Human Hepcidin Research Articles

Molecular characterization of a novel hepcidin (HepcD) from Camelus dromedarius. Synthetic peptide forms exhibit antibacterial activity.

Hepcidin is a cysteine-rich peptide widely characterized in immunological processes and antimicrobial activity in several vertebrate species. Obviously, this hormone plays a central role in the regulation of systemic iron homeostasis. However, its role in camelids' immune response and whether it is involved in antibacterial immunity have not yet been proven. In this study, we characterized the Arabian camel hepcidin nucleotide sequence with an open reading frame of 252 bp encoding an 83-amino acid preprohepcidin peptide. Eight cysteine key residues conserved in all mammalian hepcidin sequences were identified. The model structure analysis of hepcidin-25 peptide showed a high homology structure and sequence identity to the human hepcidin. Two different hepcidin-25 analogs manually synthesized by SPPS shared significant cytotoxic capacity toward the Gram-negative bacterium Escherichia coli American Type Culture Collection (ATCC) 8739 as well as the Gram-positive bacteria Bacillus subtilis ATCC 11779 and Staphylococcus aureus ATCC 6538 in vitro. The three disulfide bridges hepcidin analog demonstrated bactericidal activity, against B. subtilis ATCC 11779 and S. aureus ATCC 6538 strains, at the concentration of 15 μM (50 µg/ml) or above at pH 6.2. This result correlates with the revealed structural features suggesting that camel hepcidin is proposed to be involved in antibacterial process of innate immune response.

Inhibitory effect of the human liver-derived antimicrobial peptide hepcidin 20 on biofilms of polysaccharide intercellular adhesin (PIA)-positive and PIA-negative strains of Staphylococcus epidermidis

Staphylococcus epidermidis plays a major role in biofilm-related medical device infections. Herein the anti-biofilm activity of the human liver-derived antimicrobial peptide hepcidin 20 (hep20) was evaluated against polysaccharide intercellular adhesin (PIA)-positive and PIA-negative clinical isolates of S. epidermidis. Hep20 markedly inhibited biofilm formation and bacterial cell metabolism of PIA-positive and PIA-negative strains, but the decrease in biofilm biomass only partially correlated with a decrease in viable bacteria. Confocal microscope images revealed that, in the presence of hep20, both PIA-positive and PIA-negative strains formed biofilms with altered architectures and reduced amounts of extracellular matrix. Co-incubation of hep20 with vancomycin produced no synergistic effect, evaluated as number of viable cells, both in preventing biofilm formation and in treating preformed biofilms. In contrast, biofilms obtained in the presence of hep20, and then exposed to vancomycin, displayed an increased susceptibility to vancomycin. These results suggest that hep20 may inhibit the production/accumulation of biofilm extracellular matrix.

Total Synthesis of Human Hepcidin through Regioselective Disulfide‐Bond Formation by using the Safety‐Catch Cysteine Protecting Group 4,4′‐Dimethylsulfinylbenzhydryl

AbstractA safety‐catch cysteine protecting group, S‐4,4′‐dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine‐rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high‐resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide‐bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC50 values of 2.0 (native fold, 1) and 4.4 nM (non‐native fold, 2), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells.

Total Synthesis of Human Hepcidin through Regioselective Disulfide‐Bond Formation by using the Safety‐Catch Cysteine Protecting Group 4,4′‐Dimethylsulfinylbenzhydryl

A safety-catch cysteine protecting group, S-4,4'-dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine-rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high-resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide-bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC50 values of 2.0 (native fold, 1) and 4.4 nM (non-native fold, 2), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells.

Apoptosis induced by Fas signaling does not alter hepatic hepcidin expression

To determine the regulation of human hepcidin (HAMP) and mouse hepcidin (hepcidin-1 and hepcidin-2) gene expression in the liver by apoptosis using in vivo and in vitro experimental models. For the induction of the extrinsic apoptotic pathway, HepG2 cells were treated with various concentrations of CH11, an activating antibody for human Fas receptor, for 12 h. Male C57BL/6NCR and C57BL/6J strains of mice were injected intraperitoneally with sublethal doses of an activating antibody for mouse Fas receptor, Jo2. The mice were anesthetized and sacrificed 1 or 6 h after the injection. The level of apoptosis was quantified by caspase-3 activity assay. Liver injury was assessed by measuring the levels of ALT/AST enzymes in the serum. The acute phase reaction in the liver was examined by determining the expression levels of IL-6 and SAA3 genes by SYBR green quantitative real-time PCR (qPCR). The phosphorylation of transcription factors, Stat3, Smad4 and NF-κB was determined by western blotting. Hepcidin gene expression was determined by Taqman qPCR. The binding of transcription factors to hepcidin-1 promoter was studied using chromatin immunoprecipitation (ChIP) assays. The treatment of HepG2 cells with CH11 induced apoptosis, as shown by the significant activation of caspase-3 (P < 0.001), but did not cause any significant changes in HAMP expression. Short-term (1 h) Jo2 treatment (0.2 μg/g b.w.) neither induced apoptosis and acute phase reaction nor altered mRNA expression of mouse hepcidin-1 in the livers of C57BL/6NCR mice. In contrast, 6 h after Jo2 injection, the livers of C57BL/6NCR mice exhibited a significant level of apoptosis (P < 0.001) and an increase in SAA3 (P < 0.023) and IL-6 (P < 0.005) expression in the liver. However, mRNA expression of hepcidin-1 in the liver was not significantly altered. Despite the Jo2-induced phosphorylation of Stat3, no occupancy of hepcidin-1 promoter by Stat3 was observed, as shown by ChIP assays. Compared to C57BL/6NCR mice, Jo2 treatment (0.2 μg/g b.w.) of C57BL/6J strain mice for 6 h induced a more prominent activation of apoptosis, liver injury and acute phase reaction. Similar to C57BL/6NCR mice, the level of liver hepcidin-1 mRNA expression in the livers of C57BL/6J mice injected with a sublethal dose of Jo2 (0.2 μg/g b.w.) remained unchanged. The injection of C57BL/6J mice with a higher dose of Jo2 (0.32 μg/g b.w.) did not also alter hepatic hepcidin expression. Our findings suggest that human or mouse hepcidin gene expression is not regulated by apoptosis induced via Fas receptor activation in the liver.

LY2928057, An Antibody Targeting Ferroportin, Is a Potent Inhibitor Of Hepcidin Activity and Increases Iron Mobilization In Normal Cynomolgus Monkeys

Hepcidin, a 25-amino acid peptide hormone which is primarily synthesized and secreted from the liver, is a key regulator of iron homeostasis. It regulates dietary iron absorption, plasma iron concentrations, and tissue iron distribution through interactions with ferroportin, the only known mammalian cellular iron exporter. Hepcidin induces the internalization and subsequent degradation of ferroportin. The reduction in iron release caused by the loss of ferroportin, combined with the continuing demand for iron by erythropoietic precursors, results in a decrease in circulating iron levels. Dysregulation of the hepcidin-ferroportin axis contributes to the pathogenesis of different anemias. Decreased synthesis of hepcidin may cause systemic iron overload in iron-loading anemias such as beta-thalassemia; whereas overproduction of hepcidin may contribute to the development of anemia in inflammatory disorders, malignancies, and chronic kidney disease. LY2928057 is a novel humanized IgG4 monoclonal antibody that binds to human ferroportin with a high affinity, blocks the binding of human hepcidin to ferroportin, and is a potent inhibitor of hepcidin activity in a recombinant ferroportin expressing HEK 293 cell-based assay. In addition, this antibody was able to significantly inhibit hepcidin-induced increase in ferritin levels using Caco-2 cells, a human enterocyte cell line that naturally expresses ferroportin. LY2928057 does not block the efflux of iron from ferroportin, nor does this antibody cause the internalization of this transporter in vitro. Administration of LY2928057 results in a dose dependent increase in serum iron and hepcidin in normal cynomolgus monkeys. LY2928057 may provide therapeutic benefit for patients with hepcidin-related anemia by stabilizing ferroportin located on the cell surface, thus restoring iron export and erythropoiesis. LY2928057 is currently in clinical evaluation. Disclosures:Witcher:Eli Lilly and Company: Employment, Equity Ownership. Leung:Eli Lilly and Company: Employment, Equity Ownership. Hill:Eli Lilly and Company: Employment, Equity Ownership. De Rosa:Eli Lilly and Company: Employment, Equity Ownership. Xu:Eli Lilly and Company: Employment, Equity Ownership. Manetta:Eli Lilly and Company: Employment, Equity Ownership. Wroblewski:Eli Lilly and Company: Employment, Equity Ownership. Benschop:Eli Lilly and Company: Employment, Equity Ownership.

A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates

AbstractIron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

Cysteine Promoted C‐Terminal Hydrazinolysis of Native Peptides and Proteins

Cysteine Promoted C‐Terminal Hydrazinolysis of Native Peptides and Proteins

Antifungal Activity of the Noncytotoxic Human Peptide Hepcidin 20 against Fluconazole-Resistant Candida glabrata in Human Vaginal Fluid

Vaginal infections caused by Candida glabrata are difficult to eradicate due to this species' scarce susceptibility to azoles. Previous studies have shown that the human cationic peptide hepcidin 20 (Hep-20) exerts fungicidal activity in sodium phosphate buffer against a panel of C. glabrata clinical isolates with different levels of susceptibility to fluconazole. In addition, the activity of the peptide was potentiated under acidic conditions, suggesting an application in the topical treatment of vaginal infections. To investigate whether the peptide activity could be maintained in biological fluids, in this study the antifungal activity of Hep-20 was evaluated by a killing assay in (i) a vaginal fluid simulant (VFS) and in (ii) human vaginal fluid (HVF) collected from three healthy donors. The results obtained indicated that the activity of the peptide was maintained in VFS and HVF supplemented with EDTA. Interestingly, the fungicidal activity of Hep-20 was enhanced in HVF compared to that observed in VFS, with a minimal fungicidal concentration of 25 μM for all donors. No cytotoxic effect on human cells was exerted by Hep-20 at concentrations ranging from 6.25 to 100 μM, as shown by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide tetrazolium salt (XTT) reduction assay and propidium iodide staining. A piece of indirect evidence of Hep-20 stability was also obtained from coincubation experiments of the peptide with HVF at 37°C for 90 min and for 24 h. Collectively, these results indicate that this peptide should be further studied as a novel therapeutic agent for the topical treatment of vaginal C. glabrata infections.

HEPCIDIN. A BRIEF REVIEW

There can be no doubt that the many biological discoveries found in the research arena no matter how imperceptible, have a profound effect on the work of the clinical laboratory. To improve human health, scientific discoveries must be translated into practical applications. Such discoveries typically begin at ‘the bench’ with basic research in which scientists, including biomedical scientists study disease at a cellular or molecular level, then progress to the clinical level and eventually to the patient's ‘bedside’. This translational research has proven to be a powerful process that drives ultimately the clinical research engine to aid medical diagnosis and treatment. The never static repertoire of laboratory tests is forever being extended by translational research and the discovery and story of hepcidin that has triggered a virtual explosion of studies on iron metabolism and related disorders is an example of this process. The existence of this amino acid peptide hormone was unheard of a decade ago, but with its discovery in 2000 [1] from plasma ultrafiltrate and subsequently in urine in 2001 [2] , it not only opened the way to understand iron metabolism, but also helped in elucidating the pathomechanisms of many other diseases. In recent times international interest grew exponentially as the key role of hepcidin in body iron regulation became clearer. Confirmatory studies in regard to hepcidin’s role in a number of disorders in humans, associated either with its relative decrease or increase, have been carried out (Fig 1). [3-9] Until relatively recently the methods for the laboratory assessment of hepcidin were fairly cumbersome and somewhat technically demanding and mainly used in the research domain. They were centred on two types of assays: immunoassays based on anti-hepcidin antibodies using a reference standard of synthetic hepcidin [10, 11] and mass spectrometric assays that can detect the characteristic mass of the active 25 amino acid hepcidin species or its fragments. The analyser then quantifies the intensity of these peak(s) and compares them to a spiked internal standard (Fig 2). [12-17] Many inherent problems in regard to assaying hepcidin in biological fluids using traditional immunochemical methods based on the production of specific anti-hepcidin antibodies still exist. For example, the development of these types of assays has been hampered by several causes, including the following: (1) the compact structure of the peptide with few antigenic epitopes and its small size (only 25 amino acids); (2) the high degree of protection between animal genus, [18] with subsequent problems in developing an appropriate immunogenic response in host animals, and (3) limited availability of the necessary antigen. However, with the development of the first competitive enzyme-linked immunoassays (C-ELISA) for human hepcidin in serum, plasma [19] the block to its measurement in the clinical laboratory setting has more or less been eliminated, enabling further transitional research into this unique peptide hormone. In a recent study, [20] the authors described a prospective observational study to assess the ability of a hepcidin ELISA to ascertain the cause of anaemia among critically ill anaemic patients. In a more recent pilot study [21] , the measurement of urinary hepcidin was proposed as a means of evaluating the need for iron supplementation.

PH‐dependent disruption of Escherichia coli ATCC 25922 and model membranes by the human antimicrobial peptides hepcidin 20 and 25

The human hepcidin 25 (hep-25) and its isoform hepcidin 20 (hep-20) are histidine-containing, cystein rich, β-sheet structured peptides endowed with antimicrobial activity. We previously reported that, similar to other histidine-containing peptides, the microbicidal effects of hep-25 and hep-20 are highly enhanced at acidic pH. In the present study, we investigated whether pH influences the mode of action of hep-25 and hep-20 on Escherichia coli American Type Culture Collection 25922 and model membranes. A striking release of β-galactosidase by hepcidin-treated E. coli was observed at pH 5.0, whereas no inner membrane permeabilization capacity was seen at pH 7.4, even at bactericidal concentrations. Similar results were obtained by flow cytometry when assessing the internalization of propidium iodide by hepcidin-treated E. coli. Scanning electron microscope imaging revealed that both peptides induced the formation of numerous blebs on the surface of bacterial cells at acidic pH but not at neutral pH. Moreover, a phospholipid/polydiacetylene colourimetric vesicle assay revealed a more evident membrane damaging effect at pH 5.0 than at pH 7.4. The leakage of entrapped dextrans of increasing molecular size from liposomes was also assessed at pH 7.4. Consistent with the lack of β-galactosidase release from whole E. coli observed at such a pH value, evident leakage of only the smallest 4-kDa dextran (and not of dextrans of 20 or 70 kDa) was observed, indicating a poor ability of hepcidin peptides to permeabilize liposome vesicles at pH 7.4. Altogether, the data obtained in the present study using different approaches strongly suggest that the ability of hepcidins to perturb bacterial membranes is markedly pH-dependent.

Hepatitis C virus core+1/ARF protein decreases hepcidin transcription through an AP1 binding site

Chronic viral hepatitis C is characterized by iron accumulation in the liver, and hepcidin regulates iron absorption. Hepatitis C virus (HCV) core+1/ARFP is a novel protein produced by a second functional ORF within the core gene. Here, using reporter assays and HCV bicistronic replicons, we show that, similarly to core, core+1/ARFP decreases hepcidin expression in hepatoma cells. The activator protein 1 (AP1) binding site of the human hepcidin promoter, shown here to be relevant to basal promoter activity and to the repression by core, is essential for the downregulation by core+1/ARFP while the previously described C/EBP (CCAAT/enhancer binding protein) and STAT (signal transducer and activator of transcription) sites are not. Consistently, expression of the AP1 components c-jun and c-fos obliterated the repressive effect of core and core+1/ARFP. In conclusion, we provide evidence that core+1/ARFP downregulates AP1-mediated transcription, providing new insights into the biological role of core+1/ARFP, as well as the transcriptional modulation of hepcidin, the main regulator of iron metabolism.

Functional analyses of recombinant mouse hepcidin-1 in cell culture and animal model

Hepcidin is a peptide hormone that plays an important role in iron metabolism. We have produced a recombinant mouse hepcidin-1 by using baculovirus expression system. Its expression yield was 25 μg/ml when cell culture media were supplemented with a protease inhibitor cocktail. The recombinant mouse hepcidin-1 and synthetic human hepcidin-25 had similar effects on reducing ferroportin expression in J774A cell line and in peritoneal macrophages. However, synthetic human hepcidin-25 was more efficient than recombinant mouse hepcidin-1 in reducing iron concentration in blood circulation (p < 0.01).

A Chemical Screen Reveals New Modulators of Hepcidin Expression

Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing the human Hepcidin promoter upstream of a firefly reporter gene. We then used high throughput methods to screen 10,360 chemicals in duplicate for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change &gt;;3 standard deviations above or &gt;;1.5 standard deviations below the mean of the other chemicals, while not adversely affecting cell viability. Using these criteria, we identified 32 small molecules that upregulated and 3 that downregulated Hepcidin expression. On retesting assays, we confirmed 22 of the initial positives (69%) and 1 of the initial negatives as regulators of Hepcidin expression. Functional classification of the positive regulators indicated: 4 anti‐inflammatory agents, 4 antimicrobials, 6 antineoplastic drugs, 4 kinase inhibitors, and 4 with other or unknown functions. We are now evaluating the mechanisms of action in human hepatocytes and in a zebrafish model. The best candidates will subsequently be tested in mouse models of iron overload syndromes. Funding: NIDDK R01 DK085250–01A1.

The effects of the anti-hepcidin Spiegelmer NOX-H94 on inflammation-induced anemia in cynomolgus monkeys

AbstractAnemia of chronic inflammation is the most prevalent form of anemia in hospitalized patients. A hallmark of this disease is the intracellular sequestration of iron. This is a consequence of hepcidin-induced internalization and subsequent degradation of ferroportin, the hepcidin receptor and only known iron-export protein. This study describes the characterization of novel anti-hepcidin compound NOX-H94, a structured L-oligoribonucleotide that binds human hepcidin with high affinity (Kd = 0.65 ± 0.06 nmol/L). In J774A.1 macrophages, NOX-H94 blocked hepcidin-induced ferroportin degradation and ferritin expression (half maximal inhibitory concentration = 19.8 ± 4.6 nmol/L). In an acute cynomolgus monkey model of interleukin 6 (IL-6)–induced hypoferremia, NOX-H94 inhibited serum iron reduction completely. In a subchronic model of IL-6–induced anemia, NOX-H94 inhibited the decrease in hemoglobin concentration. We conclude that NOX-H94 protects ferroportin from hepcidin-induced degradation. Therefore, this pharmacologic approach may represent an interesting treatment option for patients suffering from anemia of chronic inflammation.

Validation and Preclinical Correlation of a New Sandwich ELISA for Measuring Murine Hepcidin.

AbstractAbstract 2100Although enzyme-linked immunosorbent assays (ELISA) exist for measuring human hepcidin, no commercially available antibodies to mouse hepcidin are validated for this purpose. Thus, we developed an ELISA to measure mouse hepcidin using low plasma volumes to allow for repeat testing of individual mice over time. To this end, human hepcidin knock-in mice (Hep2 mice) were used to generate a panel of anti mouse hepcidin antibodies. A sandwich ELISA using these antibodies was validated. Pooled plasma from iron-deficient (combining diet and phlebotomy), iron-replete, and inflamed (injecting sub-clinical amounts of lipopolysaccharide) FVB mice was used to prepare frozen aliquots of low, medium, and high reagent control plasma. These samples were tested on 5 different days (n=8 wells/sample/day). Mean hepcidin levels for the high, medium, and low reagent were 791, 504, and 117 ng/mL, respectively. The intra-assay coefficient of variation (CV) was 10, 4.5, and 15.6% for the high, medium, and low samples, respectively. Inter-assay CV was 4.7, 4.0, and 11.1% for the high, medium, and low samples, respectively. Analytical sensitivity was 0.7 ng/mL and the assay was linear (Pearson r=0.99) to 500 ng/mL at a 1:100 dilution of sample to blocking buffer (i.e. only 1 uL of sample needed per test). With 20 plasma samples spanning the range of expected hepcidin levels, there was high correlation between hepcidin determined by ELISA and the sum of hepcidin-1 and hepcidin-2 by time-of-flight mass spectrometry (Pearson r = 0.99; P<0.0001). Stability was analyzed using samples from repeated freeze-thaw cycles at −80°C or from incubation at 4°C. Each freeze-thaw cycle induced up to a 15% loss in hepcidin. Overnight refrigeration led to a 31%, 21%, and 4% decrease in hepcidin for the high, medium, and low samples, respectively. Severe hemolysis (i.e. 8 g/dL hemoglobin) caused interference, decreasing apparent hepcidin levels up to 52%; mild hemolysis (0.1 g/dL hemoglobin) decreased hepcidin levels up to 15%. To provide clinical correlation in mouse models, we used cohorts (n=5/group) of C57BL/6 females with differing iron status: 1.Iron deficient: weanling mice on a low iron diet (0–5 ppm FeSO4; AIN diet) for 8 weeks2.Iron-replete: weanling mice on a normal iron diet (45 ppm FeSO4; AIN diet) for 8 weeks3.Iron-overload: mice received weekly iron-dextran injections (5 mg for 7 weeks)4.Transfusional iron-overload: mice received weekly syngeneic RBC transfusions (1 unit for 7 weeks)Hepcidin mirrored ferritin levels and total liver iron measured at necropsy (see Table). To test whether this ELISA can track hepcidin during infection, and whether iron status affects this response, iron-deficient or -replete mice (n=10/group) were inoculated with 1000 colony forming units of Salmonella typhimurium and blood samples obtained pre-infection and 1–7 days post-infection to measure hepcidin and interleukin (IL)-6. Pre-infection, iron-deficient mice had significantly lower hepcidin levels than iron-replete mice (<0.7 vs. 136 ± 56 ng/mL, mean ± sd). On Day 7 post-infection, IL-6 was much higher in surviving iron-deficient mice (up to >2000 pg/mL) than in iron-replete mice (207 ± 70 pg/mL). Despite dramatic increases in IL-6, hepcidin increased less in iron-deficient mice (260 ± 132 ng/mL) than in iron-replete mice (636 ± 273 ng/mL). Thus, despite a stronger inflammatory stimulus, hepcidin levels are muted in iron-deficient animals. In conclusion, this validated ELISA is a new tool that can be used to study iron regulation in mice.Liver iron (μg)Ferritin (ng/mL)Hepcidin (ng/mL)Iron replete62 ± 10849 ± 100204 ± 32Iron deficient31 ± 5387 ± 1142.4 ± 1Iron overload (Fe-Dextran)11531 ± 1163109912 ± 11872633 ± 54Iron overload (Transfusion)352 ± 13302 ± 872323 ± 64Data presented as mean ± sd. Disclosures:Winters:Amgen, Inc.: Employment. Cooke:Amgen, Inc.: Employment. Sasu:Amgen, Inc.: Employment.

A Screen for Small Molecules That Regulate Hepcidin Expression

Abstract 611Hepcidin, a peptide hormone produced in the liver, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. To generate a tool for identifying small molecules that modulate Hepcidin expression, we stably transfected human hepatocytes (HepG2) cells with a reporter construct containing 3 kilobases of the human Hepcidin promoter upstream of a firefly reporter gene. We then used high throughput methods to screen 10,360 chemicals in duplicate from the Harvard Institute of Chemistry and Cell Biology library for their effect on Hepcidin expression and cell viability. Regulators were identified as chemicals that caused a change >3 standard deviations above or >1.5 standard deviations below the mean of the other chemicals (z-score >3 or <-1.5), while not adversely affecting cell viability, quantified by a nonlytic fluorescence assay. Using these criteria, we identified 32 small molecules that upregulated and 3 that downregulated Hepcidin expression. Functional classification of the positive regulators indicated: 4 anti-inflammatory agents, 4 antimicrobials, 6 antineoplastic drugs, 6 kinase inhibitors, and 12 with other or unknown function. Of the positive modulators, two were flavones, consistent with our prior discovery that the isoflavone genistein upregulates Hepcidin expression. Of the negative regulators, one was a kinase inhibitor and two were of unknown function. Experiments are underway to characterize the mechanism of action of these regulators. The best candidates will subsequently be tested in mouse models of iron overload syndromes with the intention of developing new therapies for diseases in which Hepcidin is inappropriately regulated. Disclosures:No relevant conflicts of interest to declare.

Iron Regulator Hepcidin Exhibits Antiviral Activity against Hepatitis C Virus

Hepatitis C viral infection affects 170 million people worldwide. It causes serious chronic liver diseases. HCV infection has been implicated in iron accumulation in the liver and iron overload has been shown to be a potential cofactor for HCV associated hepatocellular carcinoma progression. The underlying mechanisms are not understood. Human hepcidin, a 25 amino acid peptide mainly produced by hepatocytes, is a key regulator of iron metabolism. Alteration of hepcidin expression levels has been reported in the setting of chronic HCV infection and hepatocellular carcinoma. In this study, we aim to examine the interactions between HCV infection and hepcidin expression in liver cells. We found that hepcidin expression was suppressed in HCV infected cells. The suppressive effect appears to be regulated by histone acetylation but not DNA methylation. Moreover, we found that hepcidin had a direct antiviral activity against HCV replication in cell culture. The antiviral effect is associated with STAT3 activation. In conclusion, hepcidin can induce intracellular antiviral state while HCV has a strategy to suppress hepcidin expression. This may be a novel mechanism by which HCV circumvents hepatic innate antiviral defense.

A Key Regulator For Iron Homeostasis in chronic hepatitis C.

Background: Hepcidin is a small, cysteine-rich cationic peptide produced by hepatocytes. There is a single human hepcidin gene; whose essential role in iron homeostasis was confirmed by identifying homozygous frameshift or nonsense mutations in affected individuals with severe Juvenile hemochromatosis. IL-6 may be the mediator of hepcidin induction by inflammation. Hypoferremia is a common response to systemic infections or generalized inflammatory disorders, anemia of chronic disease occurs in patients with acute and chronic immune activation and represents an important clinical problem. Aim of the work: The study will attempt to determine the hepatic hepcidin expression levels in patients with chronic hepatitis C virus infection. Patients and methods: Fifty patients with chronic hepatitis C virus infection (CHCV), their age between (20- 55) years, selected from the National Hepatology and Tropical Medicine Research Institute were included in this study, before interferon and Ribavirin therapy, and ten healthy individuals were included to serve as controls. All the patients and controls were subjected to the following history, clinical examination, abdominal ultrasonography and collection of blood samples for routine laboratory investigations. CBCs and serological assay for serum ferritin, iron, transferrin (s-TFR) levels, Liver biopsy for hepcidin mRNA levels and iron deposits in liver by (PCR) polymerase chain reaction. All subjects gave written informed consent for enrolment in the study, which was approved by the Research Ethical Committee of the General Organization for Teaching Hospitals and Institutes. Liver biopsy was taken from healthy subjects during abdominal surgery. Results: Our results revealedthat hepatic hepcidin expression is considered highly valid marker in case of CHCV infection. Conclusion: Our study concluded thatthere’s a highly significant inverse correlation between hepcidin versus liver iron, serum iron and serum transferrin but there’s no significant correlation versus ferritin. Recommendations: Hepcidin measuring and manipulating hepcidin levels will, in the future, have a role in diagnosing and treating any number of iron related disorders.Hepcidin itself has antimicrobial properties of uncertain importance so that careful clinical trials will be required to define appropriate indications of hepcidin antagonists.

A Key Regulator For Iron Homeostasis in chronic hepatitis C.

Background: Hepcidin is a small, cysteine-rich cationic peptide produced by hepatocytes. There is a single human hepcidin gene; whose essential role in iron homeostasis was confirmed by identifying homozygous frameshift or nonsense mutations in affected individuals with severe Juvenile hemochromatosis. IL-6 may be the mediator of hepcidin induction by inflammation. Hypoferremia is a common response to systemic infections or generalized inflammatory disorders, anemia of chronic disease occurs in patients with acute and chronic immune activation and represents an important clinical problem. Aim of the work: The study will attempt to determine the hepatic hepcidin expression levels in patients with chronic hepatitis C virus infection. Patients and methods: Fifty patients with chronic hepatitis C virus infection (CHCV), their age between (20- 55) years, selected from the National Hepatology and Tropical Medicine Research Institute were included in this study, before interferon and Ribavirin therapy, and ten healthy individuals were included to serve as controls. All the patients and controls were subjected to the following history, clinical examination, abdominal ultrasonography and collection of blood samples for routine laboratory investigations. CBCs and serological assay for serum ferritin, iron, transferrin (s-TFR) levels, Liver biopsy for hepcidin mRNA levels and iron deposits in liver by (PCR) polymerase chain reaction. All subjects gave written informed consent for enrolment in the study, which was approved by the Research Ethical Committee of the General Organization for Teaching Hospitals and Institutes. Liver biopsy was taken from healthy subjects during abdominal surgery. Results: Our results revealedthat hepatic hepcidin expression is considered highly valid marker in case of CHCV infection. Conclusion: Our study concluded thatthere’s a highly significant inverse correlation between hepcidin versus liver iron, serum iron and serum transferrin but there’s no significant correlation versus ferritin. Recommendations: Hepcidin measuring and manipulating hepcidin levels will, in the future, have a role in diagnosing and treating any number of iron related disorders.Hepcidin itself has antimicrobial properties of uncertain importance so that careful clinical trials will be required to define appropriate indications of hepcidin antagonists.