Search

Professor Bruce Ames demonstrated that nutritional recommendations should be adjusted in order to 'tune-up' metabolism and reduce mitochondria decay, a hallmark of aging and many disease processes. A major subset of tunable nutrients are the minerals, which despite being integral to every aspect of metabolism are often deficient in the typical Western diet. Mitochondria are particularly rich in minerals, where they function as essential cofactors for mitochondrial physiology and overall cellular health. Yet substantial knowledge gaps remain in our understanding of the form and function of these minerals needed for metabolic harmony. Some of the minerals have known activities in the mitochondria but with incomplete regulatory detail, whereas other minerals have no established mitochondrial function at all. A comprehensive metallome of the mitochondria is needed to fully understand the patterns and relationships of minerals within metabolic processes and cellular development. This brief overview serves to highlight the current progress towards understanding mineral homeostasis in the mitochondria and to encourage more research activity in key areas. Future work may likely reveal that adjusting the amounts of specific nutritional minerals has longevity benefits for human health.

Plasma zinc concentrations (PZC) have been shown to significantly increase during zinc supplementation. This study investigated the effects of daily preventive zinc supplementation on hair and nail zinc concentrations compared with a control group. In a randomized controlled trial, 6- to 23-month-old children (n = 3407) in Lao PDR were randomly assigned to one of four groups and followed for ~ 36 weeks: daily preventive zinc dispersible tablet (7 mg/d; PZ), daily micronutrient powder (10 mg zinc/d; MNP), therapeutic zinc supplements for diarrhea treatment (20 mg/d for 10 days; TZ), or daily placebo powder (Control). Plasma, hair, and nail zinc concentrations were assessed in a sub-sample of participants (n = 457) at baseline and endline. At baseline, 75% of children had low PZC (P P = 0.99). Nail zinc concentrations were marginally higher in the PZ (115.8 (111.6, 119.9) μg/g) and the MNP (117.8 (113.3, 122.3) μg/g) groups than in the TZ group (110.4 (106.0, 114.8) μg/g; P = 0.055) at endline. This study does not support the use of hair zinc as a biomarker of zinc exposure in young children. However, it provides some evidence that zinc concentrations in nails may respond to supplemental zinc interventions and supports the need for collecting additional data on this emerging biomarker.

Lysosome function is compromised during aging and in many disease states. Interventions that promote lysosomal activity and acidification are thus of prime interest as treatments for longevity and health. Intracellular pH can be controlled by the exchange of protons for inorganic ions, and in cells from microbes to man, when potassium is restricted in the growth medium, the cytoplasm becomes acidified. Here we use a yeast model to show that potassium limited-cells exhibit hallmarks of increased acidity in the vacuole, the analog of the lysosome, and live long by a mechanism that requires the vacuolar machinery. The emerging picture is one in which potassium restriction shores up vacuolar acidity and function, conferring health benefits early in life and extending viability into old age. Against the backdrop of well-studied protein and carbohydrate restrictions that extend lifespan and healthspan, our work establishes a novel pro-longevity paradigm of inorganic nutrient limitation.

Elevated uric acid (UA) is a key risk factor for many disorders, including metabolic syndrome, gout and kidney stones. Despite frequent occurrence of these disorders, the genetic pathways influencing UA metabolism and the association with disease remain poorly understood. In humans, elevated UA levels resulted from the loss of the of the urate oxidase (Uro) gene around 15 million years ago. Therefore, we established a Drosophila melanogaster model with reduced expression of the orthologous Uro gene to study the pathogenesis arising from elevated UA. Reduced Uro expression in Drosophila resulted in elevated UA levels, accumulation of concretions in the excretory system, and shortening of lifespan when reared on diets containing high levels of yeast extract. Furthermore, high levels of dietary purines, but not protein or sugar, were sufficient to produce the same effects of shortened lifespan and concretion formation in the Drosophila model. The insulin-like signaling (ILS) pathway has been shown to respond to changes in nutrient status in several species. We observed that genetic suppression of ILS genes reduced both UA levels and concretion load in flies fed high levels of yeast extract. Further support for the role of the ILS pathway in modulating UA metabolism stems from a human candidate gene study identifying SNPs in the ILS genes AKT2 and FOXO3 being associated with serum UA levels or gout. Additionally, inhibition of the NADPH oxidase (NOX) gene rescued the reduced lifespan and concretion phenotypes in Uro knockdown flies. Thus, components of the ILS pathway and the downstream protein NOX represent potential therapeutic targets for treating UA associated pathologies, including gout and kidney stones, as well as extending human healthspan., Author summary Enzymatic purine degradation in humans ends with uric acid (UA). Multiple genetic and dietary factors raise UA levels above the norm, which is called hyperuricemia or hyperuricosuria when detected in the serum or urine, respectively. Clinical studies report a correlation between elevated UA and a plethora of chronic diseases including crystalopathies like UA kidney stones and gout, or metabolic and vascular disorders such as diabetes, obesity, and coronary artery disease. Here, we identified a regulatory role for the insulin-like signaling cascade affecting UA metabolism using a Drosophila melanogaster model. In the process we determined previously unrecognized potential drug targets to treat elevated UA levels and associated pathologies such as gout or UA kidney stones, with the potential additional benefit of extending human healthspan. Our work also establishes the fly as a model system to characterize the influence of genetic and dietary factors in gout or UA kidney stone development in a manner readily amenable for small-scale screening of drug interventions. The novelty of our findings, their broad impact, and relevance for multiple diseases opens up an important area of research to define mechanisms of UA accumulation.

The loss of NPC1 protein function is the predominant cause of Niemann-Pick type C1 disease (NP-C1), a systemic and neurodegenerative disorder characterized by late-endosomal/lysosomal accumulation of cholesterol and other lipids. Limited evidence from post-mortem human tissues, an Npc1(-/-) mouse model, and cell culture studies also suggest failure of metal homeostasis in NP-C1. To investigate these findings, we performed a comprehensive transition metal analysis of cerebrospinal fluid (CSF), plasma and tissue samples from human NP-C1 patients and an Npc1(-/-) mouse model. NPC1 deficiency in the Npc1(-/-) mouse model resulted in a perturbation of transition metal homeostasis in the plasma and key organs (brain, liver, spleen, heart, lungs, and kidneys). Analysis of human patient CSF, plasma and post-mortem brain tissues also indicated disrupted metal homeostasis. There was a disparity in the direction of metal changes between the human and the Npc1(-/-) mouse samples, which may reflect species-specific metal metabolism. Nevertheless, common to both species is brain zinc accumulation. Furthermore, treatment with the glucosylceramide synthase inhibitor miglustat, the only drug shown in a controlled clinical trial to have some efficacy for NP-C1, did not correct the alterations in CSF and plasma transition metal and ceruloplasmin (CP) metabolism in NP-C1 patients. These findings highlight the importance of NPC1 function in metal homeostasis, and indicate that metal-targeting therapy may be of value as a treatment for NP-C.

Iron overload occurs frequently in thalassemia and other disorders that require regular blood transfusions. Excess iron is toxic owing to the generation of free radicals that lead to oxidation of biomolecules and tissue damage. In order to identify compounds that reduce oxidative injury from iron, we evaluated alpha-lipoic acid (LA), a multifunctional antioxidant, in iron-overloaded primary human fibroblasts (IMR-90). Oxidant stress was measured using dichlorodihydrofluorescein diacetate that is converted to the fluorescent dichlorofluorescein (DCF) upon oxidation. Exposure to ferric ammonium citrate (FAC) increased the iron-content of IMR-90 cells and caused a rise in oxidant appearance. The addition of LA improved the cellular redox status and attenuated the iron-mediated rise in oxidants in a dose-dependent manner. The R- and RS-enantiomers of LA demonstrated similar antioxidant activity. N-tert-butyl hydroxylamine (NtBHA) treated cells also exhibited a decrease in DCF fluorescence, but at a much higher concentration compared with LA. The combination acetyl-L-carnitine (ALCAR) and LA exhibited superior antioxidant effect at all dose levels. We conclude that LA is highly effective in reversing oxidative stress arising from iron overload and that its antioxidant efficacy is further enhanced in combination with ALCAR.

Age-related macular degeneration (AMD) is the leading cause of severe visual impairment in the elderly in developed countries. AMD patients have elevated levels of iron within the retinal pigment epithelia (RPE), which may lead to oxidative damage to mitochondria, disruption of retinal metabolism, and vision impairment or loss. As a possible model for iron-induced AMD, we investigated the effects of excess iron in cultured human fetal RPE cells on oxidant levels and mitochondrial cytochrome c oxidase (complex IV) function and tested for protection by N-tert-butyl hydroxylamine (Nt-BHA), a known mitochondrial antioxidant. RPE exposure to ferric ammonium citrate resulted in a time- and dose-dependent increase in intracellular iron, which increased oxidant production and decreased glutathione (GSH) levels and mitochondrial complex IV activity. NtBHA addition to iron-overloaded RPE cells led to a reduction of intracellular iron content, oxidative stress, and partial restoration of complex IV activity and GSH content. NtBHA might be useful in AMD due to its potential to reduce oxidative stress, mitochondrial damage, and age-related iron accumulation, which may damage normal RPE function and lead to loss of vision.

Zinc is an essential trace metal that has integral roles in numerous biological processes, including enzymatic function, protein structure, and cell signaling pathways. Both excess and deficiency of zinc can lead to detrimental effects on development and metabolism, resulting in abnormalities and disease. We altered the zinc balance within Caenorhabditis elegans to examine how changes in zinc burden affect longevity and healthspan in an invertebrate animal model. We found that increasing zinc levels in vivo with excess dietary zinc supplementation decreased the mean and maximum lifespan, whereas reducing zinc levels in vivo with a zinc-selective chelator increased the mean and maximum lifespan in C. elegans. We determined that the lifespan shortening effects of excess zinc required expression of DAF-16, HSF-1 and SKN-1 proteins, whereas the lifespan lengthening effects of the reduced zinc may be partially dependent upon this set of proteins. Furthermore, reducing zinc levels led to greater nuclear localization of DAF-16 and enhanced dauer formation compared to controls, suggesting that the lifespan effects of zinc are mediated in part by the insulin/IGF-1 pathway. Additionally, zinc status correlated with several markers of healthspan in worms, including proteostasis, locomotion and thermotolerance, with reduced zinc levels always associated with improvements in function. Taken together, these data support a role for zinc in regulating both development and lifespan in C. elegans, and that suggest that regulation of zinc homeostasis in the worm may be an example of antagonistic pleiotropy.

A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.

Thalassemia (THAL) patients have a genetic anemia causing incomplete erythropoiesis and iron overload, possibly leading to cardiomyopathy and diabetes. Plasma zinc (Zn) and Copper (Cu) deficiency are also observed in some patients with THAL, however, these effects are not as well characterized. It has been suggested that oxidative stress can be caused by increased labile plasma iron (LPI), and decreased serum Zn and Cu levels, however this is unproven. LPI can initiate peroxidation releasing malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Thus, we hypothesized that that a functional Zn or Cu deficiency can increase oxidative stress and that changes in the levels of key iron trafficking proteins, such as soluble transferrin receptor (sTfR) or hemopexin (HPX) would also affect oxidative stress. 39 subjects with informed consent were enrolled (29 patients with THAL and 10 healthy controls). LPI was measured using dihydrorhodamine 123. Both MDA and 4-HNE were measured using N-methyl-2-phenylindole. Iron trafficking proteins were measured by MRM mass spectrometry. Trace mineral levels were assessed by ICP spectroscopy. Plasma levels of the iron trafficking proteins, transferrin, HPX and haptoglobin (HAP) were all decreased in THAL patients (P

Iron accumulates as a function of age in several tissues in vivo and is associated with the pathology of numerous age-related diseases. The molecular basis of this change may be due to a loss of iron homeostasis at the cellular level. Therefore, changes in iron content in primary human fibroblast cells (IMR-90) were studied in vitro as a model of cellular senescence. Total iron content increased exponentially during cellular senescence, resulting in 10-fold higher levels of iron compared with young cells. Low-dose hydrogen peroxide (H2O2) induced early senescence in IMR-90s and concomitantly accelerated iron accumulation. Furthermore, senescence-related and H2O2-stimulated iron accumulation was attenuated by N-tert-butylhydroxylamine (NtBHA), a mitochondrial antioxidant that delays senescence in vitro. However, SV40-transformed, immortalized IMR-90s showed no time-dependent changes in metal content in culture or when treated with H2O2 and/or NtBHA. These data indicate that iron accumulation occurs during normal cellular senescence in vitro. This accumulation of iron may contribute to the increased oxidative stress and cellular dysfunction seen in senescent cells.

We test whether the dysfunction with age of carnitine acetyltransferase (CAT), a key mitochondrial enzyme for fuel utilization, is due to decreased binding affinity for substrate and whether this substrate, fed to old rats, restores CAT activity. The kinetics of CAT were analyzed by using the brains of young and old rats and of old rats supplemented for 7 weeks with the CAT substrate acetyl- l -carnitine (ALCAR) and/or the mitochondrial antioxidant precursor R -α-lipoic acid (LA). Old rats, compared with young rats, showed a decrease in CAT activity and in CAT-binding affinity for both substrates, ALCAR and CoA. Feeding ALCAR or ALCAR plus LA to old rats significantly restored CAT-binding affinity for ALCAR and CoA, and CAT activity. To explore the underlying mechanism, lipid peroxidation and total iron and copper levels were assayed; all increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited lipid peroxidation but did not decrease iron and copper levels. Ex vivo oxidation of young-rat brain with Fe(II) caused loss of CAT activity and binding affinity. In vitro oxidation of purified CAT with Fe(II) inactivated the enzyme but did not alter binding affinity. However, in vitro treatment of CAT with the lipid peroxidation products malondialdehyde or 4-hydroxy-nonenal caused a decrease in CAT-binding affinity and activity, thus mimicking age-related change. Preincubation of CAT with ALCAR or CoA prevented malondialdehyde-induced dysfunction. Thus, feeding old rats high levels of key mitochondrial metabolites can ameliorate oxidative damage, enzyme activity, substrate-binding affinity, and mitochondrial dysfunction.

Parkinson's disease (PD) is a debilitating motor and cognitive neurodegenerative disorder for which there is no cure. While aging is the major risk factor for developing PD, clear environmental risks have also been identified. Environmental exposure to the manganese (Mn) metal is a prominent risk factor for developing PD and occupational exposure to high levels of Mn can cause a syndrome known as manganism, which has symptoms that closely resemble PD. In this study, we developed a model of manganism in the environmentally tractable nematode, Caenorhabditis elegans. We find that, in addition to previously described modes of Mn toxicity, which primarily include mitochondrial dysfunction and oxidative stress, Mn exposure also significantly antagonizes protein homeostasis, another key pathological feature associated with PD and many age-related neurodegenerative diseases. Mn treatment activates the ER unfolded protein response, severely exacerbates toxicity in a disease model of protein misfolding, and alters aggregate solubility. Further, aged animals, which have previously been shown to exhibit decreased protein homeostasis, are particularly susceptible to Mn toxicity when compared to young animals, indicating that the aging process sensitizes animals to metal toxicity. Mn exposure also significantly alters iron (Fe) and calcium (Ca) homeostasis, which is important for mitochondrial and ER health and which may further compound toxicity. These findings indicate that modeling manganism in C. elegans can provide a useful platform for identifying therapeutic interventions for ER stress, proteotoxicity, and age-dependent susceptibilities, key pathological features of PD and other related neurodegenerative diseases.

Zinc deficiency continues to be a major concern of international nutrition, yet there are still few sensitive and reliable biomarkers for identifying moderate zinc deficiency or monitoring changes ...

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.

Aluminium (Al) is highly abundant in the environment and can elicit a variety of toxic responses in biological systems. Here we characterize the effects of Al on Caenorhabditis elegans by identifying phenotypic abnormalities and disruption in whole-body metal homeostasis (metallostasis) following Al exposure in food. Widespread changes to the elemental content of adult nematodes were observed when chronically exposed to Al from the first larval stage (L1). Specifically, we saw increased barium, chromium, copper and iron content, and a reduction in calcium levels. Lifespan was decreased in worms exposed to low levels of Al, but unexpectedly increased when the Al concentration reached higher levels (4.8 mM). This bi-phasic phenotype was only observed when Al exposure occurred during development, as lifespan was unaffected by Al exposure during adulthood. Lower levels of Al slowed C. elegans developmental progression, and reduced hermaphrodite self-fertility and adult body size. Significant developmental delay was observed even when Al exposure was restricted to embryogenesis. Similar changes in Al have been noted in association with Al toxicity in humans and other mammals, suggesting that C. elegans may be of use as a model for understanding the mechanisms of Al toxicity in mammalian systems.

Diet-induced Obesity Decreases Liver Iron Stores in Mice Fed Iron Deficient, Adequate, or Excessive Diets

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.

Heme, a major functional form of iron in the cell, is synthesized in the mitochondria by ferrochelatase inserting ferrous iron into protoporphyrin IX. Heme deficiency was induced with N -methylprotoporphyrin IX, a selective inhibitor of ferrochelatase, in two human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astrocytoma), as well as in rat primary hippocampal neurons. Heme deficiency in brain cells decreases mitochondrial complex IV, activates nitric oxide synthase, alters amyloid precursor protein, and corrupts iron and zinc homeostasis. The metabolic consequences resulting from heme deficiency seem similar to dysfunctional neurons in patients with Alzheimer's disease. Heme-deficient SHSY5Y or U373 cells die when induced to differentiate or to proliferate, respectively. The role of heme in these observations could result from its interaction with heme regulatory motifs in specific proteins or secondary to the compromised mitochondria. Common causes of heme deficiency include aging, deficiency of iron and vitamin B6, and exposure to toxic metals such as aluminum. Iron and B6 deficiencies are especially important because they are widespread, but they are also preventable with supplementation. Thus, heme deficiency or dysregulation may be an important and preventable component of the neurodegenerative process.

SPECIFIC AIMSThe conclusion that mitochondrially derived oxidants play a role in hypoxic signal transduction has remained controversial in part because of doubts concerning methods of free radical detection and because of theoretical objections to the notion that mitochondria are capable of increasing oxidant generation in a low oxygen environment. To help resolve this controversy, the present study tested the hypothesis that hypoxia causes oxidative modifications in mtDNA or in the nuclear vascular endothelial cell growth factor (VEGF) gene, the latter of which should serve as a negative control based on the relatively insensitive nuclear genome to oxidant stress in comparison to that in the mitochondria.PRINCIPAL FINDINGS1. Hypoxia causes an oxidant stress in pulmonary artery endothelial cells as detected by fluorescence of dichlorfluorescein (DCF)We used rat cultured main pulmonary artery cells (PAECs) as the model system for these studies because they are important in the integrated pulmonary vascular...

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