Organ Iron Content Research Articles

Abdominal multi-organ iron content and the risk of Parkinson's disease: a Mendelian randomization study.

To evaluate the causal relationship between abdominal multi-organ iron content and PD risk using publicly available genome-wide association study (GWAS) data. We conducted MR analysis to assess the effects of iron content in various abdominal organs on PD risk, followed by reverse analysis. Additionally, MVMR analysis evaluated the independent effects of organ-specific iron content on PD. We utilized genetic variation data from the UK Biobank, including liver iron content (n = 32,858), spleen iron content (n = 35,324), and pancreas iron content (n = 25,617), as well as summary-level data for Parkinson's disease from the FinnGen (n = 218,473) and two other large GWAS datasets of European populations (First dataset n = 480,018; Second dataset n = 2,829). The primary MR analysis used the inverse variance-weighted (IVW) method, confirmed by MR-Egger and weighted median methods. Sensitivity analysis was performed to address potential pleiotropy and heterogeneity. Observational cohort results were validated through replication cohort analysis, followed by meta-analysis. IVW analysis revealed a causal relationship between increased liver iron content and elevated risk of PD (OR = 1.27; 95% CI: 1.05-1.53; p = 0.015). No significant causal relationship was observed between spleen (OR = 1.00; 95% CI: 0.76-1.32; p = 0.983) and pancreatic (OR = 0.93; 95% CI: 0.72-1.20; p = 0.573) iron content and increased risk of PD. Meta-analysis of GWAS data for PD from three different sources using the random-effects IVW method showed a statistically significant causal relationship between liver iron content and the occurrence of PD (OR = 1.17, 95% CI: 1.01-1.35; p = 0.012). This study presents evidence from Mendelian randomization (MR) analysis indicating a significant causal link between increased liver iron content and a higher risk of Parkinson's disease (PD). These findings suggest that interventions targeting body iron metabolism, particularly liver iron levels, may be effective in preventing PD.

Mechanism of Iron Transport in the Triticum aestivum L.–Soil System: Perception from a Pot Experiment

Iron is one of the necessary trace elements for plant growth and the human body. The ‘hidden hunger’ phenomenon in the human body caused by an imbalance of iron in soil is increasingly prominent. Addressing this issue and optimizing soil through regulatory measures to improve the absorption and utilization of iron by crops has become an urgent priority in agricultural development. This study carries out pot experiments to observe the growth process of Triticum aestivum L. under various soil iron environments. Combined with previous research results, the transport mechanism of iron in the soil—Triticum aestivum L. system was systematically explored. The results indicate that during the jointing and maturity stages of Triticum aestivum L., iron was preferentially enriched in the underground parts; at the maturity stage, the iron content in various organs of Triticum aestivum L. shows a trend of increase followed by a decrease with the soil iron content varying in the following sequence: deficient, moderately deficient, medium, moderately adequate, and adequate. The iron-deficient stress environment causes an increase in the effectiveness of rhizosphere iron, resulting in a higher level of iron in the plant stems, leaves, and seeds. Conversely, when the soil iron content is medium or adequate, the effectiveness of rhizosphere iron decreases, leading to a reduction in the iron content in each part of the plant. A concentration gradient of 7.2 mg/kg in the experimental setup is found to be the most favorable to the enrichment of iron in the shoots of Triticum aestivum L. plants. The findings of this experiment provide guidance for the fertilization strategy to mitigate iron deficiency symptoms in plants under similar acidic-alkaline conditions of soil, as well as a systematic mechanism reference and basis for studying the soil-plant-human health relationship.

The Impact of Perinatal Cobalt Chloride Exposure on Extramedullary Erythropoiesis, Tissue Iron Levels, and Transferrin Receptor Expression in Mice.

The objective of the present study was to elucidate the effect of perinatal cobalt chloride (CoCl2) exposure on extramedullary erythropoiesis in suckling mice in relation to iron (Fe) content and transferrin receptor (TfR) expression. Pregnant ICR mice were subjected to a daily dose of 75 mg CoCl2/kg body weight 2-3 days prior and 18 days after delivery. Co exposure significantly increased erythrocyte count (RBC), and reduced the erythrocytic parameters mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in the offspring. Total iron-binding capacity (TIBC) was decreased while bilirubin values were ~ 1.2-fold higher in the metal-exposed mice. Perinatal CoCl2 treatment also induced pathohistological changes in target organs (spleen, liver, and kidneys) as altered organ weight indices, leukocyte infiltration, abundant Kupffer cells in the liver, increased mesangial cellularity, and reduced capsular space in the kidney. CoCl2 administration induced significant 68-, 3.8-, 41.3-, and 162-fold increase of Co content in the kidney, spleen, liver, and RBC, respectively. Fe content in the target organs of CoCl2-treated mice was also significantly elevated. Immunohistochemical analysis demonstrated that TfR1 was well expressed in the renal tubules, hepatocytes, the red pulp, and marginal zone of white pulp in the spleen. TfR2 showed similar expression pattern, but its expression was stronger in the spleen and liver samples of Co-treated mice compared with that of the untreated controls. The results demonstrate that exposure to CoCl2 during late pregnancy and early postnatal period affects body and organ weights and alters hematological and biochemical parameters, iron content, and TfR expression in target organs.

Feasibility of the use of a handheld XRF analyzer to measure skin iron to monitor iron levels in critical organs

There exists a need for accurate, non-invasive point-of-care tests to detect body iron burden. This study investigated the use of x-ray fluorescence (XRF) measurements of skin iron as a marker for organ iron content in rats. This study also evaluated a novel application of a commercial XRF device, commonly used in mining and construction, as a rapid, portable, and non-invasive measurement tool. Rats (n = 32) were loaded with iron dextran and the iron signal of each animal’s skin, liver, and kidney was measured using a conventional XRF system. A quadratic correlation was observed between liver and skin iron signal (R2 = 0.92) and a linear correlation was observed between kidney and skin iron signal (R2 = 0.65). As such, it is concluded that skin iron content can act as a marker for both liver and kidney iron content. The same skin samples were measured using the portable XRF device and compared to the liver and kidney samples measured in the conventional XRF system. Again, a quadratic correlation was observed between liver and skin iron signal (R2 = 0.91) and a linear correlation was observed between kidney and skin iron signal (R2 = 0.83). Thus, the portable XRF device can provide rapid non-invasive, skin XRF measurements. Dosimetry was performed using the portable XRF device to assess the radiological hazard associated with its use. The average skin equivalent dose from this device is 30 ± 10 mSv/min, when the device is collimated and operated at 40 kV. In conclusion, skin iron XRF measurements can act as a surrogate marker for liver iron content, and can be measured using a commercial XRF device for a portable, fast, and non-invasive measurement.

Relationship between T2* magnetic resonance imaging-derived liver and heart iron content and serum ferritin levels in transfusion-dependent thalassemic children.

CONTEXT:T2* magnetic resonance imaging (MRI) is being increasingly used for the assessment of organ iron content in thalassemics, but cost is a major prohibitive factor for repeated measurements. If serum ferritin correlates well with the T2* MRI liver and heart, it will be economical and more simple tool to assess organ iron deposition.AIMS:The aim of this study was to find out the relationship between serum ferritin level and T2* MRI-derived liver and heart iron content in transfusion-dependent thalassemic childrenSETTINGS:Thalassemia day-care center of a teaching hospitalDESIGN:This was a cross-sectional studySUBJECTS AND METHODS:Seventy-three transfusion-dependent beta thalassemic children belonging to 2–18 years of age were subjected to T2* MRI of heart and liver to assess their iron content. Values obtained here were related to serum ferritin.STATISTICAL ANALYSIS USED:Keeping the correlation between serum ferritin and T2* MRI as primary outcome, spearman's correlation coefficient was calculated.RESULTS:We found poor (negative) correlation between serum ferritin level and T2* MRI liver (r = -0.448, P = 0.000) but no correlation between serum ferritin and T2*MRI heart (r = -0.221, P = 0.060).Conclusions:Serum ferritin cannot reliably predict the liver and heart iron content in Indian children with β thalassemia.

Administration of Minihepcidins to Animals Affected By β-Thalassemia Major Reduces Anemia and Splenomegaly

Both β-thalassemia intermedia and major are characterized by formation of hemichromes in erythroid cells, impairing their survival and the lifespan of red blood cells (RBC). Minihepcidins (MH) are novel compounds that function as hepcidin agonists and reduce iron absorption and transferrin saturation. Hbbth3/+ mice show features of β-thalassemia intermedia, such as ineffective erythropoiesis (IE), anemia and reduced hepcidin synthesis, but do not require blood transfusion for survival (non-transfusion dependent thalassemia or NTDT). As we have previously shown, the administration of MH in these animals decreased transferrin saturation, erythroid iron intake, heme synthesis and hemichrome formation, with a significant beneficial effect on RBC quality, lifespan and anemia (Casu et al, Blood 2016). In order to test if this approach could also benefit animals affected by β-thalassemia major we focused on generating a model that exhibited a low production of RBCs, severe anemia and a blood transfusion requirement for survival, as in patients affected by transfusion dependent thalassemia or TDT. We have previously shown that engraftment of Hbbth3/th3fetal liver cells (FLCs) into normal mice leads to a very severe anemia that requires blood transfusion for survival (Gardenghi et al, Blood 2007). However, Hbbth3/th3FLCs do not contain any adult or fetal-globin genes and are unable to make hemoglobin in the transplanted animals, in contrast to human β-thalassemia. Therefore, animals cannot benefit from therapies that decrease hemichrome formation and target IE such as MH. To overcome this limitation, we crossed Hbbth3/+ mice with additional models of NTDT, indicated as Hbbth1/th1 and Hbbth2/+. These mice harbor alternative mutations so that the synthesis of the mouse b-globin genes is different in each model. Hbbth1/th2 and Hbbth1/th3 pups were alive at birth, but unable to survive more than a couple of days even with the support of blood transfusion. However, recipient transgenic animals expressing GFP and engrafted with Hbbth1/th2andHbbth1/th3FLCs showed the desired phenotype 3 months post-transplant including production of GFP- RBCs (with less than 2% of host GFP+ RBC) and a different degree of anemia, respectively 5.6±0.5 g/dL and 3.1±1.5 g/dL. In the long term these animals require blood transfusion for survival. Therefore these models are useful to test drugs that have the potential to modify erythropoiesis and RBC production. Ten weeks following engraftment with Hbbth1/th2 FLCs, mice were treated for six weeks with two different doses of MH (5.25 mg/kg and 2.625 mg/kg administered every other day) in absence of blood transfusion. Animals treated with vehicleshowed severe ineffective erythropoiesis and worsening anemia over 6 weeks (from 5.6±0.5 g/dL on D0 to 5.0±0.7 g/dL on D42 of treatment). In contrast, animals treated with MH showed reversal of anemia at 3 weeks (6.6±0.3 g/dL and 6.1±0.6 g/dL in the 5.25 mg/kg and 2.625 mg/kg group, respectively, compared to 5.3±0.9 g/dL in controls), while at 6 weeks the differences were reduced compared to vehicle treated mice (6.0±0.4 g/dL and 5.7±0.5 g/dL in the 5.25 mg/kg and 2.625 mg/kg group, respectively, compared to 4.9±0.7 g/dL in controls). The RBC number followed the same trend. Furthermore, the RBC morphology of animals treated with MH was improved compared to control animals. At 6 weeks, splenomegaly was also improved in the treatment groups (13.8±2.7 mg and 16.9±2.7 mg respectively in the 5.25 mg/Kg and 2.625 mg/Kg group compared to 26.9±3.5 mg in controls). Comparing the data at 3 versus 6 weeks, we speculate that, while the MH has a positive effect on RBC quality and production, this is insufficient, in the long term, to prevent the severe splenomegaly and the consequent entrapment of the RBC, which exacerbates the anemia over time. However, we hypothesized that administration of MH could have longer lasting beneficial effects in presence of blood transfusion, which would limit the splenomegaly. Presently, we are testing this hypothesis using both the Hbbth1/th2 and Hbbth1/th3 models. Complete characterization of these models and their parameters (CBC, erythropoiesis, iron metabolism and organ iron content) is in progress. In conclusion, these models can be utilized to characterize severe thalassemia phenotypes and new drugs that have the potential to ameliorate IE and improve RBCs generation. DisclosuresMacDonald:Merganser: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

In vivo evaluation of riboflavin receptor targeted fluorescent USPIO in mice with prostate cancer xenografts.

Supplementary material is available for this article at 10.1007/s12274-016-1028-7 and is accessible for authorized users.

Combination of Tmprss6-ASO and the Iron Chelator Deferiprone Improves Erythropoiesis and Reduces Iron Overload in a Mouse Model of Beta-Thalassemia

Patients affected by non-transfusion dependent thalassemia (NTDT) do not require chronic blood transfusion for survival. However, transfusion-independence in such patients is not without side effects. Ineffective erythropoiesis (IE), the hallmark of disease, leads to a variety of serious clinical morbidities. In NTDT the master regulator of iron homeostasis, hepcidin, is chronically repressed. Consequently, patients absorb abnormally high levels of iron, which eventually requires iron chelation to prevent the clinical sequelaes associated with iron overload. It has been shown that in mice affected by NTDT (Hbbth3/+), a second-generation antisense oligonucleotide (Tmprss6-ASO) can reduce expression of transmembrane serine protease Tmprss6, the major suppressor of hepcidin expression. This leads to reduction of hemichrome formation in erythroid cells, amelioration of IE and splenomegaly, and increased hemoglobin levels (Guo et al, JCI, 2013). Now we propose the use of Tmprss6-ASO in combination with iron chelators for the treatment of NTDT using Hbbth3/+ mice as a preclinical model. Our hypothesis is that use of chelators will benefit from the positive effect of Tmprss6-ASO on erythropoiesis and iron absorption, further ameliorating organ iron content. To this end, Hbbth3/+ animals were treated with Tmprss6-ASO at 100 mg/kg/week for 6 weeks with or without the iron chelator deferiprone (DFP) at a dose of 1.25 mg/ml. Additional animals were treated with DFP alone. We fed the animals with a commercial or physiological diet, containing 200 or 35 ppm of iron, respectively.We did not observe major differences in the treated animals fed the commercial or physiological iron diet and, for this reason, the data were combined for simplicity. Administration of DFP alone was successful in decreasing organ iron content. Compared to untreated Hbbth3/+ animals, we observed a reduction of 30% and 33% in the liver and spleen, respectively, and no change in the kidney. However, erythropoiesis was not improved (looking at IE, splenomegaly, RBC production and total Hb levels). This was associated with increased serum iron levels (+25%). In Tmprss6-ASO treated Hbbth3/+ animals, we observed an improvement in liver iron content (36% reduction), amelioration of IE, and increased RBC and Hb synthesis (~2 g/dL). Compared to treatment with Tmprss6-ASO alone, combination of DFP with Tmprss6-ASO achieved the same level of suppression of Tmprss6 in the liver (~90%) and reduction of serum iron parameters. This was associated with improvement of IE, decreased reticulocyte counts and splenomegaly, and increased RBC and Hb synthesis (~2 g/dL). While we observed that both Tmprss6-ASO and DFP separately reduced liver iron content to the same extent (~30-36%), combination treatment further reduced iron concentrations in the liver and kidney (69% and 19%, respectively), with no changes in the spleen. Additional analyses are in progress to evaluate the amount of hepcidin in serum as well as expression of erythroferrone, the erythroid regulator of hepcidin.Our first conclusion is that administration of an iron chelator alone is not sufficient to improve erythropoiesis despite that organ iron content is reduced. We speculate that when iron is removed from the liver, hepcidin expression becomes more susceptible to the suppressive effect of IE rather than the enhancing effect of reduced liver organ iron concentration. In addition, the combined effect of iron mobilized from organs and unchanged (or even augmented) iron absorption leads to increased serum iron concentration. As we have shown previously, amelioration of IE in this model requires decreased erythroid iron intake and hemichrome formation. Therefore, iron chelation alone is likely insufficient to improve erythropoiesis. Additional experiments are in progress to further elucidate this mechanism.Our second conclusion is that use of Tmprss6-ASO together with DFP combines the best effects of these two drugs, in particular on erythropoiesis and organ iron content. In animals that received the combined treatment, kidney and liver iron concentrations were further decreased compared to the single treatments. This indicates that Tmprss6-ASO might be extremely helpful in the treatment of NTDT and it could further improve iron related-chelation therapies. DisclosuresCasu:Merganser Biotech LLC: Employment; Isis Pharmaceuticals, Inc.: Employment. Aghajan:Isis Pharmaceuticals, Inc.: Employment. Guo:Isis Pharmaceuticals, Inc.: Employment. Monia:Isis Pharmaceuticals, Inc.: Employment. Rivella:bayer: Consultancy, Research Funding; isis Pharmaceuticals, Inc.: Consultancy, Research Funding; merganser Biotech LLC: Consultancy, Research Funding, Stock options , Stock options Other.

Characterization of transfusion-derived iron deposition in childhood cancer survivors.

Childhood cancer survivors (CCS) receiving packed red blood cell (PRBC) transfusions may have increased risk for vital organ iron deposition causing serious late effects. This cross-sectional cohort study of a CCS cohort quantified organ iron content by magnetic resonance imaging. Iron status by serum markers and hemochromatosis gene mutation status were assessed. Seventy-five patients who had received a range (0-392 mL/kg) of cumulative PRBC transfusion volumes were enrolled (median age 14 years, range 8-25.6 years at evaluation). Median follow-up time was 4.4 years, and median time since last transfusion was 4.9 years. Cancer diagnoses included acute lymphoblastic or myelogenous leukemia (ALL/AML; n = 33) and solid tumors (n = 42). Liver and pancreatic iron concentrations were elevated in 36 of 73 (49.3%) and 19 of 72 (26.4%) subjects, respectively. Cardiac iron concentration was not increased in this cohort. In multivariate analysis, cumulative PRBC volume (P < 0.0001) and older age at diagnosis (P < 0.0001) predicted elevated liver iron concentration. Iron overload (IO) may occur in children and adolescents/young adults treated for cancer and is associated with cumulative PRBC transfusion volume and age at diagnosis. These findings have implications for development of monitoring and management guidelines for cancer patients and survivors at risk for IO, exploration of the additive risk of liver/pancreatic damage from chemotherapeutic exposures, and health education to minimize further liver/pancreatic damage from exposures such as excessive alcohol intake and hepatotoxic medications.

Relation between serum ferritin and liver and heart MRI T2* in beta thalassaemia major patients

There is a need for higly accurate non-invasive methods for assessing organ iron content in thalassaemia patients. This study evaluated the relation between serum ferritin level, liver enzyme levels and hepatitis C antibody and liver and heart iron deposition assessed by MRI T2*. Data were obtained from the medical records of 156 thalassemia major patients in Tehran. There was a moderate negative correlation between serum ferritin and liver MRI T2* relaxation time (r = -0.535) and a weak negative correlation between serum ferritin and heart MRI T2* relaxation time (r = -0.361). Hepatitis C infection and liver enzyme levels did not confound or modify the relation between ferritin and liver or heart MRI T2*. Liver and heart MRI T2* readings were poorly correlated (r = 0.281). Routine evaluation of liver and heart iron content using MRI T2* is suggested to better evaluate the haemosiderosis status in thalassemia patients.

A Long Acting Polymeric Iron Chelator for the Treatment of Transfusion Associated Iron Overload

Abstract 486 IntroductionTransfusion dependent patients with abnormal hemoglobins or bone marrow failure syndromes require treatment to prevent and reverse iron overload. Desferrioxamine (DFO) mesylate is effective in promoting iron excretion. However, its usefulness is limited by a short intravascular half-life (t1/2) which necessitates continuous 8–12 hr subcutaneous infusion, often leading to limited patient compliance. Using hyperbranched polyglycerol (HPG), we have generated DFO conjugates which have significantly prolonged t1/2, good tolerance and improved iron excretion efficiency in mice. MethodsHPGs with molecular weight (MW) of 50 to 700 kDa were conjugated to varying numbers of DFO molecules (25–400) using Schiff-base chemistry. The influence of HPG MW and DFO density on DFO iron binding was determined using UV-visible spectroscopy. The safety of HPG-DFO was evaluated by examining activated partial thromboplastin time (APTT), prothrombin time (PT), thromboelastography (TEG), platelet activation and complement activation. The effect of HPG-DFO on cell viability was evaluated in human umbilical vein endothelial cells (HUVECs). Single dose tolerability in mice was studied by injecting up to 1000 mg/kg of HPG-DFO and monitoring body weight, lactate dehydrogenase level and histological examination of organs at 14 days. Tritium labeled HPG-DFO was used to determine the circulation t1/2 in mice. Iron excretion efficacy of HPG-DFO compared to DFO was tested in iron overloaded mice by treating the animals with 150 mg/kg DFO or DFO equivalent. The iron content of organs, urine and feces was analyzed to determine the amount of iron excreted. Histological examination of the organs was performed using the Prussian blue stain. ResultsSpectroscopic analysis demonstrated that the conjugation of DFO to HPGs did not change their iron binding properties; the maximum absorption was at 429 nm for every conjugate tested. HPG-DFOs did not activate platelets, coagulation or the complement system. In vitro cell tolerance studies showed there was no increase in toxicity compared to the DFO molecule after polymer conjugation. Mice tolerance studies showed that the HPG-DFO conjugates were non toxic up to the maximum injected dose of 1000 mg/kg. The t1/2 of HPG-DFO in normal mice was 16 h for the 50 kDa conjugate and 44 h for the 500 kDa conjugate. The iron excretion efficiency as measured by urinary iron of the 50kDa HPG-DFO in iron overloaded showed significant improvement compared to placebo (58 Ìg vs 19 Ìg, p=0.0002) and DFO treated mice (58 Ìg vs 30 Ìg, p=0.0011 Figure 1). ConclusionsHPG-DFO conjugate chelators were well tolerated in mice with significantly improved t1/2 and iron excretion. This approach has the potential to significantly decrease the number and duration of injections required to offload significant amounts of organ and total body iron. Further studies to determine the optimal conjugate and dose of agent are currently ongoing. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Hepatic Iron Deposition Does Not Predict Extrahepatic Iron Loading in Mouse Models of Hereditary Hemochromatosis

Hereditary hemochromatosis is characterized by tissue iron loading and associated organ damage. However, the phenotype can be highly variable. The relationship between iron loading of different organs and the temporal nature of its deposition is still not well understood. We examined the progression of tissue iron loading in three mouse models to advance our understanding of the natural history of iron deposition in hereditary hemochromatosis. Wild-type, Hfe(-/-), Tfr2(-/-), and Hfe(-/-)/Tfr2(-/-) mice were analyzed at 3, 5, 10, 26, and 52 weeks, respectively. Hepatic, splenic, cardiac, and pancreatic iron concentrations were determined. Expression of both iron-regulatory and fibrosis genes was determined by quantitative real-time PCR in livers and hearts of 52-week-old mice. In all models, hepatic iron increased rapidly, plateauing before 10 weeks at different levels, depending on the genotype. Iron deposition in the pancreas and heart occurred after maximal iron loading of the liver was reached and was most marked in the Hfe(-/-)/Tfr2(-/-) mice. Although a significant positive correlation was identified between pancreatic and cardiac iron in all models at 52 weeks, there was no correlation between hepatic and either pancreatic or cardiac iron. There is variability in the timing and extent of tissue iron loading within a genotype, suggesting that hepatic iron levels in hereditary hemochromatosis may not accurately predict the iron content of other organs.

Effects of Oral Hyposensitization Therapy with Nickel on Haemoglobin and Haematocrit Values in Sensitized Patients

Nickel deficiency leads to reduced iron content in organs and to reduced hemoglobin levels and haematocrit counts. We carried out a clinical trial of oral hyposensitization therapy with low doses of nickel on a group of 20 patients affected by systemic allergy to this sensitizer element and with a chronic relapsing low hemoglobin level and haematocrit count. We obtained interesting results on maintaining these values in treated patients.

Macrophage Depletion Leads to Modification of Thalassemic Phenotype.

Abstract 2023Poster Board I-1045Macrophages represent an important link between erythropoiesis and iron metabolism. They support maturation and differentiation of erythroblasts and clear senescent red cells from circulation, thus recycling iron within the hematopoietic system. However, little is known about the role of macrophages in conditions of ineffective erythropoiesis like beta-thalassemia intermedia (β-TI) or sickle cell anemia (SCD). Our goal was to study the role of macrophages under such conditions, where there is a large expansion of the erythron, the lifespan of the red cells is decreased, and both iron absorption and organ iron content are increased.Clodronate liposomes have been shown to efficiently eliminate macrophages from spleen, liver and bone marrow when administered by intravenous injection. Therefore, we utilized this technique to eliminate macrophages from the spleen, liver and bone marrow of β-TI and SCD mice. Three different treatment schedules were used; a) acute high dose; b) medium term split dose; c) chronic low dose. Animals were analyzed for erythropoietic and pathological parameters.All treatments effectively depleted splenic and bone marrow macrophages as shown by flow citometric and immunohistochemical analyses. At doses that led to only a small decrease of hemoglobin (Hb) levels in normal mice, clodronate treatment induced high level of mortality in β-TI and SCD mice (70% and 75%, respectively). In contrast, administration of clodronate to mice affected by beta-thalassemia major, mice that do not make any adult or fetal Hb/mature red cells, caused no mortality suggesting that circulating abnormal red cells might be responsible for the mortality observed in β-TI and SCD mice. Pathological analysis of β-TI mice revealed the formation of thrombi in the lungs and heart to be the likely cause of death. Analysis of SCD mice is in progress. β-TI and SCD have been described as hypercoagulable states with a high risk of thrombosis. Damaged red cell membranes and ROS have been implicated as factors that increase the risk of thrombosis in β-TI and SCD patients. We hypothesize that clodronate treatment enhances this process. To test this hypothesis, we administered an anti-coagulant (heparin) and an anti-oxidant (NAC) to β-TI mice. Both these treatments resulted in increased survival from 31% to 65% compared to clodronate alone. Analysis of SCD animals is in progress. β-TI mice that survived clodronate treatment exhibited a milder thalassemic phenotype, characterized by increased Hb (∼1.5g/dL), HCT (∼7%) and RBC count (∼2e6 cells/ul), partially associated with a decreased RBC clearance. Ineffective erythropoiesis and splenomegaly were also ameliorated (∼40% decrease in spleen size). Animals depleted of macrophages also showed a marked decrease in serum iron parameters, but no difference in their organ iron concentrations. Analysis of hepcidin expression is in progress. To further corroborate these observations, β-TI mice are being crossed with mice expressing the human diphtheria toxin (CD11b-DTR) which allows for conditional depletion of macrophages.Our data suggests that macrophages play a major role in conditions of abnormal red cell production and ineffective erythropoiesis. In the former case, they have an important protective function related to the high risk of thrombosis associated with β-TI and SCD. In the latter, macrophages may act as negative modulators of erythropoietic development. The amelioration of splenomegaly and ineffective erythropoiesis observed suggests that erythroid cells might proliferate less and/or differentiate more in the absence of macrophages. One hypothesis is that macrophages sense erythroid maturation and/or differentiation and act as modulators of these processes to ensure the release of high quality red cells into the circulation. Second, macrophages, under conditions of iron overload, might produce inflammatory cytokines that further impair erythropoiesis in thalassemia. Analysis of RNA extracted from macrophages and of inflammatory cytokines in β-TI mice before and after clodronate treatment is in progress. Disclosures:No relevant conflicts of interest to declare.

Variable‐field relaxometry of iron‐containing human tissues: a preliminary study

Excess iron is found in brain nuclei from neurodegenerative patients (with Parkinson's, Alzheimer's and Huntington's diseases) and also in the liver and spleen of cirrhosis, hemochromatosis and thalassaemia patients. Ferritin, the iron-storing protein of mammals, is known to darken T(2)-weighted MR images. Understanding NMR tissue behavior may make it possible to detect those diseases, to follow their evolution and finally to establish a protocol for non-invasive measurement of an organ's iron content using MRI methods. In this preliminary work, the MR relaxation properties of embalmed iron-containing tissues were studied as well as their potential correlation with the iron content of these tissues. Relaxometric measurements (T(1) and T(2)) of embalmed samples of brain nuclei (caudate nucleus, dentate nucleus, globus pallidus, putamen, red nucleus and substantia nigra), liver and spleen from six donors were made at different magnetic fields (0.00023-14 T). The influence of the inter-echo time on transverse relaxation was also studied. Moreover, iron content of tissues was determined by inductively coupled plasma atomic emission spectroscopy. In brain nuclei, 1/T(2) increases quadratically with the field and depends on the inter-echo time in CPMG sequences at high fields, both features compatible with an outer sphere relaxation theory. In liver and spleen, 1/T(2) increases linearly with the field and depends on the inter-echo time at all fields. In our study, a correlation between 1/T(2) and iron concentration is observed. Explaining the relaxation mechanism for these tissues is likely to require a combination of several models. The value of 1/T(2) at high field could be used to evaluate iron accumulation in vivo. In the future, confirmation of those features is expected to be achieved from measurements of fresh (not embalmed) human tissues.

Hepcidin-Independent Regulation of Dietary Iron Absorption

Iron homeostasis in mammals is maintained at the level of iron absorption by the gut. Hepcidin plays a central role in homeostasis by binding to ferroportin and regulating cellular iron export. We found that mice weaned onto diets ranging from 35–350 mg Fe/kg for a period of 4 wk did not change body iron levels as measured by organ iron content and hematological parameters. Direct measure of absorption of 59Fe administered by gavage revealed an inverse correlation between dietary iron content and absorption. Gavage experiments were done following a 4h fast when the stomach and proximal small bowel were free of dietary content. Although iron absorption changed, liver expression of hepcidin mRNA did not. We measured the absorptive response in mice weaned onto diets containing 35 mg Fe/kg for 4 wk and abruptly changed to 350 mg Fe/kg. There was no change in iron absorption at day 1 but by day 3 absorption was reduced nearly 3-fold compared to controls and remained at this level for at least 7d. During this time neither liver nor spleen iron content changed but transferrin saturation increased approximately 1.5-fold. Most importantly, serum hepcidin levels, measured by a competitive binding assay (De Domenico et al. Cell Metab. 2008, 8:146–156), were unchanged. Mice were then changed from diets containing 350 mg Fe/kg to diets containing 35 mg Fe/kg. Within 24h mice increased absorption of 59Fe 3-fold. Elevated absorption continued for at least 3d, declined by 7d and at 14d was at a level found in mice maintained on a diet containing 35 mg Fe/kg. During this period there was no change in organ iron content or in transferrin saturation. Serum hepcidin did not change on day 1, but was reduced by approximately 40% on days 3 through 7. Increased iron absorption could be attributed in part to increased expression of Dmt1 but no change in ferroportin message was detected. Enterocyte ferritin levels doubled on day 1 but returned to control levels on days 3 through 7. Finally, mice with a targeted disruption of the hepcidin gene were challenged with an abrupt reduction in dietary iron content and absorption increased approximately 3-fold. These data suggest that iron absorption can respond to changes in dietary iron content independent of hepcidin and that response to changes in luminal iron content are at the level of uptake and storage in a manner intrinsic to the enterocyte.

Absence of the Hemochromatosis Gene HFE Confers Protection Under Conditions of Stress Erythropoiesis.

A single mutation in the HFE gene (C282Y), a non-classical member of the MHC-I family, may lead to hereditary hemochromatosis (HH). Although the hallmark of HH is iron overload, several lines of evidence point to a distinct hematopoietic function for HFE:HH reticuloendothelial cells are iron deficient;reconstitution of Hfe-KO mice with bone marrow (BM) from wild-type (wt) animals leads to organ iron redistribution;erythropoietic abnormalities have been reported in HH patients, including altered red cell parameters, which have been associated with iron overload.In particular, however, the HFE promoter contains several GATA.1 responsive elements suggesting that it might be expressed in erythroid cells. Our goal was to characterize erythropoiesis in a mouse model of HH, discriminating between intrinsic roles for Hfe and iron overload in these process. Various parameters in Hfe-KO mice were compared to those in wt controls. Atomic absorption was used to measure organ iron content. Erythropoietic analysis was performed by complete blood cell counts and flow cytometry at 2, 5 and 12 months of life (N≥6 mice per group). Erythropoiesis was stressed by administration of phenylhydrazine, phlebotomy (N≥8 mice per group) or elimination of macrophages using clodronate liposomes (N≥4 mice per group). Hematological parameters were followed over time. Finally, lentiviral vectors were used to overexpress Hfe in the BM or liver of Hfe-KO animals (N≥3 mice per group), erythropoietic values being analyzed as described above.Our results indicate increased erythropoiesis at steady state in Hfe-KO animals, characterized by altered red blood cell (RBC) parameters. Analysis of erythroid populations in BM and spleen revealed an increase in the proportion of immature erythroid cells in these compartments of Hfe-KO mice, especially the spleen (9.4% ± 1.8% vs 5.1% ± 1.2% in wt mice, p≤0.05). Compared to normal mice, Hfe-KO mice recovered faster from anemia or hemolytic anemia induced, respectively, by phlebotomy and administration of phenylhydrazine. By day 8 their hemoglobin and hematocrit levels returned to steady state, while normalization required 10 days in wt mice. Preliminary results showed similar behavior in wt mice transplanted with Hfe-KO BM, compared to mice transplanted with wt BM. Since macrophages play an important role in erythropoiesis, we analyzed the erythropoietic behavior of wt and Hfe-KO mice after elimination of macrophages. Anemia induced by macrophage depletion was milder in Hfe-KO mice compared to wt mice (14.0 ± 0.4 g/dL vs 11.5 ± 0.1 g/dL), and characterized by a reduction in the hemoglobin content of RBC (MCH), with little change in the RBC count compared to steady state. In contrast anemia in wt mice was characterized by a reduction in both RBC the count and the MCH. To further investigate the correlation between Hfe, erythropoiesis and the potential role of iron overload in the erythropoietic alterations observed, we generated several lentiviral vectors to express Hfe in specific compartments, including the BM and liver. High levels of Hfe expression in BM led to a lethal anemia. On the other hand, low/normal level of expression led to reversion of the alterations in erythropoiesis at steady state, with no alteration in iron content or distribution compared to Hfe-KO control animals. Lentiviral Hfe gene transfer into the liver of Hfe-KO mice led to reduction of iron in the liver and some redistribution of this metal to the spleen. These mice showed the same erythropoietic alterations as Hfe-KO controls.

The Effect of Dietary Iron on Tissue Iron Levels in Intact and Splenectomized Mice Affected by β-Thalassemia.

Patients with β-thalassemia hyper absorb dietary iron, most of which is stored in the liver. They also suffer from ineffective erythropoiesis (IE) which leads to hepatosplenomegaly, often requiring a splenectomy. We have been conducting a series of studies utilizing the th3/+ mouse model of thalassemia intermedia to investigate the absorption, distribution and erythroid utilization of iron. Here we focus on changes in the iron content of liver and spleen resulting from diets containing low (2.5 ppm), sufficient (35 ppm) and high (200 ppm) levels of iron, and assess the impact of splenectomy on its distribution. The high iron diet was standard rodent chow while the others were defined diets. Th3/+ mice were either bred or generated by transplantation of th3/+ hematopoietic stem cells from E14.5 fetal livers into lethally irradiated wild type (+/+) recipients. Wild type controls were similarly obtained. Splenectomy of bred and recipient mice was performed at 5 weeks of age and bone marrow transplantation (BMT) at 8 weeks. Non-transplanted mice were placed on the test diets at 8 weeks of age, and transplanted mice at 11 weeks. All animals were sacrificed after 4 weeks on the test diets, and livers and spleens harvested for determination of their iron content by atomic absorption. Group sizes ranged from 3 to 10 mice (median 7). In general, the mean organ iron content of mice fed the high iron diet was not significantly different from that of the animals fed the iron sufficient diet, while those fed the low iron diet had reduced levels of tissue iron. Over the course of the 4-week feeding study, the iron content of the livers and spleens of +/+ mice fed the 35-ppm diet increased 39% and 202%, respectively, while the corresponding values of those fed the 2.5-ppm diet were −21% and 30%. The changes in the liver and spleen of th3/+ mice were 79% and 32% (35-ppm diet) and 14% and 12% (2.5-ppm diet) compared to the values at baseline. The latter values, those at 8 weeks of age, were 1.8- and 30-fold higher in the th3/+ mice, the massive accumulation of iron in the spleen undoubtedly resulting from IE. Where iron intake (liver plus spleen) was low, it went preferentially to the spleen, undoubtedly to sustain erythropoiesis. Groups of splenectomized +/+ mice were also fed the three diets for 4 weeks. The mean iron content of their livers was similar to that of non-splenectomized animals. Similar studies of th3/+ mice are now in progress. A second set of studies is being conducted in transplanted +/+ and th3/+ mice, the goal being to determine whether or not the absorption and distribution of iron is the same as in bred animals. Again, the organ iron content of those mice fed the high iron diet was similar to that of the animals fed the iron sufficient diet. In the case of the transplanted +/+ animals fed iron sufficient diets, the mean iron contents of the livers and spleens were 64% and 186% increased after 4 weeks of feeding, values not markedly different from those of bred animals. The corresponding values on the 2.5-ppm diet were 27% and 72%, again the pattern being similar. The transplanted th3/+ animals accumulated significantly less iron in these organs than those that were bred. However, the rate at which they accumulated this iron was 10 to 20 times higher than that of the other groups studied, including the transplanted +/+ mice, perhaps reflecting a synergistic effect of BMT and IE on iron absorption. Mice fed the 35-ppm diet had only 75% and 46% as much iron in their livers and spleens, the animals fed the 2.5-ppm diet having even less (35% and 23%) while again showing preferential diversion of iron to the spleen. Splenectomizing the animals resulted in further increasing the liver iron, more that 2.5-fold in those fed the low iron diet. The hemoglobin levels of all the mice evaluated were unchanged as a result of the dietary studies, except for a 20% decrease seen in bred +/+ mice fed the low iron diet. We are currently studying splenectomized transplanted th3/+ mice as well as doing feeding studies of 5-months duration. In summary, a low iron diet has a marked effect on the iron levels of liver and spleen, which are accentuated under conditions of IE. Secondly, more iron is absorbed under conditions of IE than is needed for erythropoiesis, the excess being shuttled to the liver for storage.

Synthesis, characterization and MRI application of dextran-coated Fe3O4 magnetic nanoparticles

Biocompatible ferrofluid based on dextran-coated Fe3O4 magnetic nanoparticles (MNPs) was prepared through a one-step method. In contrast to the conventional co-precipitation method, hydrazine hydrate was added as reducing agent and precipitator in the present investigation. The effects of hydrazine hydrate, the weight ratio of dextran to MNPs and the molecular weight of dextran on the dispersibility of MNPs in water were investigated. Also, the particles size of modified MNP and coating efficiency of dextran on MNPs were measured. In addition, biocompatible ferrofluid was intravenously injected into rabbits, the iron content in blood and organs at different times were measured by atomic absorption spectrometer, and the bio-distribution and the bio-transportation of ferrofluid in organs was examined. Then, the magnetic resonance (MR) images of liver, marrow and lymph were acquired by MRI experiments before and after intravenous injection of ferrofluid. Image analysis revealed that the MR signal intensity of these organs notably decreased after intensified by ferrofluid. However, when there existed tumors in organs, the signal intensity of tumor did not change after injection. From that the tumor can easily be identified, which indicated a potential application of the as-prepared MNP in functional molecular imaging for biomedical research and clinical diagnosis.

Relaxation by clustered ferritin: a model for ferritin‐induced relaxation in vivo

Ferritin, the iron-storing protein of mammals, is known to darken T(2)-weighted MR images. This darkening could be used for the non-invasive measurement of an organ's iron content. Unexplained discrepancies exist between T(2) data obtained in ferritin-containing tissues and aqueous solutions of ferritin. The clustering of the protein induced by trypsin is used to evaluate the effect of ferritin agglomeration on the relaxation rates. Although the longitudinal relaxation is not significantly influenced by clustering, T(2) depends greatly on the stage of agglomeration: the transverse relaxation rate is higher for a clustered sample than for an unclustered sample. Moreover, the field and inter-echo time dependences of the relaxation rate indicate that the relaxation mechanism may be different between small clusters -- where a linear dependence of 1/T(2) on B(0) is observed -- and large clusters -- where a quadratic dependence is observed. These results help to explain the relaxation induced by ferritin in tissues.