relate to spleen removal. To better understand some of these unresolved aspects, we examined 76 thalassemia patients (35 non-transfused), 25 splenectomized non-thalassemia patients (15 with hereditary spherocytosis), and 12 healthy controls. An elevated TRV (>2.5m/s) was found in 25/76 (33%) of the patients, confined to non-transfused or those with a late start of transfusions, including patients with hemoglobin H-constant spring, a finding not previously described. These non or late-transfused patients (76% splenectomized) had significantly increased platelet activation (sCD40L), high platelet count, endothelial activation (endothelin-1), and hemolysis (LDH, plasma-free Hb), while hypercoagulable and inflammatory markers were not significantly increased. The same markers were increased in the seven patients with confirmed PH on cardiac catheterization, suggesting their possible role for screening patients at risk for PH. A combination of hemolysis and absence of spleen is necessary for developing a high TRV, as neither chronic hemolysis in the non-splenectomized thalassemia patients nor splenectomy without hemolysis, in the non-thalassemia patients, resulted in an increase in TRV.

This exploratory study assessed apoptosis in peripheral blood leucocytes (PBL) from β-thalassaemia patients receiving chronic transfusions and chelation therapy (deferasirox or deferoxamine) at baseline, 1, 6, and 12 months. At baseline, thalassaemic PBLs presented 50% greater levels of Bax (BAX), 75% higher caspase-3/7, 48% higher caspase-8 and 88% higher caspase-9 activities and 428% more nucleosomal DNA fragmentation than control subjects. Only neutrophils correlated significantly with apoptotic markers. Previously, we showed that over the treatment year, hepatic iron declined; we now show that the ratio of Bax/Bcl-2 (BCL2), (-27·3%/year), and caspase-9 activity (-13·3%/year) declined in both treatment groups, suggesting that chelation decreases body iron and indicators of PBL apoptosis.

Patients with thalassemia (Thal) have low bone mass which can lead to fracture and decreased quality of life. There are no noninvasive anabolic therapies available to improve bone health in Thal. A longitudinal cross-over pilot trial was conducted to evaluate the effectiveness of low magnitude whole body vibration (WBV) therapy on bone in 18 patients with Thal (9 adults, 10 male, 22.1 ± 10.7 years). Subjects were asked to stand on a vibrating platform (30 Hz, 0.3 g) for 20 min/day for 6 months. Areal bone mineral density (aBMD) by DXA and volumetric BMD by peripheral quantitative computed tomography (pQCT) was assessed at baseline, 6 and 12 months. Adherence in the first 3 months was greater when compared with the second 3 months (14 ± 6 vs. 10 ± 7 min/day, P=0.007). Intention to treat analysis revealed a significant increase in whole body BMC (2.6%; P = 0.021), BMC/Ht (2.6%, P = 0.02) and aBMD (1.3%; P = 0.036), as well as a net increase in serum markers of bone formation (Osteocalcin/CTx, P = 0.027) in the adult subjects. These preliminary findings suggest that vibration therapy may be an effective nonpharmacologic intervention in Thal. Future research is needed to confirm these findings in a larger sample for longer duration.

Background: Neurodegeneration with Brain Iron Accumulation (NBIA) is a group of rare genetic disorders characterized by progressive degenerative motor symptoms and the accumulation of iron in the basal ganglia. Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a form of NBIA caused by a mutation in the PANK2 gene leading to a deficiency in pantothenate kinase. Phospholipase A2G6-Associated Neurodegeneration (PLAN) is caused by a mutation in the PLA2G6 gene resulting in impaired phospholipase activity. Current understanding of systemic changes in NBIA disorders is limited, leaving no clear diagnostic biomarkers. Monitoring the systemic changes could identify candidate biomarkers for assessing disease severity and evaluating the efficacy of new therapies. Previous studies of Parkinson's disease (PD) have found a systemic burden of increased oxidative stress and chronic inflammation accompanies the neurological symptoms of the disease. Similarly, abnormal systemic iron regulation associated with brain iron accumulation as well as damage associated with neuromuscular degeneration could lead to increased oxidative stress and a state of chronic inflammation in NBIA. Our initial investigation of a patient with PLAN1, revealed elevated levels of systemic oxidative stress. We investigated a group of PKAN patients as well as continued our investigation of a patient with PLAN to evaluate the possibility of abnormal iron trafficking, increased oxidative stress and chronic inflammation in NBIA. Our aim was to expand our investigation of circulating levels of inflammatory cytokines, oxidative stress markers and iron regulatory and metabolic proteins in NBIA patients to include a group of patients with PKAN. Methods: Plasma samples from 15 PKAN patients were collected at the UCSF Benioff Children's Hospital in Oakland, California. Similarly, a plasma sample from a patient with PLAN was collected in Campbell River, British Columbia. Plasma samples from a matched group of 15 healthy controls were also collected at the University of Victoria. All patients provided informed consent to the study. The pro-inflammatory cytokines IL-6 and TNFα as well as the anti-inflammatory cytokine IL-10 were measured by ELISA. Total levels of the lipid peroxidation product malondialdehyde (MDA) were measured using N-Methyl-Phenyl-Indole (NMPI). Free, acutely generated, MDA not bound to proteins, was measured by removing plasma proteins via a 10KD spin filtration then measuring the MDA content of resulting filtrate using NMPI. Results: The levels of MDA and Free MDA were significantly elevated in PKAN patients at baseline in comparison to controls (p = 0.05, p = 0.03). IL-6 and TNFα were slightly, but not significantly elevated at baseline compared to controls. We previously demonstrated, similar elevations of oxidative stress in our case study of an NBIA patient with PLAN1. Additionally, all three inflammatory cytokines measured for this study expansion in PLAN were higher than average levels observed in the PKAN and control groups (S ee Table 1). Further analysis of systemic biomarkers in NBIA including proteomic analysis of 30 systemic blood proteins, including iron trafficking proteins is ongoing. Conclusions: We expand previous findings of elevated levels of systemic oxidative stress in other neurodegenerative diseases such as PD to include NBIA patients with PKAN and PLAN. We provide novel evidence of elevated levels of Free MDA; representative of an acute oxidative stress burden in NBIA in addition to the previously noted elevation in total MDA levels. We provide preliminary signs that of an accompanying inflammatory burden in NBIA, but a larger sample group may be needed to determine its significance. References M. Minkley, A. Jackson, D. Smith, C. Borchers, E. Vichinsky, R. Nashmi, P.B. Walter and P. M. Macloed. (2017). Neurodegeneration with Brain Iron Accumulation: PLA2G6-Associated Dystonia-Parkinsonism: Clinical and Animal Studies. Presented at the 2017 European Human Genetics Conference, Copenhagen, Denmark . Disclosures Minkley: Apopharma: Research Funding. Neumayr: Apopharma: Research Funding. 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.

Abstract 260 Sickle cell disease (SCD) is characterized by frequent painful vaso-occlusive episodes, acute life-threatening complications, and progressive vascular injury resulting in multi-organ failure. Emerging data indicate that nitric oxide (NO) plays a central role in mediating the pathogenesis of sickle-induced vascular injury. Because antisickling agents only partially ameliorate the vasculopathy of SCD, therapeutic agents that modulate NO homeostasis are rational candidates for further exploration. Simvastatin and other HMG-CoA reductase inhibitors have been shown to exert NO-mediated vasculoprotective and anti-inflammatory effects in cardiovascular disease populations. Recent experimental and clinical studies have implicated the Rho/Rho-kinase (ROCK) signaling pathway as a principal mechanism by which statins increase NO availability. We enrolled twelve SCD patients in an ongoing FDA-approved dose-escalation study of the effect of simvastatin treatment on markers of vascular dysfunction and inflammation. Patients were treated at steady-state with low-dose oral simvastatin (20 mg/day) with serial blood samples obtained prior to treatment day 0, during treatment on days 7, 14, 21, 25 and after discontinuation of treatment on day 39. Plasma samples were assayed for nitrite/nitrate (NOx) concentration using NO chemiluminescence, and high-sensitivity C-reactive protein (hs-CRP), interleukin 6 (IL-6), tissue factor (TF), vascular endothelial growth factor (VEGF) and vascular cell adhesion molecule (VCAM1) by ELISA. The effect of simvastatin on leukocyte ROCK activity was assessed by Western blot analyses in a subset of patients. Changes in biomarker levels from baseline in response to simvastatin at 21 days were assessed using matched paired t-tests. The mean age of subjects was 31.4 years (range, 13–55); 7 males and 5 females. Four subjects experienced an uneventful vaso-occlusive pain episode during the study with an acute decrease in NO levels and these data points were removed from the analysis. Short-term simvastatin treatment resulted in a 24% increase in mean plasma NOx level (p=.02). Both hs-CRP (p=.01) and IL-6 levels (p=.05) decreased dramatically by 62% and 63%, respectively. There was no difference between baseline and day 21 levels of TF, VEGF and VCAM1. Although limited to qualitative assessment in a subset of patients, our pilot data also demonstrated a decrease in leukocyte ROCK activity after treatment with simvastatin. These preliminary data showing increased NO availability and anti-inflammatory effects of simvastatin treatment in SCD are consistent with studies in cardiovascular disease populations documenting the cholesterol-independent benefits of statins and provide a compelling rationale to further investigate the potential therapeutic efficacy of statins in SCD. Correlation of the effect of simvastatin on ROCK inhibition may provide further evidence for a possible mechanism by which statins modulate NO bioavailability in SCD. Disclosures: Off Label Use: Simvastatin (Zocor)is a cholesterol-lowering agent that has been shown to confer vascular protection beyond its lipid lowering effects. This drug is being studied in patients with sickle cell disease to obtain preliminary safety and efficacy data of its potential vasculoprotective effects.

Abstract 4071 Poster Board III-1006 Introduction Infection is the second most common cause of death in thalassemia. The innate immune system provides a 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) are important for transducing the signal for bacterial Lipopolysaccharide (LPS), resulting not only in cytokine production, but also in the control of extracellular iron levels through production of neutrophil gelatinase associated Lipocalin (NGAL). However, the exact role that NGAL plays and the expression level of PRRs are unknown in thalassemia. Thus, the goal in these studies is to investigate the relationship of iron overload to the innate immune cell expression of PRRs and NGAL in thalassemia. Patients and Methods Fifteen transfusion dependent thalassemia patients (11 – 29 yrs old) participating in the combination trial of deferasirox (an oral iron chelator) and deferoxamine were enrolled (Novartis sponsored CICL670AUS24T). Fasting blood samples were obtained i) at baseline after a 72 hr washout of chelator, and ii) at 6 and 12 months on study. Five healthy controls (13 - 18 yrs old) were also enrolled. Fresh monocytes were isolated using antibody-linked magnetic microbeads (Miltenyi Biotec Inc). Highly enriched populations of CD14+ monocytes were verified by flow cytometry. The expression of TLR4, also examined by flow cytometry is reported as the mean fluorescent intensity (MFI). In patients with thalassemia, liver iron concentration (LIC) was analyzed by biomagnetic susceptibility (“SQUID”, Ferritometer®). The plasma levels of NGAL were analyzed by ELISA. Results At baseline the expression of monocyte TLR4 (mean 18.8 ± 3.5 MFI) was reduced 30% compared to the healthy controls (mean 26.9 ± 7.6 MFI, p Conclusions These preliminary studies support the hypothesis that iron burden has a negative impact on the innate immune response in thalassemia as demonstrated by the decreased expression of TLR4. After intensive chelation, the levels of TLR4 increased, indicating that decreased iron overload with chelation may improve innate immune responsiveness. Finally, the iron transport protein NGAL is significantly elevated in thalassemia possibly acting to prevent essential iron uptake by pathogenic bacteria. Disclosures: Harmatz: Novartis: Research Funding; Apotex : Membership on an entity's Board of Directors or advisory committees; Ferrokin: Membership on an entity's Board of Directors or advisory committees. Vichinsky:Novartis: Consultancy, Research Funding.