Hemochromatosis classification: update and recommendations by the BIOIRON Society.

Hemochromatosis (HC) is a genetically heterogeneous disorder in which uncontrolled intestinal iron absorption may lead to progressive iron overload (IO) responsible for disabling and life-threatening complications such as arthritis, diabetes, heart failure, hepatic cirrhosis, and hepatocellular carcinoma. The recent advances in the knowledge of pathophysiology and molecular basis of iron metabolism have highlighted that HC is caused by mutations in at least 5 genes, resulting in insufficient hepcidin production or, rarely, resistance to hepcidin action. This has led to an HC classification based on different molecular subtypes, mainly reflecting successive gene discovery. This scheme was difficult to adopt in clinical practice and therefore needs revision. Here we present recommendations for unambiguous HC classification developed by a working group of the International Society for the Study of Iron in Biology and Medicine (BIOIRON Society), including both clinicians and basic scientists during a meeting in Heidelberg, Germany. We propose to deemphasize the use of the molecular subtype criteria in favor of a classification addressing both clinical issues and molecular complexity. Ferroportin disease (former type 4a) has been excluded because of its distinct phenotype. The novel classification aims to be of practical help whenever a detailed molecular characterization of HC is not readily available.

Reticulocyte Antioxidant Enzymes mRNA Levels versus Reticulocyte Maturity Indices in Hereditary Spherocytosis, ß-Thalassemia and Sickle Cell Disease.

The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and peroxiredoxin 2 (Prx2) are particularly important in erythroid cells. Reticulocytes and other erythroid precursors may adapt their biosynthetic mechanisms to cell defects or to changes in the bone marrow environment. Our aim was to perform a comparative study of the mRNA levels of CAT, GPX1, PRDX2 and SOD1 in reticulocytes from healthy individuals and from patients with hereditary spherocytosis (HS), sickle cell disease (SCD) and ß-thalassemia (ß-thal), and to study the association between their transcript levels and the reticulocyte maturity indices. In controls, the enzyme mRNA levels were significantly correlated with reticulocyte maturity indices for all genes except for SOD1. HS, SCD and ß-thal patients showed younger reticulocytes, with higher transcript levels of all enzymes, although with different patterns. ß-thal and HS showed similar reticulocyte maturity, with different enzyme mRNA levels; SCD and HS, with different reticulocyte maturity, presented similar enzyme mRNA levels. Our data suggest that the transcript profile for these antioxidant enzymes is not entirely related to reticulocyte maturity; it appears to also reflect adaptive mechanisms to abnormal erythropoiesis and/or to altered erythropoietic environments, leading to reticulocytes with distinct antioxidant potential according to each anemia.

Haemochromatosis patients' research priorities: Towards an improved quality of life.

BACKGROUND: Chronic diseases are associated with a range of functional and psychosocial consequences that can adversely affect patients' quality of life (QoL). Haemochromatosis (HC) is a genetically heterogeneous disorder characterized by chronic iron overload that can ultimately lead to multiple organ dysfunction. Clinical diagnosis remains challenging due to the nonspecificity of symptoms and a lack of confirmatory genotyping in a substantial proportion of patients. Illness perception among HC patients has not been extensively investigated, lacking relevant information on how to improve their QoL. METHODS: We present the results of the first worldwide survey conducted in nearly 1500 HC respondents, in which we collected essential demographic information and identified the aspects that concern HC patients the most. RESULTS: Out of all the participants, 45.3% (n = 676) voiced their concern about physical and psychological consequences such as HC-related arthropathies, which can ultimately affect their social functioning. A similar proportion of patients (n = 635, 42.5%) also consider that better-informed doctors are key for improved HC disease management. Taking a patient-centred approach, we expose differences in patients' disease perspective by social and economic influences. CONCLUSIONS: We identify potential targets to improve patients' health-related QoL and reflect on strategic measures to foster gender equity in access to health resources. Finally, we make a call for a highly coordinated effort across a range of public policy areas to empower participants in the HC research process and design. PATIENT OR PUBLIC CONTRIBUTION: Nearly 1500 patients with hereditary HC responded to an anonymized online survey in which research and clinical priorities were addressed regarding this chronic and rare disease.

The effect of a natural polyphenol supplement on iron absorption in adults with hereditary hemochromatosis.

OBJECTIVES: We developed a natural polyphenol supplement that strongly chelates iron in vitro and assessed its effect on non-heme iron absorption in patients with hereditary hemochromatosis (HH). METHODS: We performed in vitro iron digestion experiments to determine iron precipitation by 12 polyphenol-rich dietary sources, and formulated a polyphenol supplement (PPS) containing black tea powder, cocoa powder and grape juice extract. In a multi-center, single-blind, placebo-controlled cross-over study, we assessed the effect of the PPS on iron absorption from an extrinsically labelled test meal and test drink in patients (n = 14) with HH homozygous for the p.C282Y variant in the HFE gene. We measured fractional iron absorption (FIA) as stable iron isotope incorporation into erythrocytes. RESULTS: Black tea powder, cocoa powder and grape juice extract most effectively precipitated iron in vitro. A PPS mixture of these three extracts precipitated ~ 80% of iron when 2 g was added to a 500 g iron solution containing 20 µg Fe/g. In the iron absorption study, the PPS reduced FIA by ~ 40%: FIA from the meal consumed with the PPS was lower (3.01% (1.60, 5.64)) than with placebo (5.21% (3.92, 6.92)) (p = 0.026)), and FIA from the test drink with the PPS was lower (10.3% (7.29 14.6)) than with placebo (16.9% (12.8 22.2)) (p = 0.002). CONCLUSION: Our results indicate that when taken with meals, this natural PPS can decrease dietary iron absorption, and might thereby reduce body iron accumulation and the frequency of phlebotomy in patients with HH. TRIAL REGISTRY: clinicaltrials.gov (registration date: 9.6.2019, NCT03990181).

Atherosclerosis is aggravated by iron overload and ameliorated by dietary and pharmacological iron restriction.

AIMS: Whether and how iron affects the progression of atherosclerosis remains highly debated. Here, we investigate susceptibility to atherosclerosis in a mouse model (ApoE-/- FPNwt/C326S), which develops the disease in the context of elevated non-transferrin bound serum iron (NTBI). METHODS AND RESULTS: Compared with normo-ferremic ApoE-/- mice, atherosclerosis is profoundly aggravated in iron-loaded ApoE-/- FPNwt/C326S mice, suggesting a pro-atherogenic role for iron. Iron heavily deposits in the arterial media layer, which correlates with plaque formation, vascular oxidative stress and dysfunction. Atherosclerosis is exacerbated by iron-triggered lipid profile alterations, vascular permeabilization, sustained endothelial activation, elevated pro-atherogenic inflammatory mediators, and reduced nitric oxide availability. NTBI causes iron overload, induces reactive oxygen species production and apoptosis in cultured vascular cells, and stimulates massive MCP-1-mediated monocyte recruitment, well-established mechanisms contributing to atherosclerosis. NTBI-mediated toxicity is prevented by transferrin- or chelator-mediated iron scavenging. Consistently, a low-iron diet and iron chelation therapy strongly improved the course of the disease in ApoE-/- FPNwt/C326S mice. Our results are corroborated by analyses of serum samples of haemochromatosis patients, which show an inverse correlation between the degree of iron depletion and hallmarks of endothelial dysfunction and inflammation. CONCLUSION: Our data demonstrate that NTBI-triggered iron overload aggravates atherosclerosis and unravel a causal link between NTBI and the progression of atherosclerotic lesions. Our findings support clinical applications of iron restriction in iron-loaded individuals to counteract iron-aggravated vascular dysfunction and atherosclerosis.

Chromosome 6p SNP microhaplotypes and IgG3 levels in hemochromatosis probands with HFE p.C282Y homozygosity.

Subnormal IgG1 or IgG3 levels occurred in 30% of hemochromatosis probands with HFE p.C282Y homozygosity and were concordant in HLA-identical siblings. We sought to identify factors associated with IgG subclasses in Alabama probands with p.C282Y homozygosity evaluated for 500 kb microhaplotypes AAT and GGG defined by SNPs in chromosome 6p genes PGBD1, ZNF193, and ZNF165. In regressions on IgG subclasses, we used: age; sex; GGG (dichotomous); iron removed to achieve depletion; CD8+ T-lymphocytes; and other IgG subclasses. Among 49 probands, AAT and GGG occurred in 95.9% and 16.3%, respectively. Thirteen probands (26.5%) had subnormal IgG1; 11 probands (22.4%) had subnormal IgG3. Mean IgG3 was higher in probands with than without GGG (75 mg/dL [95% confidence interval 63, 89] vs. 58 mg/dL [49, 71], respectively; p = 0.0321). Regression on IgG3 revealed: GGG positivity (p = 0.0106); and IgG1 (p = 0.0015). In a replication cohort of 22 Portugal probands with p.C282Y homozygosity, mean IgG3 was higher in probands with than without GGG (46 ±â€¯16 vs. 31 ±â€¯12 mg/dL, respectively; p = 0.0410). We conclude that mean IgG3 levels are higher in hemochromatosis probands with p.C282Y homozygosity with chromosome 6p microhaplotype GGG than in probands homozygous for microhaplotype AAT.

Local iron homeostasis in the breast ductal carcinoma microenvironment.

BACKGROUND: While the deregulation of iron homeostasis in breast epithelial cells is acknowledged, iron-related alterations in stromal inflammatory cells from the tumor microenvironment have not been explored. METHODS: Immunohistochemistry for hepcidin, ferroportin 1 (FPN1), transferrin receptor 1 (TFR1) and ferritin (FT) was performed in primary breast tissues and axillary lymph nodes in order to dissect the iron-profiles of epithelial cells, lymphocytes and macrophages. Furthermore, breast carcinoma core biopsies frozen in optimum cutting temperature (OCT) compound were subjected to imaging flow cytometry to confirm FPN1 expression in the cell types previously evaluated and determine its cellular localization. RESULTS: We confirm previous results by showing that breast cancer epithelial cells present an 'iron-utilization phenotype' with an increased expression of hepcidin and TFR1, and decreased expression of FT. On the other hand, lymphocytes and macrophages infiltrating primary tumors and from metastized lymph nodes display an 'iron-donor' phenotype, with increased expression of FPN1 and FT, concomitant with an activation profile reflected by a higher expression of TFR1 and hepcidin. A higher percentage of breast carcinomas, compared to control mastectomy samples, present iron accumulation in stromal inflammatory cells, suggesting that these cells may constitute an effective tissue iron reservoir. Additionally, not only the deregulated expression of iron-related proteins in epithelial cells, but also on lymphocytes and macrophages, are associated with clinicopathological markers of breast cancer poor prognosis, such as negative hormone receptor status and tumor size. CONCLUSIONS: The present results reinforce the importance of analyzing the tumor microenvironment in breast cancer, extending the contribution of immune cells to local iron homeostasis in the tumor microenvironment context.

Polyacrylic acid-coated and non-coated iron oxide nanoparticles induce cytokine activation in human blood cells through TAK1, p38 MAPK and JNK pro-inflammatory pathways.

Iron oxide nanoparticles (ION) can have a wide scope of applications in biomedicine, namely in magnetic resonance imaging, tissue repair, drug delivery, hyperthermia, transfection, tissue soldering, and as antimicrobial agents. The safety of these nanoparticles, however, is not completely established, namely concerning their effect on immune system and inflammatory pathways. The aim of this study was to evaluate the in vitro effect of polyacrylic acid (PAA)-coated ION and non-coated ION on the production of six cytokines [interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 8 (IL-8), interferon gamma (IFN-γ) and interleukin 10 (IL-10)] by human peripheral blood cells, and to determine the inflammatory pathways involved in this production. The obtained results showed that PAA-coated and non-coated ION were able to induce all the tested cytokines and that activation of transforming growth factor beta (TGF-ß)-activated kinase (TAK1), p38 mitogen-activated protein kinases (p38 MAPK) and c-Jun N-terminal kinases (JNK) were involved in this effect.

Strengthening the perception-assessment tools for dengue prevention: a cross-sectional survey in a temperate region (Madeira, Portugal).

BACKGROUND: Community participation is mandatory in the prevention of Dengue outbreaks. Taking public views into account is crucial to guide more effective planning and quicker community participation in preventing campaigns. This study aims to assess community perceptions of Madeira population in order to explore their involvement in the A. aegypti's control and reinforce health-educational planning. Due to the lack of accurate methodologies for measuring perception, a new tool to assess the community's perceptions was built. METHODS: A cross-sectional survey was performed in the Island's aegypti-infested area, exploring residents' perceptions regarding most critical community behaviour: aegypti-source reduction and their domestic aegypti-breeding sites. A novel tool defining five essential topics which underlie the source reduction's awareness and accession was built, herein called Essential-Perception (EP) analysis. RESULTS: Of 1276 individuals, 1182 completed the questionnaire (92 · 6%). EP-Score analysis revealed that community's perceptions were scarce, inconsistent and possibly incorrect. Most of the population (99 · 6%) did not completely understood the five essential topics explored. An average of 54 · 2% of residents only partially understood each essential topic, revealing inconsistencies in their understanding. Each resident apparently believed in an average of four false assumptions/myths. Significant association (p<0.001) was found between both the EP-Score level and the domestic presence of breeding sites, supporting the validity of this EP-analysis. Aedes aegypti's breeding sites, consisting of décor/leisure containers, presented an atypical pattern of infestation comparing with dengue prone regions. CONCLUSIONS: The studied population was not prepared for being fully engaged in dengue prevention. Evidences suggest that EP-methodology was efficient and accurate in assessing the community perception and its compliance to practices. Moreover, it suggested a list of myths that could persist in the community. This is the first study reporting an aegypti-entomological pattern and community's perception in a developed dengue-prone region. Tailored messages considering findings of this study are recommended to be used in future campaigns in order to more effectively impact the community perception and behaviour.

Insufficient referral for genetic counseling in the management of hereditary haemochromatosis in portugal: a study of perceptions of health professionals requesting HFE genotyping.

There is a general consensus that HFE- related Hereditary Haemochromatosis (HFE-HH) should be diagnosed at early stages in pre-symptomatic individuals, in order to prevent the most severe consequences of iron overload. In Portugal, despite an increasing number of requests for genetic diagnosis of this rare disease, there is not a corresponding increase in requests for genetic counselling. The objective of the present study was to evaluate physicians' main motivations for requesting HFE genotyping or genetic counselling for HFE-HH. We assessed current medical practices regarding family testing and diagnosis and discuss whether these can be improved in order to increase the effectiveness of disease prevention. Our results show there is a general lack of knowledge about the selection of patient cases that should be sent for genetic counseling or for molecular testing of HFE-HH by physicians (especially by general practitioners). The lack of family-based screening may indirectly compromise the efficiency of disease prevention in terms of early diagnosis and treatment. We concluded it is necessary to circulate more information about Hereditary Haemochromatosis among health professionals in order to improve strategies for its early diagnosis.

Catalase, Glutathione Peroxidase, and Peroxiredoxin 2 in Erythrocyte Cytosol and Membrane in Hereditary Spherocytosis, Sickle Cell Disease, and ß-Thalassemia.

Catalase (CAT), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2) can counteract the deleterious effects of oxidative stress (OS). Their binding to the red blood cell (RBC) membrane has been reported in non-immune hemolytic anemias (NIHAs). Our aim was to evaluate the relationships between CAT, GPx, and Prx2, focusing on their role at the RBC membrane, in hereditary spherocytosis (HS), sickle cell disease (SCD), ß-thalassemia (ß-thal), and healthy individuals. The studies were performed in plasma and in the RBC cytosol and membrane, evaluating OS biomarkers and the enzymatic activities and/or the amounts of CAT, GPx, and Prx2. The binding of the enzymes to the membrane appears to be the primary protective mechanism against oxidative membrane injuries in healthy RBCs. In HS (unsplenectomized) and ß-thal, translocation from the cytosol to the membrane of CAT and Prx2, respectively, was observed, probably to counteract lipid peroxidation. RBCs from splenectomized HS patients showed the highest membrane-bound hemoglobin, CAT, and GPx amounts in the membrane. SCD patients presented the lowest amount of enzyme linkage, possibly due to structural changes induced by sickle hemoglobin. The OS-induced changes and antioxidant response were different between the studied NIHAs and may contribute to the different clinical patterns in these patients.

Iron overload induces dysplastic erythropoiesis and features of myelodysplasia in Nrf2-deficient mice.

Iron overload (IOL) is hypothesized to contribute to dysplastic erythropoiesis. Several conditions, including myelodysplastic syndrome, thalassemia and sickle cell anemia, are characterized by ineffective erythropoiesis and IOL. Iron is pro-oxidant and may participate in the pathophysiology of these conditions by increasing genomic instability and altering the microenvironment. There is, however, lack of in vivo evidence demonstrating a role of IOL and oxidative damage in dysplastic erythropoiesis. NRF2 transcription factor is the master regulator of antioxidant defenses, playing a crucial role in the cellular response to IOL in the liver. Here, we crossed Nrf2-/- with hemochromatosis (Hfe-/-) or hepcidin-null (Hamp1-/-) mice. Double-knockout mice developed features of ineffective erythropoiesis and myelodysplasia including macrocytic anemia, splenomegaly, and accumulation of immature dysplastic bone marrow (BM) cells. BM cells from Nrf2/Hamp1-/- mice showed increased in vitro clonogenic potential and, upon serial transplantation, recipients disclosed cytopenias, despite normal engraftment, suggesting defective differentiation. Unstimulated karyotype analysis showed increased chromosome instability and aneuploidy in Nrf2/Hamp1-/- BM cells. In HFE-related hemochromatosis patients, NRF2 promoter SNP rs35652124 genotype TT (predicted to decrease NRF2 expression) associated with increased MCV, consistent with erythroid dysplasia. Our results suggest that IOL induces ineffective erythropoiesis and dysplastic hematologic features through oxidative damage in Nrf2-deficient cells.

Protective effect of an ERAP1 haplotype in ankylosing spondylitis: investigating non-MHC genes in HLA-B27-positive individuals.

OBJECTIVE: The association of non-MHC genes with AS has been recently suggested. We aimed to investigate the association of the ERAP1, IL23R and TNFSF15 regions and the susceptibility to and protection from AS in HLA-B27-positive individuals. METHODS: A total of 200 unrelated AS patients and 559 healthy unrelated subjects, all HLA-B27 positive, were tested. Twenty single nucleotide polymorphisms (SNPs) were investigated in and near IL23R (nine SNPs), in ERAP1 (five SNPs) and in TNFSF15 (six SNPs). RESULTS: ERAP1 rs30187 [odds ratio (OR) = 1.5, P = 4.7 × 10(-3)] had the strongest association with AS susceptibility. A protective effect was found in three of the ERAP1 SNPs: rs17482078 (OR = 0.7, P = 2.8 × 10(-2)), rs10050860 (OR = 0.7, P = 2.3 × 10(-2)), rs2287987 (OR = 0.6, P = 1.3 × 10(-2)). The ERAP1 haplotype rs17482078/rs10050860/rs30187/rs2287987-CCTT showed an association with AS susceptibility (P = 6.8 × 10(-3)) and a protective effect was identified in rs17482078/rs10050860/rs30187/rs2287987-TTCC (P = 3.1 × 10(-2)). Significant association with AS susceptibility was found in one IL23R marker (rs1004819, P = 4.3 × 10(-2), OR = 1.3). No associations were observed in the TNFSF15 region. CONCLUSION: The identification of a new protection haplotype in ERAP1 and the lack of association of the TNFSF15 region can provide new insights into the understanding of the mechanisms underlying the susceptibility to and protection from AS.

Hemochromatosis and Xeroderma Pigmentosum: Two (Un)Suspicious Neighbors.

A 51-year-old woman, clinically diagnosed with Xeroderma pigmentosum (XP), showed abnormalities in liver enzymes, high ferritin and transferrin saturation levels, with ultrasonographic features of chronic liver disease, in addition to skin hyperpigmentation. Genetic testing confirmed the clinical hypothesis of hereditary hemochromatosis (HH). Due to the known proximity of HFE (6p22.2) and POLH (6p21.1) genes, accountable for HH and the XP-V variant, respectively, a genetic test was offered and a rare variant of the POLH gene was identified. We report the first confirmed case, to our knowledge, of a patient diagnosed both with XP and HH, in whom two mutated neighbor genes - POLH and HFE - were identified, possibly the result of genetic linkage.

Uma mulher de 51 anos, com antecedentes pessoais de Xeroderma pigmentosum (XP), apresentava, além de hiperpigmentação cutânea, alterações nas enzimas hepáticas, elevação da ferritina sérica e da saturação da transferrina, bem como alterações ecográficas compatíveis com doença hepática crónica. A realização de um teste genético permitiu confirmar a hipótese diagnóstica de Hemocromatose Hereditária (HH). Pela proximidade conhecida dos genes HFE (6p22.2) e POLH (6p21.1), responsáveis pela HH e pelo XP-V, respetivamente, foi realizado um teste genético que detetou um polimorfismo raro do gene POLH. Reportamos o primeiro caso de uma paciente diagnosticada com XP e HH, na qual foram identificados dois genes vizinhos mutados ­ POLH e HFE ­, possivelmente como resultado de ligação genética.

Iron Related Biomarkers Predict Disease Severity in a Cohort of Portuguese Adult Patients during COVID-19 Acute Infection.

Large variability in COVID-19 clinical progression urges the need to find the most relevant biomarkers to predict patients' outcomes. We evaluated iron metabolism and immune response in 303 patients admitted to the main hospital of the northern region of Portugal with variable clinical pictures, from September to November 2020. One hundred and twenty-seven tested positive for SARS-CoV-2 and 176 tested negative. Iron-related laboratory parameters and cytokines were determined in blood samples collected soon after admission. Demographic data, comorbidities and clinical outcomes were recorded. Patients were assigned into five groups according to severity. Serum iron and transferrin levels at admission were lower in COVID-19-positive than in COVID-19-negative patients. The levels of interleukin (IL)-6 and monocyte chemoattractant protein 1 (MCP-1) were increased in COVID-19-positive patients. The lowest serum iron and transferrin levels at diagnosis were associated with the worst outcomes. Iron levels negatively correlated with IL-6 and higher levels of this cytokine were associated with a worse prognosis. Serum ferritin levels at diagnosis were higher in COVID-19-positive than in COVID-19-negative patients. Serum iron is the simplest laboratory test to be implemented as a predictor of disease progression in COVID-19-positive patients.

Loss of erythroblasts in acute myeloid leukemia causes iron redistribution with clinical implications.

Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis and limited treatment strategies. Determining the role of cell-extrinsic regulators of leukemic cells is vital to gain clinical insights into the biology of AML. Iron is a key extrinsic regulator of cancer, but its systemic regulation remains poorly explored in AML. To address this question, we studied iron metabolism in patients with AML at diagnosis and explored the mechanisms involved using the syngeneic MLL-AF9-induced AML mouse model. We found that AML is a disorder with a unique iron profile, not associated with inflammation or transfusion, characterized by high ferritin, low transferrin, high transferrin saturation (TSAT), and high hepcidin. The increased TSAT in particular, contrasts with observations in other cancer types and in anemia of inflammation. Using the MLL-AF9 mouse model of AML, we demonstrated that the AML-induced loss of erythroblasts is responsible for iron redistribution and increased TSAT. We also show that AML progression is delayed in mouse models of systemic iron overload and that elevated TSAT at diagnosis is independently associated with increased overall survival in AML. We suggest that TSAT may be a relevant prognostic marker in AML.

Hepcidin messenger RNA expression in human lymphocytes.

Hepcidin regulates intracellular iron levels by interacting with and promoting the degradation of ferroportin, a membrane protein and the only known cellular iron exporter. Studies of hepcidin expression and regulation have focused on its effects in innate immunity and as a regulator of systemic iron metabolism. In the present study we characterized the expression of hepcidin messenger RNA (mRNA) in human peripheral blood mononuclear cells (PBMCs) with a focus on peripheral blood lymphocytes (PBLs). We found that (1) all human PBMCs analyzed express basal hepcidin mRNA levels; (2) hepcidin mRNA expression increases after T-lymphocyte activation; (3) expression by PBLs increases in response to challenge by holotransferrin (Fe-TF) and by ferric citrate in vitro; (4) the Fe-TF-mediated up-regulation of hepcidin decreases ferroportin expression at the cytoplasmic membrane of PBLs; and (5) silencing of tumour necrosis factor-alpha (TNF-alpha) abrogates the effect of Fe-TF. In summary, we show that hepcidin expression determines intracellular iron levels by regulating the expression of ferroportin, as described in other cells, and that inappropriately low expression of hepcidin impairs normal lymphocyte proliferation. The results establish hepcidin as a new player in lymphocyte biology.

Hemochromatosis and pregnancy: iron stores in the Hfe-/- mouse are not reduced by multiple pregnancies.

Hereditary hemochromatosis (HH), a widespread hereditary iron metabolism disorder, is characterized by an excessive absorption of dietary iron, resulting in increased body iron stores. Some studies indicate a sex difference in disease expression, with women showing a slower disease progression and a less severe clinical profile. This is usually attributed to iron loss during menstruation and pregnancy. However, this link has not been clearly demonstrated. The Hfe-/- mouse model recapitulates key aspects of HH, including an iron overload phenotype similar to that observed in human patients. In this study, we use it to test the impact of multiple pregnancies in the iron stores. One-year-old nulliparous and pluriparous (averaging 29 weaned pups per female) C57BL/6 (B6) and Hfe-/- mice were euthanized, and blood and tissues were collected. Several serological and erythroid parameters were evaluated, as well as tissue nonheme iron content and serum ferritin. Hepcidin 1, hepcidin 2, and bone morphogenetic protein 6 (BMP6) expressions in the liver were determined by real-time PCR. No significant differences were observed for many serological and erythroid parameters although differences occurred in transferrin saturation and mean corpuscular volume in Hfe-/- mice and total iron-binding capacity in B6 mice. Hepatic iron concentration was similar for nulliparous and pluriparous mice of both genotypes, but total iron per organ (liver, spleen, heart, and pancreas) was higher overall in pluriparous females than nulliparous. Hepcidin 1 and 2 and BMP6 expressions were significantly decreased in pluriparous females, when compared with nulliparous, in both genotypes. In conclusion, multiple pregnancies do not reduce body iron stores in Hfe-/- mice.

Erythropoietin mediates hepcidin expression in hepatocytes through EPOR signaling and regulation of C/EBPalpha.

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. Recent in vivo studies have shown that hepcidin is down-regulated by erythropoiesis, anemia, and hypoxia, which meets the need of iron input for erythrocyte production. Erythropoietin (EPO) is the primary signal that triggers erythropoiesis in anemic and hypoxic conditions. Therefore, a direct involvement of EPO in hepcidin regulation can be hypothesized. We report here the regulation of hepcidin expression by EPO, in a dose-dependent manner, in freshly isolated mouse hepatocytes and in the HepG2 human hepatocyte cell model. The effect is mediated through EPOR signaling, since hepcidin mRNA levels are restored by pretreatment with an EPOR-blocking antibody. The transcription factor C/EBPalpha showed a pattern of expression similar to hepcidin, at the mRNA and protein levels, following EPO and anti-EPOR treatments. Chromatin immunoprecipitation experiments showed a significant decrease of C/EBPalpha binding to the hepcidin promoter after EPO supplementation, suggesting the involvement of this transcription factor in the transcriptional response of hepcidin to EPO.