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Regulation of erythropoiesis is achieved by integration of distinct signals. Among these, macrophages are emerging as erythropoietin-complementary regulators of erythroid development, particularly under stress conditions. We investigated the contribution of macrophages for physiological and pathological conditions of enhanced erythropoiesis. We utilized mouse models of induced anemia, Polycythemia vera and β-thalassemia in which macrophages were chemically depleted. Our data indicate that macrophages contribute decisively for recovery from induced anemia as well as the pathological progression of Polycythemia vera and β-thalassemia by modulating erythroid proliferation and differentiation. We validated these observations in primary human cultures, showing a critical direct impact of macrophages on proliferation and enucleation of erythroblasts from healthy individuals and Polycythemia vera or β-thalassemic patients. In summary, we identify a new mechanism that we named “Stress Erythropoiesis Macrophage-supporting Activity” (SEMA) that contributes to the pathophysiology of these disorders and will have critical scientific and therapeutic implications in the near future.

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 β-thalassemia require iron chelation therapy to protect against progressive iron overload and non-transferrin-bound iron. Some patients fail to respond adequately to deferoxamine and deferasirox monotherapy while others have side effects which limit their use of these drugs. Since combining deferiprone and deferoxamine has an additive effect, placing all patients into net negative iron balance, we investigated the possibility that combining deferasirox and deferoxamine would lead to similar results. We conducted 34-day metabolic iron balance studies in six patients in whom the relative effectiveness of deferasirox (30 mg/kg/day) and deferoxamine (40 mg/kg/day) was compared, alone and in combination. Patients consumed fixed low-iron diets; daily urinary and stool iron excretion were determined by atomic absorption. Red blood cell transfusions were given prior to each drug treatment to minimize the effects of ineffective erythropoiesis. Serial safety measures, hematologic parameters, serum chemistries, ferritin levels and urinalyses were determined. All patients were in negative iron balance when treated with deferoxamine alone while four of six patients remained in positive balance when deferasirox monotherapy was evaluated. Daily use of both drugs had a synergistic effect in two patients and an additive effect in three others. Five of six patients would be in negative iron balance if they used the combination of drugs just 3 days a week. No significant or drug-related changes were observed in the blood work-ups or urinalyses performed. We conclude that supplementing the daily use of deferasirox with 2 - 3 days of deferoxamine therapy would place all patients into net negative iron balance thereby providing a convenient way to tailor chelation therapy to the individual needs of each patient.

In hereditary hemochromatosis, mutations in HFE lead to iron overload through abnormally low levels of hepcidin. In addition, HFE potentially modulates cellular iron uptake by interacting with transferrin receptor, a crucial protein during erythropoiesis. However, the role of HFE in this process was never explored. We hypothesize that HFE modulates erythropoiesis by affecting dietary iron absorption and erythroid iron intake. To investigate this, we used Hfe-KO mice in conditions of altered dietary iron and erythropoiesis. We show that Hfe-KO mice can overcome phlebotomy-induced anemia more rapidly than wild-type mice (even when iron loaded). Second, we evaluated mice combining the hemochromatosis and β-thalassemia phenotypes. Our results suggest that lack of Hfe is advantageous in conditions of increased erythropoietic activity because of augmented iron mobilization driven by deficient hepcidin response. Lastly, we demonstrate that Hfe is expressed in erythroid cells and impairs iron uptake, whereas its absence exclusively from the hematopoietic compartment is sufficient to accelerate recovery from phlebotomy. In summary, we demonstrate that Hfe influences erythropoiesis by 2 distinct mechanisms: limiting hepcidin expression under conditions of simultaneous iron overload and stress erythropoiesis, and impairing transferrin-bound iron uptake by erythroid cells. Moreover, our results provide novel suggestions to improve the treatment of hemochromatosis.

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene and characterized by chronic anemia caused by ineffective erythropoiesis, and accompanied by a variety of serious secondary complications such as extramedullary hematopoiesis, splenomegaly, and iron overload. In the past few years, numerous studies have shown that such secondary disease conditions have a genetic basis caused by the abnormal expression of genes with a role in controlling erythropoiesis and iron metabolism. In this article, the most recent discoveries related to the mechanism(s) responsible for anemia/ineffective erythropoiesis and iron overload are discussed in detail. Particular attention is paid to the pathway(s) controlling the expression of hepcidin, which is the main regulator of iron metabolism, and the Epo/EpoR/Jak2/Stat5 signaling pathway, which regulates erythropoiesis. Better understanding of how these pathways function and are altered in β-thalassemia has revealed several possibilities for development of new therapeutic approaches to treat of the complications of this disease.

Patients with thalassemia major accumulate body iron over time as a consequence of continuous red blood cell transfusions which cause hepatic, endocrine, and cardiac complications. Despite the availability of three iron chelators, some patients fail to respond adequately to monotherapy with any of them. Combination therapy, consisting in the use of two chelators on the same day, has been introduced to increase the efficacy and to induce negative iron balance in patients with severe iron overload. Extensive long-term experience has shown that combined chelation with deferiprone and deferoxamine (DFO) rapidly reduces liver iron, serum ferritin, and myocardial siderosis, improves cardiac function, reverses and prevents endocrine complications, reduces cardiac mortality, and improves survival. Side effects, though significant, are manageable if properly monitored. Preliminary promising results have been obtained using combined chelation with deferasirox and DFO. As more drug combination regimes are evaluated, it should be possible to better tailor iron chelation to the needs of the patients, minimizing toxicity and maximizing efficacy throughout life.

Hepcidin (HAMP) negatively regulates iron absorption, degrading the iron exporter ferroportin at the level of enterocytes and macrophages. We showed that mice with β-thalassemia intermedia (th3/+) have increased anemia and iron overload. However, their hepcidin expression is relatively low compared to their iron burden. We also showed that the iron metabolism gene Hfe is down-regulated in concert with hepcidin in th3/+ mice. These observations suggest that low hepcidin levels are responsible for abnormal iron absorption in thalassemic mice and that down-regulation of Hfe might be involved in the pathway that controls hepcidin synthesis in β-thalassemia. Therefore, these studies suggest that increasing hepcidin and/or Hfe expression could be a strategy to reduces iron overload in these animals. The goal of this paper is to review recent findings that correlate hepcidin, Hfe, and iron metabolism in β-thalassemia and to discuss potential novel therapeutic approaches based on these recent discoveries.

Summary This study aimed to determine differences in the rates of growth, endocrine- and calcium-related abnormalities in the various thalassemia syndromes in North America treated with current therapies. Medical history, physical examinations and blood and urine collections were obtained from patients with all thalassemia syndromes age 6 years and older in the Thalassemia Clinical Research Network. 361 subjects, 49% male, mean age 23·2 years (range 6·1–75 years) were studied. Approximately 25% of children and adults, regardless of the thalassemia syndrome, had short stature. Overall growth in children was mildly affected. Final height was close to midparental height (z = −0·73 ± 1·24). Patients with beta thalassemia major (TM) had higher rates of hypogonadism, multiple endocrinopathies, worse hyperglycaemia, subclinical hypoparathyroidism and hypercalciuria. Hypogonadism remained the most frequent endocrinopathy and was frequently under-treated. 12·8% of the subjects had 25 vitamin D concentrations less than 27 nmol/l and 82% less than 75 nmol/l, regardless of the thalassemia syndrome. Adolescents had lower 25 vitamin D levels than children and adults. Compared to patients with other thalassemia syndromes, those with beta TM suffered from higher rates of multiple endocrinopathies, abnormal calcium metabolism and hypercalciuria. Vitamin D abnormalities were high among adolescents.

Adults with beta thalassemia major frequently have low BMD, fractures, and bone pain. The purpose of this study was to determine the prevalence of low BMD, fractures, and bone pain in all thalassemia syndromes in childhood, adolescence, and adulthood, associations of BMD with fractures and bone pain, and etiology of bone disease in thalassemia. Patients of all thalassemia syndromes in the Thalassemia Clinical Research Network, > or =6 yr of age, with no preexisting medical condition affecting bone mass or requiring steroids, participated. We measured spine and femur BMD and whole body BMC by DXA and assessed vertebral abnormalities by morphometric X-ray absorptiometry (MXA). Medical history by interview and review of medical records, physical examinations, and blood and urine collections were performed. Three hundred sixty-one subjects, 49% male, with a mean age of 23.2 yr (range, 6.1-75 yr), were studied. Spine and femur BMD Z-scores < -2 occurred in 46% and 25% of participants, respectively. Greater age, lower weight, hypogonadism, and increased bone turnover were strong independent predictors of low bone mass regardless of thalassemia syndrome. Peak bone mass was suboptimal. Thirty-six percent of patients had a history of fractures, and 34% reported bone pain. BMD was negatively associated with fractures but not with bone pain. Nine percent of participants had uniformly decreased height of several vertebrae by MXA, which was associated with the use of iron chelator deferoxamine before 6 yr of age. In patients with thalassemia, low BMD and fractures occur frequently and independently of the particular syndrome. Peak bone mass is suboptimal. Low BMD is associated with hypogonadism, increased bone turnover, and an increased risk for fractures.

In β-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by β-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle–promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-XL. The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

The most widely used drug for iron chelation is deferoxamine (DFO) mesylate. While effective in promoting iron excretion, it requires prolonged daily infusions, often resulting in poor compliance. A clinical trial was conducted using starch-conjugated DFO (S-DFO; 40SD02), a high-molecular-weight iron chelator possessing prolonged vascular retention. Single doses of S-DFO were infused intravenously into groups of four transfusion-dependent patients with beta-thalassaemia at doses of 150, 300, 600 and 900 mg/kg. Urinary iron excretion and various pharmacologic parameters were evaluated for 1 week and safety for 3 weeks. No drug-related effects were observed on clinical chemistries, haematological and coagulation parameters, urinalyses, vital signs or electrocardiograms. Drug-related adverse events were limited to four urticarial reactions, none requiring termination of the infusion. The drug stimulated clinically significant urinary iron excretion, with the highest dose (900 mg/kg) inducing excretion of 1.31 mg of iron/kg (range 0.79-1.90 mg/kg) over 1 week, with residual iron-binding capacity present in the plasma for over 6 d. In summary, treatment with S-DFO, administered weekly, has the potential to achieve iron balance in the poorly compliant patient.

Progressive iron overload is the most salient and ultimately fatal complication of β-thalassemia. However, little is known about the relationship among ineffective erythropoiesis (IE), the role of iron-regulatory genes, and tissue iron distribution in β-thalassemia. We analyzed tissue iron content and iron-regulatory gene expression in the liver, duodenum, spleen, bone marrow, kidney, and heart of mice up to 1 year old that exhibit levels of iron overload and anemia consistent with both β-thalassemia intermedia (th3/+) and major (th3/th3). Here we show, for the first time, that tissue and cellular iron distribution are abnormal and different in th3/+ and th3/th3 mice, and that transfusion therapy can rescue mice affected by β-thalassemia major and modify both the absorption and distribution of iron. Our study reveals that the degree of IE dictates tissue iron distribution and that IE and iron content regulate hepcidin (Hamp1) and other iron-regulatory genes such as Hfe and Cebpa. In young th3/+ and th3/th3 mice, low Hamp1 levels are responsible for increased iron absorption. However, in 1-year-old th3/+ animals, Hamp1 levels rise and it is rather the increase of ferroportin (Fpn1) that sustains iron accumulation, thus revealing a fundamental role of this iron transporter in the iron overload of β-thalassemia.

Transfusional iron overload is a potentially fatal complication of the treatment of thalassaemia. We aimed to investigate short-term efficacy, pharmacokinetic/pharma- codynamic (PK/PD) relations, and safety of ICL670, a novel, tridentate, orally active iron chelator.We enrolled 24 patients and divided them into three cohorts consisting of a minimum of seven individuals. Patients were admitted to a metabolic unit and consumed a diet with a defined content of iron. Two patients in each cohort were randomly allocated placebo. Five or more patients received one daily dose of ICL670 at 10, 20, or 40 mg x kg(-1) x day(-1), from day 1 to 12. Net iron excretion (NIE) was measured between days 1 and 12. Primary objectives included assessment of safety and tolerability (measured by adverse events and clinical laboratory monitoring), pharmacokinetics (measured as drug and drug-iron complex), and cumulative net iron excretion (measured by faecal and urine output minus food input). Analysis was for efficacy.ICL670 was absorbed promptly and was detectable in the blood for 24 h. Exposure (area under the curve of plasma concentration) to ICL670 at pharmacokinetic steady state was proportional to dose. All three doses resulted in positive NIE. The NIE achieved at 20mg x kg(-1) day(-1) would prevent net iron accumulation in most patients transfused with 12-15 mL packed red-blood-cells kg(-1) month(-1), equivalent to 0.3-0.5 mg iron kg(-1) x day(-1). A linear relation (PK/PD) was recorded between exposure to ICL670 and total iron excretion, by contrast with placebo (r2=0.54, p0.0001). Skin rashes were noted in four patients treated at 20 and 40 mg x kg(-1) x day(-1), and one patient also developed grade 2 transaminitis.ICL670 given once daily at 20 mg/kg seems to be an effective orally active iron chelator and is reasonably well tolerated. Long-term studies are now necessary to establish the practical contribution of this drug.

The 17-electron complex (C5Ph5)Cr(CO)3 reacts with halogens (C6H5I·Cl2, Br2, and I2) in C6H6 to yield the dimeric oxidation products [(C5Ph5)Cr(μ-X)X]2 as thermally stable solids. Reactions with other chlorinating agents similarly yield [(C5Ph5)CrCl2]2. An X-ray crystal structure of [(C5Ph5)Cr(μ-Cl)Cl]2 was obtained. The magnetic properties of the Cl2-bridged dimer have been determined and modeled using the usual isotropic Hamiltonian H = −2JŜ1·Ŝ2, which yields J/k = −30 K. Low-temperature (77 K) Raman spectra of solid [(C5Ph5)CrX2]2 (X = Cl, I) allow assignments to be made for the metal-ring and metal halogen stretching modes in the low-frequency region (

The HIV-1 protein Rev, critical for translation of incompletely spliced retroviral mRNAs encoding capsid elements, requires a host cell protein termed “eukaryotic initiation factor 5A” (eIF-5A). This is the only protein containing hypusine, a lysine-derived hydroxylated residue that determines its proposed bioactivity, the translation of a subset of cellular mRNAs controlling G 1 -to-S transit of the cell cycle. We postulated that inhibiting the hypusine-forming deoxyhypusyl hydroxylase (DOHH) should, by depleting eukaryotic initiation factor 5A, compromise Rev function and thus reduce HIV-1 multiplication. We now report that the α-hydroxypyridones, specifically mimosine, a natural product, and deferiprone, an experimental drug, inhibited deoxyhypusyl hydroxylase in T-lymphocytic and promonocytic cell lines and, in a concentration-dependent manner, suppressed replication of HIV-1. However, the α-hydroxypyridones did not affect the formation of unspliced or multiply spliced HIV-1 transcripts. Rather, these agents caused Rev-dependent incompletely spliced HIV-1 mRNA such as gag, but not cellular “housekeeping” mRNAs, to disappear from polysomes. Consequently, α-hydroxypyridone-mediated depletion of eIF-5A decreased biosynthesis of structural HIV-1 protein encoded by gag , measured as p24, whereas the induced formation of cellular protein like tumor necrosis factor alpha remained unaffected. By interfering with the translation of incompletely spliced retroviral mRNAs, these compounds restrict HIV-1 to the early, nongenerative phase of its reproductive cycle. In the inducibly HIV-1 expressing T-cell line ACH-2, the deoxyhypusyl hydroxylase inhibitors triggered extensive apoptosis, particularly of cells that actively produce HIV-1. Selective suppression of retroviral protein biosynthesis and preferential apoptosis of retrovirally infected cells by α-hydroxypyridones point to a novel mode of antiretroviral action.

Serum ferritin protein is an acute phase reactant. We hypothesized that serum ferritin protein generated in response to an inflammatory process would have much less iron (Fe) in it than would “normal” ferritin protein, and therefore measuring serum ferritin iron would assess human body iron status unconfounded by inflammation. Basic Methods. We measured serum ferritin iron in 140 clinical samples obtained from the serum banks of Bronx VA Medical Center Hematology and Nutrition Laboratory (Bronx, NY), the CDC Nutritional Biochemistry serum sample bank (Atlanta, GA), and the sample bank from patients with thalassemia and iron overload treated at New York Hospital (New York, NY). Each was analyzed for three conventional criteria of iron status: serum iron, percentage of transferrin saturation and ferritin protein. In addition, tests for inflammation were also performed: C-reactive protein, WBC and transaminases. Seventy-seven patients' sera from 140 screened met each of three consistent criteria for stages of iron status. Serum ferritin was immobilized by immunoprecipitation with rabbit antihuman polyclonal antibody bound to agarose and separated from other iron-containing proteins, digested with 0.2 ml of 3N nitric acid and analyzed for iron content by atomic absorption spectroscopy. Results. Serum ferritin iron ranged in normal controls from 10 ng to 35 ng Fe/ml. The patients with iron deficiency (4/4) and those in negative iron balance (5/6) had values ≤10 ng. Positive iron balance (8/9) and iron overload (22/22) values were >35 ng/ml, in contrast to 11/19 with inflammation. Seventeen of twenty-two with overload had values >100 ng/ml while only 1/19 with inflammation had such a value. Ferritin iron in ferritin protein was >15% by weight in 14/22 with iron overload but in 0/19 with inflammation. Implications of the Work. Serum ferritin iron is a simple, direct measure of iron stores that we propose, in conjunction with measuring serum ferritin protein, as a minimally invasive screening procedure for accurately assessing the whole range of human body iron status, unconfounded by inflammation.

The trypanosome alternative oxidase (TAO) is an attractive target for chemotherapy for the diseases caused by African trypanosomes because there is no equivalent enzyme in mammalian hosts. Many inhibitors of this enzyme have been described, but there have been no data on the mechanism of inhibition. In the present study, reduced 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (decyl-CoQ-H2) was used as a substitute for the natural substrate CoQ9-H2 to allow direct measurements of the TAO in crude mitochondrial preparations from Trypanosoma brucei brucei. A Km value of 3.8 microM was obtained for this substrate. The following five compounds that have alkyl side chains from 1 to 4 carbons and belong to three classes of inhibitors showed a competitive inhibition pattern with respect to decyl-CoQ-H: p-methoxybenzhydroxamic acid, p-ethoxybenzhydroxamic acid,p-n-butyloxybenzhydroxamic acid, methyl 3,4-dihydroxybenzoate and N-n-butyl-3,4-dihydroxybenzamide. The following four compounds belonging to the same chemical classes but having alkyl side chains from 10 to 12 carbons showed uncompetitive inhibition patterns: p-n-dodecyloxybenzhydroxamic acid, n-decyl 3,4-dihydroxybenzoate, n-dodecyl 3,4-dihydroxybenzoate, and N-n-decyl-3,4-dihydroxybenzamide. Clearly, the first group of inhibitors compete with CoQ-H2 for the active site of the TAO. We propose that the uncompetitive patterns produced by the second group of inhibitors are due to the greater lipophilicity of these compounds and the resulting change in the interaction of the inhibitors and the membrane containing the TAO, thus affecting the local concentration of the inhibitors at the active site.

To the editor: β-thalassemia is a genetic disorder of hemoglobin production characterized by ineffective erythropoiesis and anemia.[1][1] Iron overload, a major source of morbidity, results from inappropriately low expression of the gene encoding hepcidin ( Hamp1 ).[1][1] Hamp1 controls plasma

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients. We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S). Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

The effectiveness of deferoxamine (DFO), deferiprone (DFP), or deferasirox (DFX) in thalassemia major was assessed. Outcomes were reported as means±SD, mean differences with 95% CI, or standardized mean differences. Statistical heterogeneity was tested using χ2 (Q) and I2. Sources of bias and Grading of Recommendations Assessment, Development and Evaluation system (GRADE) were considered. Overall, 1520 patients were included. Only 7.4% of trials were free of bias. Overall measurements suggest low trial quality (GRADE). The meta-analysis suggests lower final liver iron concentrations during associated versus monotherapy treatment (p

Hepcidin (HAMP) negatively regulates iron absorption, degrading the iron exporter ferroportin at the level of enterocytes and macrophages. We showed that mice with beta-thalassemia intermedia (th3/+) have increased anemia and iron overload. However, their hepcidin expression is relatively low compared to their iron burden. We also showed that the iron metabolism gene Hfe is down-regulated in concert with hepcidin in th3/+ mice. These observations suggest that low hepcidin levels are responsible for abnormal iron absorption in thalassemic mice and that down-regulation of Hfe might be involved in the pathway that controls hepcidin synthesis in beta-thalassemia. Therefore, these studies suggest that increasing hepcidin and/or Hfe expression could be a strategy to reduces iron overload in these animals. The goal of this paper is to review recent findings that correlate hepcidin, Hfe, and iron metabolism in beta-thalassemia and to discuss potential novel therapeutic approaches based on these recent discoveries.

Osteoporosis is a frequent problem in disorders characterized by iron overload, such as the thalassemias and hereditary hemochromatosis. The exact role of iron in the development of osteoporosis in these disorders is not established. To define the effect of iron excess in bone, we generated an iron-overloaded mouse by injecting iron dextran at 2 doses into C57/BL6 mice for 2 months. Compared with the placebo group, iron-overloaded mice exhibited dose-dependent increased tissue iron content, changes in bone composition, and trabecular and cortical thinning of bone accompanied by increased bone resorption. Iron-overloaded mice had increased reactive oxygen species and elevated serum tumor necrosis factor-α and interleukin-6 concentrations that correlated with severity of iron overload. Treatment of iron-overloaded mice with the antioxidant N-acetyl-L-cysteine prevented the development of trabecular but not cortical bone abnormalities. This is the first study to demonstrate that iron overload in mice results in increased bone resorption and oxidative stress, leading to changes in bone microarchitecture and material properties and thus bone loss.

Myocardial iron overload is the leading cause of death in patients with β-thalassemia major. An intensification monotherapy with deferoxamine (DFO) as well as a combination therapy with DFO and deferiprone (DFP) reduces myocardial iron and improves cardiac function. However, the prognosis for thalassemia major patients with established cardiac disease switched from DFO monotherapy to combined DFP/DFO chelation is unknown. Twenty-eight thalassemia major patients with cardiac disease were enrolled in a prospective study lasting 42 ± 6 months. Fifteen (9 high-ferritin and 6 low-ferritin) were placed on DFP/DFO (DFP, 75 mg/kg t.i.d.; DFO, 40–50 mg/kg over 8–12 h at night 5–7 days/week), while 13 (5 high- and 8 low-ferritin) received DFO alone. No cardiac events were observed among high-ferritin patients on combination therapy, whereas 4 cardiac events ( p = 0.0049), including three deaths, occurred in high-ferritin patients on DFO monotherapy. These findings demonstrate that in thalassemia major patients with well-established cardiac disease combined iron-chelation therapy with DFP/DFO is superior to DFO monotherapy.

Osteoporosis and fractures occur frequently in patients with beta-thalassemias, a group of congenital hemolytic anemias characterized by decreased synthesis of the beta chain of hemoglobin. In this study, we determined the bone abnormalities of the th3 thalassemia mouse, generated by deletion of the mouse beta-chain genes. The heterozygous th3/+ mouse has moderate anemia and serves as a model of beta-thalassemia intermedia, which represents the mild thalassemia phenotype. The th3/th3 mouse has lethal anemia and is a model of beta-thalassemia major, which is characterized by life-threatening anemia requiring regular transfusions to sustain life. Compared to controls, (1) microCT of trabecular bone showed decreased bone volume fraction, number of trabeculae, and trabecular thickness in both th3/+ and th3/th3 (P0.05); (2) cortical bone analysis showed thinner cortices and increased marrow area in th3/+ (P0.05); (3) microCT abnormalities in th3/+ mice were present by 2 months and did not worsen with age; (4) histomorphometry was significant for decreased bone formation and resorption in both th3/+ and th3/th3, and expression of cathepsin K and osteocalcin from bone of both th3/+ and th3/th3 animals was reduced (P0.05); (5) biomechanics showed reduced maximum load, maximum moment, and structural stiffness in both th3/+ and th3/th3 (P0.01). In conclusion, the th3 mouse model of thalassemia manifests bone changes reminiscent of those in humans and can be used for further bone studies in thalassemia. Bone changes are associated with decreased bone turnover and develop early during the period of bone accrual.

Intermediate and short stumpy bloodstream forms of Trypanosoma brucei brucei are transitional stages in the differentiation of mammal-infective long slender bloodstream forms into the procyclic forms found in the midgut of the tsetse vector. Although the mitochondria of the proliferative long slender forms do not accumulate rhodamine 123, the mitochondria of the transitional forms attain this ability thus revealing the development of an electromotive force (EMF) across the inner mitochondrial membrane. The EMF is inhibited by 2,4-dinitrophenol, rotenone and salicylhydroxamic acid but not by antimycin A or cyanide. Consequently, NADH dehydrogenase, site I of oxidative phosphorylation, is the source of the EMF and the plant-like trypanosome alternative oxidase (TAO) supports the electron flow serving as the terminal oxidase of the chain. Although the TAO is present in the long slender forms as well, it serves only as the terminal oxidase for electrons from glycerol-3-phosphate dehydrogenase. The data presented here, combined with older data, lead to the conclusion that the mitochondria of transitional intermediate and short stumpy forms likely produce ATP. This putative production is either by F1F0 ATPase driven by the complex I proton pump or by mitochondrial substrate level phosphorylation, or most likely by both. These conclusions contrast with the previously held dogma that all bloodstream form mitochondria are incapable of ATP production.

To develop new treatments for beta-thalassemia, it is essential to identify the genes involved in the relevant pathophysiological processes. Iron metabolism in thalassemia mice being investigated, focusing on the expression of a gene called hepcidin (Hamp), which is expressed in the liver and whose product (Hamp) is secreted into the bloodstream. In mice, iron overload leads to overexpression of Hamp, while Hamp-knockout mice suffer from hemochromatosis. The aim of this study is to investigate Hamp in the mouse model of beta-thalassemia and to address the potential gene transfer of Hamp to prevent abnormal iron absorption.

Iron chelation therapy with desferrioxamine (DFO) has dramatically improved the outlook in beta-thalassemia. Parenteral DFO reduces tissue iron stores, prevents iron-induced organ damage, and reduces morbidity and mortality, with little serious toxicity. However, the burden of prolonged subcutaneous portable pump infusions, high cost, and patient noncompliance have prompted the development of new methods of administration and new formulations of DFO as well as oral iron chelators. Deferiprone (L1), the only oral iron chelator studied in large long-term clinical trials, is less effective and more toxic than DFO and may not adequately control iron overload; however, compliance and quality of life are improved. Combinations of two iron chelators (such as parenteral DFO plus oral L1, or 2,3-DHB; or oral L1 plus HBED) have been shown to produce additive and synergistic effects, explained by the shuttle hypothesis. Iron bound to a "shuttle"--an oral agent that mobilizes tissue iron--is exchanged in the bloodstream with a "sink"--such as parenteral DFO--and excreted via the kidneys, while the shuttle is reutilized. Combination therapy may produce enhanced iron excretion, target specific iron compartments, minimize side effects, increase treatment options, improve compliance, and facilitate individualization of therapy. Better understanding of the kinetics of iron metabolism, iron overload, and chelation should improve therapeutic strategies.

Increased susceptibility to infectious disease is observed in persons with transfusion-dependent thalassemia and iron overload who experience increased exposure to pathogens and chronic immune stimulation. An abnormal low CD8 + T (LT8) immune phenotype defines a subgroup of patients. The CD8 + T cell immunophenotype is stable despite continued blood transfusion and is independent of age. CD8 + T cells, but not CD4 + T cells, were modulated during intravenous chelation with deferoxamine. Return to characteristic pretreatment levels of CD8 was observed in both the low and the normal groups, suggesting the possibility of a set point. Proliferative response to mitogens and antigens was increased by chelation. Because CD8 + T cells are important in immune response to infectious disease, these studies suggest that intrinsic CD8 + T cell subset differences may be a critical factor in determining susceptibility to infection independent of transfusional iron overload or alloantigen exposure.

In a rat model of Pneumocystis carinii pneumonia, a 3-week infusion of deferoxamine producing concentrations in plasma of > or = 1.5 micrograms m-1 eliminated the trophozoite life cycle stage. Since this concentration is well below that routinely achieved in patients treated for iron overload, deferoxamine has promise as a therapy for AIDS-associated P.carinii pneumonia.

The iron-chelating drug deferoxamine (DFO) has been shown to be active in animal models of Pneumocystis carinii pneumonia (PCP), with effective daily intraperitoneal bolus dosages being 400 and 1,000 mg of DFO mesylate kg of body weight-1 in mouse and rat models, respectively. Continuous infusion produced a moderately improved response in a rat model. The data reported here demonstrate that the response achieved by continuous infusion of 195 and 335 mg of DFO mesylate kg-1 day-1 in the rat model is associated with mean concentrations in plasma of 1.3 and 2.5 micrograms of DFO ml-1 and mean concentrations in lung tissue of 4.9 and 6.0 micrograms of DFO g of lung tissue-1, respectively. Since current clinical use of DFO mesylate for the treatment of iron overload produces higher concentrations in the plasma of patients, DFO may prove to be a useful anti-PCP treatment. The 2.4- to 3.8-fold higher DFO concentration observed in lung tissue compared with that observed in plasma may be important in the response of PCP to DFO.

The iron-chelating drug deferoxamine mesylate (DFO) is active against Pneumocystis carinii in vitro and in rat and mouse models of P. carinii pneumonia. Because DFO has a short half-life, daily divided or continuous dosage was expected to improve the dose response, as is the case with DFO treatment of malaria. Therefore, results of single daily intraperitoneal injections were compared with results of an evenly divided four-times-daily dosage and the efficacy of delivery with implanted infusion pumps. The highest bolus dosage (1,000 mg kg-1 of body weight day-1) was as effective as the standard combination of trimethoprim with sulfamethoxazole. Unexpectedly, very little improvement was observed with the divided or continuous dosage, and several mechanisms that could account for this are discussed.

Background Understanding patients’ views about medication is crucial to maximize adherence. Thalassemia is a congenital blood disorder requiring chronic blood transfusions and daily iron chelation therapy. Methods The Beliefs in Medicine Questionnaire (BMQ) was used to assess beliefs in chelation in thalassemia patients from North America and London in the Thalassemia Longitudinal Cohort (TLC) of the Thalassemia Clinical Research Network (TCRN). Chelation adherence was based on patient report of doses administered out of those prescribed in the last four weeks. Results Of 371 patients (ages 5-58y, mean 24y), 93% were transfused and 92% receiving chelation (26% deferoxamine (DFO; a slow subcutaneous infusion via portable pump), 63% oral, 11% combination). Patients expressed high “necessity” for transfusion (96%), DFO chelation (92%) and oral chelation (89%), with lower “concern” about treatment (48%, 39%, 19% respectively). Concern about oral chelation was significantly lower than that of DFO (p Conclusions Despite their requirement for multimodal therapy, thalassemia patients have positive views about medicine, more so than in other disease populations. Patients may benefit from education about the tolerability of chelation and strategies to effectively cope with side effects, both of which might be beneficial in lowering body iron burden. Clinicaltrials.gov identifier NCT00661804

The experience of the past dozen years clearly indicates that iron plays a significant role in the pathogenesis of a wide variety of disease states apart from hemochromatosis. 1 In cases of rheumatoid arthritis and reperfusion injury, iron catalyzes the formation of free radicals which destroy membranes and thereby lead to tissue damage. Sequestration of this iron would obviate much of this damage. In lymphopro-liferative disorders and parasitic diseases such as malaria, iron serves as an essential nutrient for dividing cells. Withholding iron would inhibit cell division and hence slow the progression of such diseases. While unlikely to affect cures, chelation of iron would undoubtedly be of benefit as adjunctive therapy in these and related disorders.

Abstract 901 β-Thalassemia is a genetic disorder characterized by reduced or absent β-globin synthesis. Complications include anemia, ineffective erythropoiesis (IE), splenomegaly and iron overload. Homeostatic iron levels are maintained by the hepatic hormone hepcidin (HAMP), which degrades the iron exporter ferroportin. Iron-responsive transcriptional control of HAMP is normally mediated by the bone morphogenetic protein (BMP)/SMAD pathway. Despite the high organ iron load associated with β-thalassemia, low Hamp levels have been reported in patients with this disorder. HAMP levels are also low relative to organ iron load in mouse models of β-thalassemia. Although iron overload is a central component of the pathogenesis of this disease, the factors contributing to inadequate HAMP expression have not been fully described. We previously showed that moderate transgenic overexpression of HAMP increased hemoglobin levels, improved erythropoiesis, and decreased both splenomegaly and iron overload in a mouse model of β-thalassemia intermedia (th3/+). In order to define the pathways controlling HAMP transcription in β-thalassemia, initial studies examined HAMP expression in the context of BMP/SMAD pathway members and targets genes. qRT-PCR analyses confirm decreased HAMP expression in 2 month old th3/+ mice and indicate a significantly larger decrease in mice with β-thalassemia major (th3/th3) relative to wild-type (+/+) controls. A significant decrease in the common SMAD pathway member, SMAD4, is also evident in both strains. TMPRSS6, the transcript encoding the protease matriptase-2, is also decreased in th3/th3 mice. In contrast, at 5 months of age the transcriptional profile of these genes in th3/+ mice is indistinguishable from that of wild-type mice. By 12 months of age, there is a trend toward increased HAMP transcription corresponding to increases in the BMP/SMAD targets ID1 and ATOH8. Examination of the effect of a low-iron diet on the transcriptional levels of these genes over time in th3/+ mice indicates no significant differences from +/+ mice after the data were normalized to liver iron concentrations. In contrast, comparison of the transcriptional profile of these genes with +/+ mice fed a high-iron diet indicates that HAMP expression is blunted in thalassemic mice. Preliminary Western blot analyses indicate that phosphorylation of SMAD 1/5/8 in th3/+ and th3/th3 liver samples, although comparable to that in +/+ samples, is lower than the levels observed in mice fed a high-iron diet. These data indicate that iron sensing through the BMP/SMAD pathway is intact, but somewhat blunted in thalassemic mice. Thus an alternative mechanism, possibly mediated by the IE that is characteristic of this disease, is likely responsible for the suppression of HAMP. In order to gain support for this hypothesis, qRT-PCR analyses were performed on mice overexpressing transgenic erythropoietin (TgEpo) to examine the effect of erythropoiesis on HAMP expression. The data indicate suppression of HAMP transcription despite clear activation of the BMP/SMAD pathway, as evidenced by increases in BMP6 and ID1. Of note, SMAD7 transcription was not detected in these animals. Compared to +/+ mice, phlebotomized mice also show decreased transcription of HAMP and SMAD4 without corresponding decreases in the BMP/SMAD pathway targets ID1 and ATOH8. TMPRSS6 transcription was also decreased, but FURIN expression was increased. FURIN, a known hypoxia target gene, was also transcribed at a significantly higher rate in th3/th3 mice than in either th3/+ or +/+ mice. qRT-PCR data indicate that this is mediated by HIF-2α, as evidenced by decreased transcription of the HIF-1α target, pyruvate dehydrogenase kinase 1, and increased transcription of the HIF-2α target, erythropoietin (EPO), in th3/th3 liver samples. HIF2-α expression in th3/th3 liver lysates was confirmed by Western blot analysis. Collectively, these data indicate that HAMP expression in thalassemia is suppressed through a mechanism distinct from a BMP/SMAD feedback loop and provide support for the existence of an ‘erythroid factor'. Results also suggest the increased suppression of HAMP observed in th3/th3 mice is attributable to the activation of HIF2-α, which in turn would be expected to aggravate the production of an erythroid factor through the upregulation of EPO. Disclosures: No relevant conflicts of interest to declare.

Abstract 3187 ß-Thalassemia and sickle cell disease (SCD) are the most common genetic red blood cell (RBC) disorders characterized respectively by limited production of aberrant ß-globin chains. In both cases, chronic transfusions and iron chelation are required to treat the anemia and/or formation of abnormal RBC. In ß-thalassemia, anemia stimulates erythropoietin (Epo) synthesis, which in turn leads to increased erythropoiesis and development of hepatosplenomegaly, often resulting in the need for splenectomy. Recently, we demonstrated that erythroid cells from ß-thalassemic mice have a hyper-activation of Jak2, a kinase that mediates the signaling triggered by the binding of Epo to the Epo receptor. This led us to hypothesize that Jak2 inhibitors could be utilized to minimize erythroid expansion in this disorder, limiting splenomegaly. A Jak2 inhibitor (Tg101209 or Tg) was first tested in mice affected by ß-thalassemia intermedia (th3/+). Two doses of Tg (150 and 100 mg/Kg/day) were given orally for 10 days. Tg administration induced a mild decrease of hemoglobin levels (8.8±0.2, 8±0.2 and 7.8±0.2g/dL for placebo, Tg 100 mg/Kg and Tg 150 mg/Kg treated mice, respectively. p We then evaluated the outcome of combining Tg with blood transfusion, a common therapy in b-thalassemia and SCD. In b-thalassemia, massively enlarged spleens are believed to sequester a significant proportion of circulating RBC, thereby limiting their lifespan and the efficacy of transfusion regimens. We hypothesize that decreasing splenomegaly by administration of Jak2 inhibitors could increase the efficacy of transfusion. This was first tested in th3/+ animals. In this case, transfusion alone was sufficient to increase the hemoglobin (Hb) levels approximately 3 g/dL and reduce the spleen size to 65% of that seen in non-transfused controls. In this model, the combined effect of transfusion and administration of Tg was more effective, the spleen size been 50% of non-transfused controls (p Lastly, we tested combination therapy in a mouse model of SCD. Mice treated with Tg and transfusion exhibited slightly lower levels of Hb than transfused controls (Hb=9.7±0.2g/dL versus Hb=10.9±0.2g/dL). However, compared to the control, mice receiving combination therapy exhibited a larger percentage of donor RBCs, while endogenous erythropoiesis was markedly suppressed along with the production of sickle RBCs (1.3±0.3×106 RBC/ul compared to transfused-controls exhibiting 2.7±0.3×106 RBC/ul). In summary, administration of Jak2 inhibitors might reduce the production of pathological cells that, together with preservation of the splenic architecture, could minimize the propensity of patients to thrombotic events. Furthermore, suppression of endogenous erythropoiesis and reduction of the transfusion regimen would be expected to also reduce iron accumulation, making it easier to prevent its toxic effects through chelation therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 1035 β-Thalassemia is a disorder associated with abnormal β-globin production, leading to anemia, extramedullary hematopoiesis (EMH), a decreased lifespan of the red cells and iron overload. In this disorder erythropoiesis is ineffective due to increased erythroid apoptosis and erythroblast proliferation, as well as deficient differentiation. Recent evidence suggests that erythroid development, especially under conditions of anemia (stress erythropoiesis), is highly dependent on microenvironmental factors within the erythroid niche, potentially mediated by the interaction of erythroblasts with macrophages. However, little is known about the function of these cells in pathological anemias associated with abnormal erythropoiesis. Our goal was to study the role of macrophages in normal, stress and ineffective erythropoiesis (IE). Macrophages were eliminated by intravenous administration of clodronate-containing liposomes. Treatment was carried out for up to 12 weeks, serial measurements being made of erythropoietic and pathological parameters. As a model of stress erythropoiesis, phlebotomized wt mice were used. To study IE we utilized th3/+ mice, a model of β-thalassemia intermedia (TI). Clodronate treatment effectively depleted splenic and bone marrow (BM) macrophages as shown by FACS and immunohistochemical analyses. Depletion of macrophages in wt mice had little effect on steady state erythropoiesis. In contrast, clodronate treatment drastically impaired the response to stress erythropoiesis in these mice, as shown by the slow recovery from phlebotomy-induced anemia. This was associated with a very slow rate of RBC and reticulocyte production, suggesting that erythroid activity was markedly impaired. Accordingly, mice depleted of macrophages were unable to expand their pool of erythroid progenitors in the BM and spleen in response to anemia, suggesting that macrophages play a critical role in this process. A similar defect was observed in response to Epo stimulation, suggesting that an intact erythroid niche is essential for normal activity of Epo in promoting erythroid expansion. Interestingly, TI mice treated with clodronate exhibited an improvement of the thalassemic phenotype. Within 40 hours of clodronate treatment, mice showed an increase in hemoglobin (Hb), RBC and reticulocyte counts in the peripheral blood, and a reduction of extra-medullary hematopoiesis (increased ratio of mature to immature erythroid cells) and splenomegaly (P In conclusion, our data suggests that macrophages have two major roles in β-thalassemia: 1) to modulate iron availability for erythroid cells; 2) to impair erythroid development, as suggested by the amelioration of splenomegaly and EMH observed after clodronate treatment. We hypothesize that the macrophages within erythroblastic islands control erythropoiesis, acting as modulators of this process. Under conditions of stress erythropoiesis they positively influence erythroid development, promoting proliferation to increase the pool of erythroid cells. However, under conditions of chronic stress such as in TI, macrophages limit differentiation and promote excessive expansion of the erythron, contributing to IE. Disclosures: No relevant conflicts of interest to declare.

Abstract 1046 Anemia of inflammation (AI) is a widespread multi-factorial form of anemia characterized by hepcidin-induced iron restricted erythropoiesis as well as direct cytokine effects on the bone marrow, blunted erythropoietin production and efficacy, and shortened red blood cell (RBC) lifespan. Our aim is to perform an in depth study of AI, identifying the components and mechanisms associated with its pathophysiology. We generated a mouse model of AI using a single intraperitoneal injection of heat-killed Brucella abortus (HKBA). In this model we explored the role played by interleukin-6 and hepcidin in the onset of anemia. We utilized wild-type (WT), interleukin-6 knockout (IL-6 KO) and hepcidin knockout (Hamp KO) mice (n ≥ 6/group) injected with HKBA, and conducted weekly CBC's for 7 weeks to follow the progression and resolution of anemia. Anemia started developing one week after HKBA administration and reached a nadir after 2 weeks in all mice. Hemoglobin values from WT mice were lowest 2 weeks after injection (6.4 ± 1.2 g/dl) but slowly recovered over 7 weeks. Initially, IL-6 KO mice were equally affected with similar hemoglobin values at 2 weeks (6.9 ± 1.3 g/dl). However, these mice recovered after 3 weeks. Hamp KO mice were less anemic throughout the course of the study, with hemoglobin values of 10.3 ± 0. 9 g/dl at 2 weeks and resolution after 4 weeks. These data demonstrate that while both interleukin-6 and hepcidin contribute to AI, lack of either molecule alone is not sufficient to prevent AI. Therefore, additional factors likely play an important role in the etiology of AI. In order to rule out the effect of iron overload on the reduced severity of anemia observed in Hamp KO mice injected with HKBA, 1 week-old mice were fed an iron-deficient diet in order to first deplete their iron stores, and then returned to the normal diet before HKBA injection. We observed that iron-depleted Hamp KO mice were still less sensitive to HKBA administration, suggesting that this effect was independent of iron overload and dependent on the intrinsic lack of hepcidin expression. We further investigated the erythropoiesis in WT, IL-6 KO, and Hamp KO mice one week after HKBA injection. We performed FACS analyses of BM and spleen using CD44 and Ter119 antibodies. Both the mature RBCs (CD44−/Ter119+) and erythroid progenitor cells (CD44+/Ter119+) were dramatically reduced in the BM of HKBA-treated WT mice compared to controls (CD44+/Ter119+ cells diminished from 35.5 ± 0.2% to 2.8 ± 0.8%; CD44−/Ter119+ cells from 17.2 ± 0.2% to 8.2 ± 0.8%). The reduction of erythroid cells was attenuated in HKBA-treated IL-6 KO mice (CD44+/Ter119+ cells diminished from 32.8 ± 0.1% to 7.5 ± 6.0%; CD44−/Ter119+ cells from 22.1 ± 0.5% to 10.4 ± 3.8%). Hamp KO mice, on the other hand, showed a dramatic reduction of the CD44+/Ter119+ population in their BM (from 24.1 ± 2.5% to 1.8 ± 0.3%), while mature CD44−/Ter119+ cells were less affected (from 15.4 ± 2.3% to 14.1 ± 2.6%). Erythropoiesis was altered in the spleen as well. However, while the CD44+/Ter119+ cells were reduced in all the mice strains, the CD44−/Ter119+ population was increased one week after HKBA injection. This profile was more similar to ineffective erythropoiesis than iron-restricted erythropoiesis. Splenomegaly was also observed in all HKBA-treated mice. In addition, we measured increased apoptosis and production of reactive oxygen species (ROS) in the reticulocytes and orthochromatic erythroblasts of the spleen and BM of all mice. Overall, these data suggest that, in addition to iron restricted-erythropoiesis, an acute inflammatory effect on erythropoiesis is occurring in the HKBA model of AI, affecting erythroid cell survival and/or proliferation. Further analyses aimed at determining the RBC life span and survival in these mice are in progress. Moreover, we are analyzing iron-related gene expression in all groups of mice, along with measurement of their serum iron levels, iron stores, and serum cytokine levels, at different time points. Preliminary data indicate that numerous cytokine mRNAs (including IL-1α, IL-1β, TNF-α, INF-γ) are elevated in the spleen of WT mice 6 hours after HKBA injection. We are investigating the role that these cytokines might have on erythropoiesis, and the anemia observed in IL-6 KO and Hamp KO mice after injection of HKBA. Disclosures: Cooke: Amgen: Employment. Sasu:Amgen: Employment.

On the basis of our previous demonstration of the high inhibitory activity of a series of p-n-alkyloxybenzhydroxamic acids and n-alkyl esters of 3,4-dihydroxybenzoic acid against the trypanosome alternative oxidase in a cell-free mitochondrial preparation of Trypanosoma brucei brucei, we synthesized a series of N-n-alkyl-3,4-dihydroxybenzamides for evaluation as inhibitors of this enzyme. This class of compounds was selected with the expectation of their having similar inhibitory activity to but greater solubility than the esters and hydroxamic acids noted above and greater resistance to serum hydrolases in vivo. We predicted that such properties would allow an inhibitor of the trypanosome alternative oxidase to be coadministered with glycerol as a means of providing treatment for infections by African trypanosomes. As expected, such benzamides were both more soluble and more stable, some being more active against the target enzyme than the corresponding ester. One, N-n-butyl-3,4-dihydroxybenzamide, was selected for evaluation in vivo against T. brucei brucei. When combined with glycerol, this benzamide was found to be curative. A regimen wherein 450 mg of N-n-butyl-3,4-dihydroxybenzamide per kg and 15 g of glycerol per kg were given hourly in three divided doses cured 17 of 19 mice with established T. brucei brucei infections. This combination is more active in vivo than any other designed to block simultaneously both the unique respiratory electron transport system and the anaerobic glycolytic pathways of these pathogenic protozoa.

Abstract 2063 ß-Thalassemia is a genetic disorder characterized by decreased or absent production of ß-globin chains, leading to ineffective erythropoiesis, anemia and iron overload. Hepcidin, the hormone that controls iron homeostasis, is regulated by several mechanisms, including erythropoiesis, iron overload, inflammation and hypoxia. In the absence of transfusion therapy, patients with ß-thalassemia major exhibit a severe ineffective erythropoiesis that suppresses hepcidin expression. However, in patients or animal affected by ß-thalassemia intermedia (th3/+), iron overload is associated with a milder form of ineffective erythropoiesis. In this study we investigated whether th3/+ mice retain the ability to modulate hepcidin expression in response to iron load, despite their increased erythropoietic activity. We analyzed some of the genes involved in the regulation of hepcidin, in particular, genes that are upregulated by iron overload in wt mice. These included Bmp6, a strong modulator of Hamp in response to iron, and Id1, Atoh8 and Smad7, other targets of the Bmp/Smad pathway. Analysis of the phosphorylation of the Smad protein complex is in progress. In addition, we generated mice affected by ß-thalassemia intermedia lacking the Hfe gene (Hfe-th3/+), in an attempt to determine whether or not this gene is involved in hepcidin regulation in this disorder. We analyzed th3/+ mice at 2, 5 and 12 months of age. In 2-month-old th3/+ mice hepcidin expression was significantly low compared to wt mice. As th3/+ mice age and their iron overload worsens, hepcidin expression increases showing similar and elevated levels in th3/+ compared to wt animals, respectively at 5 and 12 months. At 2 months, hepcidin expression normalized to liver iron concentration exhibited even lower levels in th3/+ mice compared to wt animals. This ratio did not change in aging th3/+ animals, despite the fact that their liver iron concentration increased over time (0.66, 1.24, and 1.45 ug/mg of dry weight at 2, 5 and 12 months, respectively). The expression levels of Bmp6, Id1, Atoh8 and Smad7 followed a similar pattern, being generally downregulated at 2 months compared to wt mice. However, as iron overload progressed, th3/+ mice exhibited increased expression of these genes compared to wt mice. Similar to what was observed with hepcidin, their expression was low in th3/+ mice at all ages when normalized to liver iron concentration. These observations indicate that hepcidin expression in ß-thalassemia increases over time and is regulated by the relative levels of ineffective erythropoiesis and iron overload. We also investigated the relationship between Hfe and hepcidin in response to iron in ß-thalassemia. We transplanted the ß-thalassemic phenotype into lethally irradiated wt or Hfe-KO mice, generating th3/+ and Hfe-th3/+ animals, respectively. Compared to th3/+ mice, we observed that Hfe-th3/+ animals had increased hepatic iron (3.09 vs 1.29 ug/mg of dry weight, p≤0.05) and serum iron (232 vs 162 ug/dL, p≤0.05), with no significant changes in splenic iron concentration. The Hfe-th3/+ mice also exhibited increased hemoglobin levels (9.4 vs 7.8 g/dL, p≤0.001) due to an increase in both red cell counts (8.9 vs 8.0 ×106 cells/uL, p≤0.01) and mean corpuscular hemoglobin levels (10.6 vs 9.7 pg, **p≤0.05). However, this did not reduce splenomegaly or ineffective erythropoiesis. We also analyzed the levels of hepcidin, Bmp6, Id1, Smad7 and Atoh8 in 5-month-old mice. At his time point expression of most of these genes was similar between wt, th3/+ and Hfe-th3/+ mice. Only expression of Bmp6 was elevated in the two thalassemic groups compared to wt mice. When the levels of hepcidin, Bmp6, Id1, Smad7 and Atoh8 expression were normalized to liver iron content, we observed significant reductions in Hfe-th3/+ mice compared to th3/+ animals. Taken together, these observations indicate that iron overload can partially counteract the repressive effect of ineffective erythropoiesis on hepcidin expression in th3/+ mice. Moreover, lack of Hfe further impairs the ability of hepcidin and other iron regulated genes to respond to iron overload, aggravating this feature in thalassemic mice. Overall, this indicates that Hfe plays a positive role in the regulation of hepcidin in ß-thalassemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 4251 A deficient hepcidin response to iron is the principal mechanism responsible for increased iron uptake from the diet leading to iron overload. In hereditary hemochromatosis (HH), mutations in the HFE gene lead to iron overload through abnormally low levels of hepcidin. Interestingly, hepcidin has been shown to respond to a variety of stimuli, including iron, hypoxia, erythropoiesis and inflammation, requiring integration of the respective signals for its regulation. Further studies showed that HFE/Hfe could also modulate cellular iron uptake by associating with the transferrin receptor-1 (Tfrc), a crucial protein for iron uptake by erythroid cells. In addition, some studies have reported altered erythropoietic values in HH patients. Despite these findings, the role of Hfe in erythropoiesis was never explored. We hypothesized that Hfe influences erythropoiesis by two distinct mechanisms: 1) limiting hepcidin expression, thereby increasing iron availability, under conditions of simultaneous iron overload and stress erythropoiesis; 2) participating directly in the control of transferrin-bound iron uptake by erythroid cells. To test this hypothesis we investigated the role of Hfe in erythropoiesis, aiming to uncover the relative contribution of each of the aforementioned mechanisms. When erythropoiesis was challenged by phlebotomy, Hfe-KO animals were able to recover faster from anemia (p≤0.05) than either normal or iron overloaded wt mice. In Hfe-KO mice, despite their increased iron load, downregulation of hepcidin in response to phlebotomy or erythropoietin administration was comparable to that seen in wt mice. In contrast, iron overloaded wt mice showed increased hepcidin expression both at steady state and after erythropoietic stimulation compared to wt or Hfe-KO mice. In phlebotomized mice fed a standard diet, analysis of serum iron and transferrin saturation indicated that wt mice on the standard diet were able to increase their serum iron very rapidly. After 24 hours, both wt and Hfe-KO mice had similar serum iron and transferrin saturation levels. On the other hand, wt mice kept on an iron deficient diet over the course of phlebotomy, were unable to overcome the phlebotomy-induced anemia. In contrast, Hfe-KO mice fed the low iron diet were able to recover from anemia, although at a slower pace than either Hfe-KO or wt mice on a standard diet. These data indicate that gastrointestinal iron absorption in both wt and Hfe-KO mice is a major factor leading to recovery from anemia, although the excess iron in the liver of Hfe-KO mice contributes to restoration of the red blood cell reservoir. Phlebotomy is the main tool utilized to treat iron overload in HH patients. However, our data suggests that this treatment leads to both mobilization of iron from stores and increased gastrointestinal iron absorption. These observations suggest that patients might benefit from a controlled iron diet or from supplementation with hepcidin or an hepcidin agonist to limit iron absorption. Next, we determined that Hfe is expressed in erythroid cells and that it interacts with Tfrc in murine erythroleukemia cells. Moreover, we discovered that the level of Tfrc expression in Hfe-KO cells is 80% of that seen in wt cells, as measured by flow cytometry. This observation, together with measurement of iron uptake using 59Fe-saturated transferrin, indicated that Hfe-KO erythroid cells take up significantly more iron than wt cells. To confirm that Hfe plays a role in erythropoiesis independent from that in the liver, we transplanted Hfe-KO or wt bone marrow cells into lethally irradiated wt recipients and analyzed their recovery from phlebotomy. We observed that recovery from anemia was faster in Hfe→wt than in wt→wt and was associated with increased mean corpuscular hemoglobin levels, suggesting that lack of Hfe in the hematopoietic compartment can lead to increased hemoglobin production. In summary, our results indicate that lack of Hfe enhances iron availability for erythropoiesis by two distinct mechanisms. On the one hand, Hfe plays an important role in maintaining erythroid iron homeostasis by limiting the response of hepcidin to iron, particularly under conditions of erythropoietic stimulation. On the other hand, lack of Hfe contributes directly to increased iron intake by erythroid progenitors, even in the absence of iron overload. Disclosures: No relevant conflicts of interest to declare.

Abstract 1009 The principal regulator of iron homeostasis, the hepatic peptide hepcidin (Hamp), degrades the iron-transport protein ferroportin (Fpn) localized on absorptive enterocytes, hepatocytes and macrophages. Low Hamp expression has been associated with iron overload in patients and mice affected by β-thalassemia intermedia (th3/+). Our hypothesis is that more iron is absorbed than required for erythropoiesis in β-thalassemia. Therefore, we propose that limiting the dietary iron intake of th3/+ mice either by feeding them a low iron diet or increasing their Hamp expression will have a beneficial effect on iron overload with no effects on erythropoiesis. In particular, since Hamp expression is low in β-thalassemia, a moderate increase of Hamp expression should not interfere with erythropoiesis by preventing release of iron from macrophages. However, we predict that very high levels of Hamp expression will limit the recycling of iron from macrophages, thereby exacerbating the anemia. We first analyzed wt and th3/+ mice fed diets containing a physiological amount of iron (35 ppm) or low iron (2.5 ppm) for 1 and 5 months. These mice were then compared to wt and th3/+ mice expressing a transgenic Hamp (THamp and THamp/th3, respectively). In wt mice, the low-iron diet decreased tissue iron levels leading to anemia (Hb: 14.6±0.7 g/dL and 8.6±2.4 g/dL at 1 and 5 months, respectively). In th3/+ mice fed the low-iron diet, the amount of iron in the liver and spleen decreased over time and after 5 months was 10 times lower than at the beginning of treatment. However, in this case the low-iron diet did not worsen the anemia, (Hb: 8.2±1.3 g/dL vs. 7.8±1.8 g/dL at 1 and 5 months, respectively). In the case of THamp and THamp/th3 mice, we stratified those animals whose transgenic Hamp expression was moderate (2-4 higher) or high (>4 times higher) compared to the endogenous Hamp expression in control mice. In THamp animals expressing a moderate level of Hamp, the total iron content of the liver was decreased (65±21 μg vs. 131±31 μg in wt controls) while no significant changes were detected in the spleen. THamp mice also exhibited anemia (Hb: 11.2±1.8 g/dL vs. 13.9±1.1 g/dL at 1 month). The iron content of the liver and spleen was reduced in THamp/th3 (127±86 μg vs. 234±49 μg and 131±88 μg vs. 271±74 μg, respectively, compared to th3/+ controls), while their hematological values were dramatically improved. Splenomegaly was also significantly reduced. Similar findings were observed at 5 months. Looking at animals expressing high levels of transgenic Hamp, both THamp and THamp/th3 mice exhibited vast accumulations of iron in macrophages, profound anemia, reticulocytosis and increased splenomegaly, confirming that high levels of Hamp block iron recycling and are detrimental to erythropoiesis. Interestingly, in THamp/th3 mice expressing a moderate level of Hamp we observed that the increase in hemoglobin levels was associated with increased red cell numbers but reduced mean corpuscular hemoglobin levels. Paradoxically, this could indicate that reduction of the anemia in THamp/th3 mice is mediated by decreased heme synthesis. α-Globin/heme aggregates lead to ineffective erythropoiesis and a limited red cell life span by producing reactive oxygen species and altering the structure of red cell membranes. Compared to th3/+ mice, THamp/th3 mice exhibited reduced heme contents, insoluble membrane-bound α-globins and reactive oxygen species resulting in an increased life span and more normal morphology of their red blood cells. While the number of red blood cells was increased, the number of reticulocytes, and the total number of erythroid precursors in the spleen were reduced. This was associated with a reduction in reactive oxygen species. Cell cycle analysis of the erythroid cells at different stages of differentiation, expression of heme related proteins and synthesis of α- and β-globin chains in THamp/th3 mice is in progress. Overall, this study indicates that use of hepcidin might be effective in reducing iron overload and improving erythropoiesis in β-thalassemia thereby limiting toxicity due to heme not incorporated into the adult hemoglobin tetramer. In conclusion, we believe this study provides the first evidence that hepcidin could be utilized for the treatment of abnormal iron absorption in β-thalassemia and other related disorders, with additional beneficial effects on ineffective erythropoiesis, splenomegaly and anemia. Disclosures: Nemeth: Intrinsic Life Sciences: Employment, Membership on an entity's Board of Directors or advisory committees.

Abstract 5163 Introduction: Deferasirox (Exjade, ICL670) is an orally effective iron chelating agent approved for use in patients 2 years of age and older with transfusional iron overload. While it is effective as a single agent, there are patients who do not attain net negative iron balance despite being at the upper limit of approved dosing (40 mg/kg/day). Deferiprone (Ferriprox, L1) is another orally effective iron chelator. Through a series of metabolic iron balance studies we were able to demonstrate that various regimens involving the combined use of deferiprone and deferoxamine (Desferal, DFO) were capable of placing all patients into net negative iron balance with the potential to adjust dosing schedules and the ratio of drugs to maximize effectiveness while minimizing toxicity. A variety of such regimens are now in widespread clinical use. We have taken the same approach in order to optimize the use of deferasirox. Methods: Six patients with thalassemia major were enrolled in a 34-day metabolic iron balance study wherein deferasirox and deferoxamine were evaluated alone and in combination, each patient serving as his/her own control. Deferoxamine (40 mg/kg) was administered on days 5 – 10 as an 8-hour subcutaneous infusion during the night. On days 15 – 20, deferasirox (30 mg/kg) was given orally, 30 minutes prior to breakfast. Both drugs were given on days 25 – 30, the same doses and dosing schedules being employed. Non drug days allowed for washout of stool iron induced by the previous treatment. The patients consumed a fixed low-iron diet consisting of four individualized meal plans. Daily collections of urine and stool were made and their iron content determined by atomic absorption, a correction being made for all uneaten food. Results: As in previous studies, there was significant patient to patient variability in terms of the amount of drug-induced iron excretion. Combination therapy placed all patients into iron balance exceeding 200% (206% - 270%, mean 251%). The combination was synergistic in two patients (35% and 57%), additive in three patients, and less than additive in one patient. Where iron excretion was more than additive, all of the excess appeared in the urine (a 2.1- and 3.4-fold increase). Deferasirox proved to be less effective than deferoxamine in all six patients (relative iron excretion: 23% - 59%, mean 42%), stool iron excretion in response to the respective drugs being 94% -100% (mean 98%) and 49% - 74% (mean 59%). Net negative iron balance was achieved in 2/6 patients when on deferasirox (iron balance 28% - 129%, mean 72%) and 6/6 patients when infusing deferoxamine (iron balance 125% - 219%, mean 167%). Discussion: These results suggest that chelation therapy can be tailored to the individual needs of each patient, selectively removing iron from different pools while minimizing any side effects. Deferasirox appears to shuttle iron to deferoxamine for excretion in the urine, not unlike the situation when deferiprone and deferoxamine are combined, although the source of the iron may be somewhat different. It is hoped that these results will provide patients with further options to optimize their chelation regimens. Disclosures: Grady: Novartis Pharmaceuticals: Research Funding. Off Label Use: Deferasirox and Deferoxamine are both iron chelating agents. Their use in combination is not indicated on their labeling. Galanello:Novartis Pharmaceuticals: Speakers Bureau. Paley:Novartis Pharmaceuticals: Employment. Giardina:Novartis Pharmaceuticals: Research Funding.