We developed and validated the first serum enzyme-linked immunosorbent assay for hepcidin, the principal iron-regulatory hormone that has been very difficult to measure. In healthy volunteers, the 5% to 95% range of hepcidin concentrations was 29 to 254 ng/mL in men (n ‫؍‬ 65) and 17 to 286 ng/mL in women (n ‫؍‬ 49), with median concentrations 112 versus 65 (P < .001). The lower limit of detection was 5 ng/mL. Serum hepcidin concentrations in 24 healthy subjects correlated well with their urinary hepcidin (r ‫؍‬ 0.82). Serum hepcidin appropriately correlated with serum ferritin (r ‫؍‬ 0.63), reflecting the regulation of both proteins by iron stores. Healthy volunteers showed a diurnal increase of serum hepcidin at noon and 8 pm compared with 8 am, and a transient rise of serum hepcidin in response to iron ingestion. Expected alterations in hepcidin levels were observed in a variety of clinical conditions associated with iron disturbances. Serum hepcidin concentrations were undetectable or low in patients with iron deficiency anemia (ferritin < 10 ng/mL), iron-depleted HFE hemochromatosis, and juvenile hemochromatosis. Serum hepcidin concentrations were high in patients with inflammation (C-reactive protein > 10 mg/ dL), multiple myeloma, or chronic kidney disease. The new serum hepcidin enzymelinked immunosorbent assay yields accurate and reproducible measurements that appropriately reflect physiologic, pathologic, and genetic influences, and is informative about the etiology of iron disorders. IntroductionHepcidin is the principal iron-regulatory hormone that mediates the homeostasis of extracellular iron concentrations. 1,2 Although the peptide is initially synthesized as an 84-amino acid preprohepcidin, it is processed in hepatocytes by a signal peptidase and the prohormone convertase furin 3 to its bioactive form, a 25-amino acid peptide circulating in plasma 4,5 and excreted in urine. 5 Hepcidin acts by regulating iron influx into plasma from tissues engaged in iron storage or transport: duodenal enterocytes that absorb dietary iron, hepatocytes that store iron, and macrophages that recycle iron from senescent erythrocytes. At the molecular level, hepcidin binds to the sole known cellular iron efflux channel, ferroportin, and induces its internalization and lysosomal degradation 6 by mechanisms similar to those that inactivate other more conventional membrane receptors. N-terminally truncated breakdown products of hepcidin are detectable in plasma 7 and urine 5,8 but show impaired ability to internalize ferroportin. 9 Hepcidin synthesis is physiologically increased by elevated plasma iron concentration, 10,11 decreased by erythropoietic activity, 12 and pathologically increased by inflammation. 10,13 Hepcidin excess plays the major role in anemia of inflammation [14][15][16] and iron-resistant iron-deficiency anemia. [17][18][19][20] At the opposite extreme, hepcidin deficiency is the cause of iron overload in most hereditary hemochromatoses 21 and contributes to iron overload in ␤-thalassemia and other ir...

Background and objectives: Hepcidin is a key regulator of iron homeostasis, but its study in the setting of chronic kidney disease (CKD) has been hampered by the lack of validated serum assays.Design, setting, participants, & measurements: This study reports the first measurements of bioactive serum hepcidin using a novel competitive ELISA in 48 pediatric (PCKD2-4) and 32 adult (ACKD2-4) patients with stages 2 to 4 CKD along with 26 pediatric patients with stage 5 CKD (PCKD5D) on peritoneal dialysis.Results: When compared with their respective controls (pediatric median ‫؍‬ 25.3 ng/ml, adult ‫؍‬ 72.9 ng/ml), hepcidin was significantly increased in PCKD2-4 (127.3 ng/ml), ACKD2-4 (269.9 ng/ml), and PCKD5D (652.4 ng/ml). Multivariate regression analysis was used to assess the relationship between hepcidin and indicators of anemia, iron status, inflammation, and renal function. In PCKD2-4 (R 2 ‫؍‬ 0.57), only ferritin correlated with hepcidin. In ACKD2-4 (R 2 ‫؍‬ 0.78), ferritin and soluble transferrin receptor were associated with hepcidin, whereas GFR was inversely correlated. In PCKD5D (R 2 ‫؍‬ 0.52), percent iron saturation and ferritin were predictors of hepcidin. In a multivariate analysis that incorporated all three groups (R 2 ‫؍‬ 0.6), hepcidin was predicted by ferritin, C-reactive protein, and whether the patient had stage 5D versus stages 2 to 4 CKD.Conclusions: These findings suggest that increased hepcidin across the spectrum of CKD may contribute to abnormal iron regulation and erythropoiesis and may be a novel biomarker of iron status and erythropoietin resistance.

The antibacterial protein hepcidin regulates the absorption, tissue distribution, and extracellular concentration of iron by suppressing ferroportin-mediated export of cellular iron. In CKD, elevated hepcidin and vitamin D deficiency are associated with anemia. Therefore, we explored a possible role for vitamin D in iron homeostasis. Treatment of cultured hepatocytes or monocytes with prohormone 25-hydroxyvitamin D or active 1,25-dihydroxyvitamin D decreased expression of hepcidin mRNA by 0.5-fold, contrasting the stimulatory effect of 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D on related antibacterial proteins such as cathelicidin. Promoterreporter and chromatin immunoprecipitation analyses indicated that direct transcriptional suppression of hepcidin gene (HAMP) expression mediated by 1,25-dihydroxyvitamin D binding to the vitamin D receptor caused the decrease in hepcidin mRNA levels. Suppression of HAMP expression was associated with a concomitant increase in expression of the cellular target for hepcidin, ferroportin protein, and decreased expression of the intracellular iron marker ferritin. In a pilot study with healthy volunteers, supplementation with a single oral dose of vitamin D (100,000 IU vitamin D 2 ) increased serum levels of 25D-hydroxyvitamin D from 2762 ng/ml before supplementation to 4463 ng/ml after supplementation (P,0.001). This response was associated with a 34% decrease in circulating levels of hepcidin within 24 hours of vitamin D supplementation (P,0.05). These data show that vitamin D is a potent regulator of the hepcidin-ferroportin axis in humans and highlight a potential new strategy for the management of anemia in patients with low vitamin D and/or CKD.

We isolated a novel 22-residue, C-terminally amidated antimicrobial peptide, moronecidin, from the skin and gill of hybrid striped bass. Two isoforms, differing by only one amino acid, are derived from each parental species, white bass (Morone chrysops) and striped bass (Morone saxatilis). Molecular masses (2543 and 2571 Da), amino acid sequences (FFHHIFRGIVHVGKTIH(K/R) LVTGT), cDNA, and genomic DNA sequences were determined for each isoform. A predicted 79-residue moronecidin prepropeptide consists of three domains: a signal peptide (22 amino acids), a mature peptide (22 amino acids), and a C-terminal prodomain (35 amino acids). The synthetic, amidated white bass moronecidin exhibited broad spectrum antimicrobial activity that was retained at high salt concentration. An ␣-helical structure was confirmed by circular dichroism spectroscopy. The moronecidin gene consists of three introns and four exons. Peptide sequence and gene organization were similar to pleurocidin, an antimicrobial peptide from winter flounder. A TATA box and several consensus-binding motifs for transcription factors were found in the region 5 to the transcriptional start site. Moronecidin gene expression was detected in gill, skin, intestine, spleen, anterior kidney, and blood cells by kinetic reverse transcription (RT)-PCR. Thus, moronecidin is a new ␣-helical, broad spectrum antimicrobial peptide isolated from the skin and gills of hybrid striped bass.

We report the isolation of a novel antimicrobial peptide, bass hepcidin, from the gill of hybrid striped bass, white bass (Morone chrysops) · striped bass (M. saxatilis). After the intraperitoneal injection of Micrococcus luteus and Escherichia coli, the peptide was purified from HPLC fractions with antimicrobial activity against Escherichia coli. Sequencing by Edman degradation revealed a 21-residue peptide (GCRFCCNCCPNMSGCGVCCRF) with eight putative cysteines. Molecular mass measurements of the native peptide and the reduced and alkylated peptide confirmed the sequence with four intramolecular disulfide bridges. Peptide sequence homology to human hepcidin and other predicted hepcidins, indicated that the peptide is a new member of the hepcidin family. Nucleotide sequences for cDNA and genomic DNA were determined for white bass. A predicted prepropeptide (85 amino acids) consists of three domains: a signal peptide (24 amino acids), prodomain (40 amino acids) and a mature peptide (21 amino acids). The gene has two introns and three exons. A TATA box and several consensus-binding motifs for transcription factors including C/EBP, nuclear factor-jB, and hepatocyte nuclear factor were found in the region upstream of the transcriptional start site. In white bass liver, hepcidin gene expression was induced 4500-fold following challenge with the fish pathogen, Streptococcus iniae, while expression levels remained low in all other tissues tested. A novel antimicrobial peptide from the gill, bass hepcidin, is predominantly expressed in the liver and highly inducible by bacterial exposure.Keywords: antimicrobial peptide; fish; hepcidin; innate immunity; Streptococcus iniae.Antimicrobial peptides (AMPs) are a broadly distributed group of molecules that are important in host defense against microbial invasion. A growing number of peptides involved in innate immunity have been isolated from plants, invertebrates, and higher vertebrates. Human hepcidin and liver-expressed antimicrobial peptide (LEAP-1) are identical AMPs, which were isolated independently from urine and human blood ultrafiltrate, respectively [1,2]. Peptide sequences of additional hepcidins have been predicted from expressed sequence tag databases from the liver of mouse Fish have evolved to thrive in an aqueous environment with a rich microbial flora, and several AMPs have been isolated from fish [7]. During our search for AMPs from gills of hybrid striped bass, three RP-HPLC fractions with antimicrobial acitivity were found [8]. One contained moronecidin, a 22-residue AMP with an amphipathic a-helical structure. From two other adjacent fractions, we isolated another novel AMP, bass hepcidin, a 21-residue, cysteine-rich peptide, which is a homologue of human hepcidin. We report here the first hepcidin to be isolated from a nonhuman vertebrate, the first cysteine-rich AMP isolated from fish, and the first demonstration of hepcidin gene expression induced by live bacterial challenge. M A T E R I A L S A N D M E T H O D STissue collection and purification of ba...

Streptococcus iniae represents a major health and economic problem in fish species worldwide. Random Tn917 mutagenesis and high-throughput screening in a hybrid striped bass (HSB) model of meningoencephalitis identified attenuated S. iniae mutants. The Tn917 insertion in one mutant disrupted an S. iniae homologue of a phosphoglucomutase (pgm) gene. Electron microscopy revealed a decrease in capsule thickness and cell wall rigidity, with ⌬PGM mutant cells reaching sizes ϳ3-fold larger than those of the wild type (WT). The ⌬PGM mutant was cleared more rapidly in HSB blood and was more sensitive to killing by cationic antimicrobial peptides including moronecidin from HSB. In vivo, the ⌬PGM mutant was severely attenuated in HSB, as intraperitoneal challenge with 1,000 times the WT lethal dose produced only 2.5% mortality. Reintroduction of an intact copy of the S. iniae pgm gene on a plasmid vector restored antimicrobial peptide resistance and virulence to the ⌬PGM mutant. In analysis of the aborted infectious process, we found that ⌬PGM mutant organisms initially disseminated to the blood, brain, and spleen but were eliminated by 24 h without end organ damage. Ninety to 100% of fish injected with the ⌬PGM mutant and later challenged with a lethal dose of WT S. iniae survived. We conclude that the pgm gene is required for virulence in S. iniae, playing a role in normal cell wall morphology, surface capsule expression, and resistance to innate immune clearance mechanisms. An S. iniae ⌬PGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.With increased development of intensive operations, disease has become a significant hurdle to the profitable culture of fish and shellfish. Streptococcal infections in fish, in particular those produced by the pathogen Streptococcus iniae, have increased markedly with intensification of aquaculture practices (37). S. iniae causes a fatal meningoencephalitis and is associated with large-scale mortality in a wide variety of marine and freshwater cultured fish species, as well as in wild species (5,39,46). More than 30 species of fish have documented susceptibility to S. iniae disease, including trout (10), yellowtail (19), tilapia (39), barramundi (6), and hybrid striped bass (HSB) (11). S. iniae is distributed globally and is estimated to cause yearly economic losses of hundreds of millions of dollars. Occasionally, S. iniae can cause serious zoonotic infections in humans who injure themselves while handling infected fish (20,42).Relatively little is known of the genetics of S. iniae or of the pathogenic mechanisms underlying its virulence. Here, we describe a severely attenuated mutant of S. iniae, identified through random transposon mutagenesis and direct screening for virulence in HSB. Analysis of the transposon insertion site revealed a disruption of an open reading frame (ORF) with similarity to bacterial phosphoglucomutase (pgm) genes. The enzyme phosphoglucomutase (PGM) interconverts glucose-6-phosphate an...

BackgroundHepcidin plays a key role in body iron metabolism by preventing the release of iron from macrophages and intestinal cells. Defective hepcidin synthesis causes iron loading, while overproduction results in defective reticuloendothelial iron release and iron absorption.

At term pregnancy, hepcidin concentrations are very low, allowing maximal availability of iron for the fetus. Maternal and cord blood hepcidin levels were independently associated with either maternal or cord blood iron status.