Extracellular Iron Concentrations Research Articles

SMARCB1 regulates a TFCP2L1-MYC transcriptional switch promoting renal medullary carcinoma transformation and ferroptosis resistance

Renal medullary carcinoma (RMC) is an aggressive tumour driven by bi-allelic loss of SMARCB1 and tightly associated with sickle cell trait. However, the cell-of-origin and oncogenic mechanism remain poorly understood. Using single-cell sequencing of human RMC, we defined transformation of thick ascending limb (TAL) cells into an epithelial-mesenchymal gradient of RMC cells associated with loss of renal epithelial transcription factors TFCP2L1, HOXB9 and MITF and gain of MYC and NFE2L2-associated oncogenic and ferroptosis resistance programs. We describe the molecular basis for this transcriptional switch that is reversed by SMARCB1 re-expression repressing the oncogenic and ferroptosis resistance programs leading to ferroptotic cell death. Ferroptosis resistance links TAL cell survival with the high extracellular medullar iron concentrations associated with sickle cell trait, an environment propitious to the mutagenic events associated with RMC development. This unique environment may explain why RMC is the only SMARCB1-deficient tumour arising from epithelial cells, differentiating RMC from rhabdoid tumours arising from neural crest cells.

Investigations into the Mechanisms and Clinical Implications of Modulation of Hepcidin Levels By Luspatercept in TD MDS and TD b-Thalassemia

Introduction: Multiple blood transfusions lead to significant iron overload in transfusion dependent (TD) thalassemia (TDT) and TD-MDS. This excess iron is deposited in various organs leading to end organ damage, morbidity, and mortality. Iron overload patients are treated with iron chelation therapy (ICT) owing to lack of effective physiological iron removal mechanisms. Furthermore, chronic transfusions lead to paradoxical changes in hepcidin, the master regulator of iron homeostasis. While hepcidin is upregulated under conditions of increased intracellular and extracellular iron concentrations through BMP6 and IL6 dependent signaling; it is downregulated during expanded erythropoiesis through Erythroferrone (ERFE) to increase iron availability for developing erythron. Increased levels of hepcidin sequesters iron within organs leading to inefficiency of iron chelation therapy in TD-MDS and TDT patients. Luspatercept is a modified activin receptor fusion protein (modified ActRIIB-Fc) that sequesters certain members of TGF-b superfamily ligands, thus enhancing late-stage erythroid-maturation. Luspatercept is approved for TDT and TD-MDS patient populations-based on significant reduction in transfusion burden in two independent phase 3 trails (BELIEVE and MEDALIST, respectively). Here, we evaluated modulation of hepcidin levels with luspatercept treatment and its clinical implications in anemias due to elevated levels of hepcidin including TD-MDS and TDT patients. Methods: Luspatercept was administered sub-cutaneously every three weeks. Hepcidin and ERFE levels were measured in serum or plasma samples from BELIEVE (NCT02604433) and MEDALIST (NCT02631070) trial patients using specific ELISA and or chemiluminescence assay kits at week 25 following treatment. The regulation of hepcidin by luspatercept, BMP6 and IL6 in HepG2 cell-line model was determined by ELISA (in conditioned media) and q-PCR at increasing doses and time points. Results: Serum hepcidin levels at baseline across both arms in MEDALIST and BELIEVE were 80.65 ng/ml and 43.1 ng/ml respectively. These were significantly higher than normal healthy individuals (range of 5-10 ng/ml; P < 0.001), reported previously (Ilkovska etal JIMB 2016). Patients treated with luspatercept in both TDT and TD-MDS showed significant reduction in serum levels of hepcidin by 49% and 53% respectively (P<0.001) at week 25 compared to baseline. Hepcidin levels in the placebo group in TD-MDS patients demonstrated an increase of 119% (P<0.001) and a decrease of 12% in TDT patients (P=0.004) at the same time point. In addition, ERFE protein levels in serum, were elevated in response to Luspatercept treatment in both TD-MDS (30%) and TDT (51%) (P<0.001) in comparison to the placebo arms of decrease of 28% in TD-MDS and an increase of 2% in TDT. In HepG2 cells, treatment with recombinant BMP6 and IL6 increased hepcidin protein expression in a dose dependent manner. Luspatercept treatment inhibited this increase in hepcidin expression mediated by BMP6 and IL6. Time course analyses (2, 6, 24 and 72 hours), revealed that luspatercept inhibited hepcidin transcription and secretion levels starting at 24 hours. Conclusions: Luspatercept treatment reduced transfusion burden and iron loading rate due to improved erythropoiesis and release of ERFE by maturing erythroblasts consequently leading to reductions in elevated hepcidin levels in both TD-MDS and TDT patients. Our data provides a mechanistic rationale for downregulation of hepcidin levels with luspatercept treatment. Interestingly, cell-based assay data suggest that luspatercept directly modulates hepcidin transcription suggesting that in diseased conditions with abnormally higher levels of hepcidin and TGFβ family ligands, luspatercept might also modulate hepcidin transcription by sequestering TGFβ family ligands implicated in hepcidin synthesis. We postulate that decreased hepcidin levels results in release of excess iron into peripheral blood, making it available for effective chelation. These observations implicate sequencing of luspatercept, and ICT for effective management of iron overload in these TD patients. Furthermore, the data provide a mechanistic rationale for evaluating luspatercept in hepcidin dependent anemia of inflammation and iron refractory iron deficiency anemia (IRIDA) disease indications.

Cover Feature: Iron Oxide Nanoparticles Functionalized with Fucoidan: a Potential Theranostic Nanotool for Hepatocellular Carcinoma (ChemBioChem 16/2022)

Iron oxide nanoparticles (NPs) were functionalized with fucoidan, a natural sulfated polysaccharide. NP cellular uptake was quantified by magnetic measurement at various extracellular iron concentrations. Cell invasion and migration were reduced with NPs. Concomitantly, a high decrease of reactive oxygen species production related with a decrease of the matrix metalloproteinase-9 activity and an increase of its expression was observed. Proteomic analysis showed that some fucoidan regulated proteins appeared related to protein synthesis, N-glycan processing, and cellular stress. These results pave the way for USPIO-fucoidan-NPs as potential theranostic nanotools for hepatocellular carcinoma treatment. More information can be found in the Research Article by F. Geinguenaud et al.

Iron Oxide Nanoparticles Functionalized with Fucoidan: a Potential Theranostic Nanotool for Hepatocellular Carcinoma.

Fucoidan is a natural sulfated polysaccharide with a large range of biological activities including anticancer and anti-oxidation activities. Hepatocellular carcinoma is the fourth most common aggressive cancer type. The aim of this study was to investigate the bioactivity of free fucoidan versus its vectorization using nanoparticles (NPs) in human hepatoma cells, Huh-7. Iron oxide NPs were functionalized with fucoidan by a one-step surface complexation. NP cellular uptake was quantified by magnetic measurement at various extracellular iron concentrations. Cell invasion and migration were reduced with NPs while free fucoidan increases these events at low fucoidan concentration (≤0.5 μM). Concomitantly, a high decrease of reactive oxygen species production related with a decrease of the matrix metalloproteinase-9 activity and an increase of its expression was observed with NPs compared to free fucoidan. A proteomic analysis evidenced that some fucoidan regulated proteins appeared, which were related to protein synthesis, N-glycan processing, and cellular stress. To our knowledge, this is the first study which reveals such activity induced by fucoidan. These results pave the way for USPIO-fucoidan-NPs as potential theranostic nanotools for hepatocellular carcinoma treatment.

Hepcidin Protects against Lethal Escherichia coli Sepsis in Mice Inoculated with Isolates from Septic Patients.

Iron is an essential micronutrient for most microbes and their hosts. Mammalian hosts respond to infection by inducing the iron-regulatory hormone hepcidin, which causes iron sequestration and a rapid decrease in the plasma and extracellular iron concentration (hypoferremia). Previous studies showed that hepcidin regulation of iron is essential for protection from infection-associated mortality with the siderophilic pathogens Yersinia enterocolitica and Vibrio vulnificus However, the evolutionary conservation of the hypoferremic response to infection suggests that not only rare siderophilic bacteria but also common pathogens may be targeted by this mechanism. We tested 10 clinical isolates of Escherichia coli from children with sepsis and found that both genetic iron overload (by hepcidin-1 knockout [HKO]) and iatrogenic iron overload (by intravenous iron) potentiated infection with 8 out of the 10 studied isolates: after peritoneal injection of E. coli, iron-loaded mice developed sepsis with 60% to 100% mortality within 24 h, while control wild-type mice suffered 0% mortality. Using one strain for more detailed study, we show that iron overload allows rapid bacterial multiplication and dissemination. We further found that the presence of non-transferrin-bound iron (NTBI) in the circulation is more important than total plasma or tissue iron in rendering mice susceptible to infection and mortality. Postinfection treatment of HKO mice with just two doses of the hepcidin agonist PR73 abolished NTBI and completely prevented sepsis-associated mortality. We demonstrate that the siderophilic phenotype extends to clinically common pathogens. The use of hepcidin agonists promises to be an effective early intervention in patients with infections and dysregulated iron metabolism.

Pseudomonas aeruginosa AlgR Phosphorylation Status Differentially Regulates Pyocyanin and Pyoverdine Production.

ABSTRACTPseudomonas aeruginosa employs numerous, complex regulatory elements to control expression of its many virulence systems. The P. aeruginosa AlgZR two-component regulatory system controls the expression of several crucial virulence phenotypes. We recently determined, through transcriptomic profiling of a PAO1 ΔalgR mutant strain compared to wild-type PAO1, that algZR and hemCD are cotranscribed and show differential iron-dependent gene expression. Previous expression profiling was performed in strains without algR and revealed that AlgR acts as either an activator or repressor, depending on the gene. Thus, examination of P. aeruginosa gene expression from cells locked into different AlgR phosphorylation states reveals greater physiological relevance. Therefore, gene expression from strains carrying algR alleles encoding a phosphomimetic (AlgR D54E) or a phosphoablative (AlgR D54N) form were compared by microarray to PAO1. Transcriptome analyses of these strains revealed 25 differentially expressed genes associated with iron siderophore biosynthesis or heme acquisition or production. The PAO1 algR D54N mutant produced lower levels of pyoverdine but increased expression of the small RNAs prrf1 and prrf2 compared to PAO1. In contrast, the algR D54N mutant produced more pyocyanin than wild-type PAO1. On the other hand, the PAO1 algR D54E mutant produced higher levels of pyoverdine, likely due to increased expression of an iron-regulated gene encoding the sigma factor pvdS, but it had decreased pyocyanin production. AlgR specifically bound to the prrf2 and pvdS promoters in vitro. AlgR-dependent pyoverdine production was additionally influenced by carbon source rather than the extracellular iron concentration per se. AlgR phosphorylation effects were also examined in a Drosophila melanogaster feeding, murine acute pneumonia, and punch wound infection models. Abrogation of AlgR phosphorylation attenuated P. aeruginosa virulence in these infection models. These results show that the AlgR phosphorylation state can directly, as well as indirectly, modulate the expression of iron acquisition genes that may ultimately impact the ability of P. aeruginosa to establish and maintain an infection.

Does Iron Modulate Cytokine Production by Desferrioxamine Treated Mononuclear Cells?

Iron is an essential element for maintaining the normal function of cellular and multicellular organisms and it is closely associated with inflammation and immunity. Macrophages and lymphocytes play an important role in its homeostasis. Hereby we have evaluated cytokine production by iron depleted and iron supplemented human peripheral blood mononuclear cells (PBMC) from healthy adult donors. Non-stimulated or stimulated PBMC (lipopolysaccharide-LPS or phorbol-myristate acetate-PMA and ionomycin) were incubated for 24 h without or with desferrioxamine (DFO) and the quantity of TNFα, IL-1β, IL-6, IFNγ, IL-2, IL-10 and IL-1ra in the supernatants was evaluated using ELISA kits. In addition, cytokine levels were determined in supernatants of cultures to which DFO was added for 2 h followed by 24 h of incubation with iron. The secretion of IL-1β, IL-6 and IL-1ra by non-stimulated PBMC and that of IL-1β by LPS-stimulated cells was dose-dependently enhanced following incubation with increasing concentrations of DFO between 25 and 100 µM. On the other hand LPS-induced production of IL-10 and PMA/ionomycin stimulated IL-2 and IFNγ secretion was dose dependently reduced. The inhibited IFNγ, IL-2 and IL-10 secretion by stimulated PBMC caused by DFO was reversed after incubation with iron at concentrations similar to those in human serum. However, the inhibition of LPS-induced IL-1ra or IL-1β production induced by DFO was not affected by iron supplementation. The results of the study underline the importance of intra and/or extracellular iron concentration and DFO for the release of inflammatory cytokines by human peripheral blood mononuclear cells.

Expression of hepcidin and ferroportin in full term placenta of pregnant cows

Hepcidin (HEP) and ferroportin (FPN) play a central role in systemic iron homeostasis. The HEP/FPN axis controls both extracellular iron concentration and total body iron levels. HEP is synthesized mainly by hepatocytes and controls the absorption of dietary iron and the distribution of iron to the various cell types; its synthesis is regulated by both iron and innate immunity. FPN is a membrane protein and the major exporter of iron from mammalian cells, including iron recycling macrophages, iron absorbing duodenal enterocytes, and iron storing hepatocytes. HEP limits the pool of extracellular iron by binding FPN and mediating its degradation, thus preventing its release from intracellular sources. Here we investigated, for the first time, the molecular and morphological expression of HEP and FPN in placenta of pregnant cows at term. Their expression has been evaluated investigating their mRNAs by reverse transcriptase PCR (RT-PCR). Sequencing of related amplicons revealed a 100% identity with HEP and FPN sequences from Bos taurus as reported in the GeneBank (mRNASequence ID: NM_001114508.2 and ID: NM_001077970.1, respectively). HEP and FPN proteins have also been revealed by Western blot analysis and immunohistochemistry. The strongest immunoreactivity for both proteins was observed in the cytoplasm of the trophoblastic cells of the villi and the caruncular crypts of the placentome. Hep mRNA was more representative in caruncular rather cotyledonar areas; on the contrary, Fpn mRNA was more expressed in cotyledonar rather than in caruncular areas. Transcripts of ferritin, transferrin and its receptor have been also documented by real time RT-PCR.HEP and FPN placental proteins may play a dual role. HEP/FPN axis seems to have a central role in infections, with microorganisms within macrophages or that survive in the bloodstream or other cellular spaces. In addition, HEP may be responsible for iron flux regulation as a molecular bridge for iron trafficking and response to infection. FPN may also have a significant role for embryonic development, growth and organogenesis.

Iron Chelators Dictate Immune Cells Inflammatory Ability: Potential Adjuvant Therapy for IBD.

The importance of hemoproteins for life lies largely in their iron-mediated chemical properties. In the human body, there are about 4 g of iron, a precious resource preserved by sophisticated recycling mechanisms. Iron is also important for pathogen growth, so it is not surprising that immune cells developed mechanisms to reduce iron availability in cases of inflammation. In healthy conditions, macrophages degrade hemoproteins and export iron, while if inflammation develops, they retain cytoplasmic iron to reduce extracellular iron concentrations. Iron-rich macrophages possess a stronger inflammatory ability, which explains the chronic inflammatory response observed in states of iron overload. Inflammatory bowel syndromes are often characterized by intestinal blood loss and consequent anemia, but iron-supplementation therapies may exacerbate the inflammatory response. In chronically transfused patients iron overload is frequently observed; the iron can become toxic and in excess, even fatal if not treated with iron-chelating drugs. In the present review, we discuss the importance of iron homeostasis in states of health and inflammation, focusing on iron and iron-chelation treatment for IBD patients. Oral administration of natural ironchelating chemicals may be an effective adjuvant therapy for IBD patients, acting on numerous aspects of chronic inflammatory syndromes.

Molecular regulation of cellular iron balance.

Handling a life-supporting yet redox-active metal like iron represents a significant challenge to cells and organisms that must not only tightly balance intra- and extracellular iron concentrations but also chaperone it during its journey from its point of entry to final destinations, to prevent inappropriate generation of damaging reactive oxygen species. Accordingly, regulatory mechanisms have been developed to maintain appropriate cellular and body iron levels. In intracellular compartments, about 95% of iron is protein-bound and the expression of the major proteins of iron metabolism is controlled by an integrated and dynamic system involving multilayered levels of regulation. However, dysregulation of iron homeostasis, which could result from both iron-related and unrelated effectors, may occur and have important pathological consequences in a number of human disorders. In this review, we describe the current understanding of the mechanisms that keep cellular iron balance and outline recent advances that increased our knowledge of the molecular physiology of iron metabolism. © 2017 IUBMB Life, 69(6):389-398, 2017.

Метаболизм железа в норме и при патологии

ABSTRACT This review describes modern conceptions of the physi-ological and pathological roles of iron, as well as the main mechanisms of iron metabolism regulation. In recent years, it has been shown that both deficiency and excess of iron can have damaging effects on the body, and the existence of homeostatic mechanisms controlling the total iron con-tent of the body has been proved. The body of an average healthy adult human contains 3 to 5 g iron, most of which is contained in blood cells, bone marrow and liver; it is bound to proteins and this is important for prevention of cytotoxic effects of free iron ions. This review summarizes data on the main proteins involved in iron metabolism and their role in iron homeostasis. The processes of iron recirculation and the functional role of hepcidin, the key protein regulating ex-tracellular iron concentration, are emphasized. The review provides brief data on pathogenic mechanisms of functional iron deficiency development and its role in anemia of chron-ic disease, as well as the pathogenesis, diagnostics and management of secondary iron overload.

Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis.

The reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacterium Francisella tularensis strain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslA mutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoB mutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment of F. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicin in vitro and in vivo.

The iron-regulatory hormone hepcidin: a possible therapeutic target?

The maintenance of stable extracellular and intracellular iron concentrations requires the coordinated regulation of iron transport into plasma. Iron is a fundamental cofactor for several enzymes involved in oxidation-reduction reactions. The redox ability of iron can lead to the production of oxygen free radicals, which can damage various cellular components. Therefore, the appropriate regulation of systemic iron homeostasis is decisive in vital processes. Hepcidin has emerged as the central regulatory molecule of systemic iron homeostasis. It is synthesized in hepatocytes and in other cells and released into the circulation. It inhibits the release of iron from enterocytes of the duodenum and from macrophages by binding to the iron exporter protein, ferroportin (FPN). FPN is a transmembrane protein responsible for iron export from cells into the plasma. Hepcidin is internalized with FPN and both are degraded in lysosomes. The hepcidin-FPN axis is the principal regulator of extracellular iron homeostasis in health and disease. Its manipulation via agonists and antagonists is an attractive and novel therapeutic strategy. Hepcidin agonists include compounds that mimic the activity of hepcidin and agents that increase the production of hepcidin by targeting hepcidin-regulatory molecules. The inhibition of hepcidin could be a potentially attractive therapeutic strategy in patients suffering from anaemia or chronic inflammation. In this review, we will summarize the role of hepcidin in iron homeostasis and its contribution to the pathophysiology of inflammation and iron disorders. We will examine emerging new strategies that modulate hepcidin metabolism.

Hepcidin–Minireview

Iron related disorders are encountered in daily clinical settings. Maintenance of stable extracellular iron concentrations requires the coordinate regulation of iron transport into plasma from dietary sources in the duodenum, from recycled senescent red cells in macrophages and from storage in hepatocytes. Hepcidin acts as a systemic iron-regulatory hormone. Many human diseases are associated with alterations in hepcidin concentrations. This review has focused on hepcidin structure, kinetics and function, its correlation with iron metabolism disorders, the therapeutic potential for modulating hepcidin expression and the diagnostic potential of hepcidin measurements in clinical practice.

A Pseudo-tRNA Modulates Antibiotic Resistance in Bacillus cereus

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNAOther, which does not function in translation. Deletion of tRNAOther led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNAOther. This suggested that tRNAOther might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3′ and 5′-ends of tRNAOther. tRNAOther expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5′ leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNAOther may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell.

Accumulation of Non-Transferrin-Bound Iron by Neurons, Astrocytes, and Microglia

Neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and hemorrhagic stroke are associated with increased levels of non-transferrin-bound iron (NTBI) in the brain, which can promote Fenton chemistry. While all types of brain cells can take up NTBI, their efficiency of accumulation and capacity to withstand iron-mediated toxicity has not been directly compared. The present study assessed NTBI accumulation in cultures enriched in neurons, astrocytes, or microglia after exposure to ferric ammonium citrate (FAC). Microglia were found to be the most efficient in accumulating iron, followed by astrocytes, and then neurons. Exposure to 100μM FAC for 24h increased the specific iron content of cultured neurons, astrocytes, and microglial cells by 30-, 80-, and 100-fold, respectively. All cell types accumulated iron against the concentration gradient, resulting in intracellular iron concentrations that were several orders of magnitude higher than the extracellular iron concentrations. Accumulation of these large amounts of iron did not affect the viability of the cell cultures, indicating a high resistance to iron-mediated toxicity. These findings show that neurons, astrocytes and microglia cultured from neonatal mice all have the capacity to accumulate and safely store large quantities of iron, but that glial cells do this more efficiently than neurons. It is concluded that neurodegenerative conditions involving iron-mediated toxicity may be due to a failure of iron transport or storage mechanisms, rather than to the presence of high levels of NTBI.

Anemia in Hodgkin Lymphoma: The Role of Interleukin-6 and Hepcidin.

Abstract 3656Poster Board III-592Anemia is present at diagnosis in approximately 40% of patients with Hodgkin lymphoma (HL). The anemia is typically the normochromic, normocytic anemia of chronic disease seen in a wide variety of inflammatory states. It is more commonly observed in advanced stages of disease and when B-symptoms are present. Local production of cytokines by the Hodgkin and Reed-Sternberg cells and the surrounding microenvironment are supposed to mediate the systemic inflammatory reactions. Studies in humans and mice suggest that the liver-produced acute-phase peptide hepcidin is the principal regulator of extracellular iron concentration and may be the principal mediator of anemia of chronic disease and/or inflammation. As IL-6 is a potent inducer of hepcidin expression, we studied the role of hepcidin in HL analyzing for correlations between plasma hepcidin levels, cytokine levels, parameters of inflammation, iron metabolism and patient characteristics. We studied 65 patients with HL at diagnosis. Anemia defined as hemoglobin level <12 g/dl was present in 31 patients. Hemoglobin levels were lower in female patients, patients with age > 45 years, in the presence of B-symptoms, stage IV disease and with a higher IPS score (> 2). Plasma samples at diagnosis were analyzed for levels of the cytokines IL-6, IL-10, and the chemokine TARC using ELISA techniques (R&D Dignostics), while hepcidin levels were determined using a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (TOF MS), as described previously (Swinkels et al, PLOS ONE 2008; 3:e2706). The mean hepcidin level was 7.9 nmol/L, ranging from below the detection limit of 0.5 nmol/l in 3 patients to 27.4 nM. Hepcidin levels were higher in patients aged over 45 years (p=0.03), and there was a borderline significance for higher hepcidin levels in male patients in comparison with females (p=0.06). In anemic patients hepcidin levels inversely correlated with haemoglobin values (r=-0.45, p=0.01). We found a highly significant correlation between levels of hepcidin and IL-6 (r=0.55, p=0.0001). IL-6 was elevated in 40/55 patients, and IL-6 inversely correlated with haemoglobin values (r=-0.49, p=0.0001). No significant correlation was observed between IL-10 or TARC levels and hepcidin arguing against a role for these cytokines in hepcidin up-regulation in HL patients. The analysis of hepcidin with parameters of iron metabolism in patients with HL showed significant correlations with ferritin (r=0.62, p= 0.0001), while iron and iron-binding capacity inversely correlated with hepcidin (r=-0.42, p=0.009; and r=-0.43, p=0.02, respectively). Increases in serum ferritin, low serum iron and reduced iron-binding capacity are characteristic for anemia of chronic disease. In a multivariate regression analysis, IL-6 and ferritin levels independently correlated with hepcidin (p=0.003 and p=0.02, respectively), while haemoglobin levels were not independently associated with hepcidin in this model. Hepcidin levels were significantly associated with stage IV disease (p=0.01), the presence of B-symptoms (p=0.03), and IPS score > 2 (p=0.005). In conclusion, our findings support the hypothesis, that in HL elevated levels of circulating IL-6 are associated with upregulation of hepcidin, resulting in hypoferremia and development of anemia with the typical signs of anemia of chronic disease. In addition, associations of hepcidin levels with patient characteristics of prognostic significance suggest that hepcidin should be studied as a new potential biomarker in HL. Disclosures:Tjalsma:hepcidinanalysis.com: Managing Director, Membership on an entity's Board of Directors or advisory committees. Swinkels:hepcidinanalysis.com: Medical Director, Membership on an entity's Board of Directors or advisory committees.

Hepcidin in acute iron toxicity

Background Hepcidin regulates extracellular iron concentration by inhibiting iron release from macrophages and preventing iron absorption in the intestine. Our objective was to evaluate the expression of hepcidin in the liver in acute iron poisoning in a rat model. Methods Male Wistar rats were assigned to group 1, who received 750 mg/kg elemental iron (LD 50) by gavage, and group 2 (control), who received distilled water. Iron concentrations and liver transaminases were measured in the serum. Hepcidin messenger RNA levels were measured in the liver. Results Mean serum iron levels, aspartate aminotransferase, alanine aminotransferase, and uric acid were significantly higher in group 1 compared to group 2 ( P < .0001, P = .01, P < .0001, and P = 0.0001, respectively). Hepcidin messenger RNA levels in the liver were significantly higher in the study group ( P = .005). Conclusions In acute iron intoxication, hepcidin expression in the liver significantly increased. Further studies are needed to determine whether hepcidin levels correlate with the severity of the intoxication.

Ferrous iron transport protein B gene ( feoB1) plays an accessory role in magnetosome formation in Magnetospirillum gryphiswaldense strain MSR-1

To investigate the role of ferrous iron transport (Feo) systems in magnetosome formation, the gene for protein FeoB ( feoB1), encoding 704 amino acids, was cloned from magnetotactic bacterium Magnetospirillum gryphiswaldense strain MSR-1. feoB1 constitutes a putative operon with feoA1, and the interval between the two genes is 36 base pairs. A feoB1-deficient mutant (Δ feoB1) was constructed, and compared with wild-type in terms of iron uptake, iron content and functional complementation. Ferrous iron and ferric iron uptake in wild-type were respectively 1.8-fold and 1.3-fold higher than in the Δ feoB1 mutant. Iron content (w/w) of Δ feoB1 mutant was enhanced only slightly as extracellular iron concentration (either ferrous or ferric citrate) increased, whereas iron content of wild-type increased about 2-fold as extracellular iron concentration rose from 20 to 80 μM. Transmission electron microscopy revealed that Δ feoB1 cells grown with either ferrous or ferric citrate produced fewer magnetosomes, with smaller diameter, compared to wild-type cells. Assay of feoAB1 promoter– lacZ transcriptional fusions indicated that the feoAB1 putative operon was downregulated when MSR-1 cells were grown under iron-rich condition. Magnetosome formation was reduced but not abolished in the feoB1 mutant, indicating that FeoB1 protein plays a significant role in this process. Other iron transport systems are presumed to be involved in iron uptake in MSR-1.

Regulation of AQP4 protein expression in rat brain astrocytes: Role of P2X7 receptor activation

ATP has been recognized as an important extracellular signaling molecule and P2X receptors are membrane ion channels activated by the binding of extracellular ATP. Since both AQP4 and P2X7 receptor (P2X7R) are known to be present in astrocytes, we examined whether P2X7R activation plays a role in the regulation of AQP4 expression in astrocytes. Immunoblotting and immunocytochemistry confirmed the expression of both P2X7R and AQP4 in primary cultured rat astrocytes. Co-immunoprecipitation assays of the HEK293 cells expressing both proteins revealed no protein–protein interaction. An activation of P2X7R in primary cultured astrocytes by a P2X7R agonist significantly decreased the AQP4 protein expression, which was abolished by the pre-treatment of a P2X7R antagonist. In addition, AQP4 expression was not affected by high extracellular copper, zinc, or iron concentrations. In a rat model with anoxia-induced brain injury where extracellular ATP levels could be increased, whole brain AQP4 expression was significantly decreased, whereas P2X7R expression was unchanged. Importantly, pre-treatment of P2X7R antagonist in rats significantly inhibited the AQP4 down-regulation in anoxic brain injury, consistent with the in vitro results observed in astrocytes. In conclusion, P2X7R activation in astrocytes was associated with down-regulation of AQP4 in rat brain astrocytes in vitro and in vivo, and this was prevented by P2X7 receptor blockade. Thus, an activation of P2X7R in astrocytes in response to brain injury is likely to play a role in the protective down-regulation of AQP4, which might inhibit water influx to the cells and attenuate the acute cytotoxic brain edema after acute brain injury.