Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material.

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

Iron-related markers are associated with infection after liver transplantation.

Though serum iron has been known to be associated with an increased risk of infection, hepcidin, the major regulator of iron metabolism, has never been systematically explored in this setting. Finding early biomarkers of infection, such as hepcidin, could help identify patients in whom early empiric antimicrobial therapy would be beneficial. We prospectively enrolled consecutive patients (n = 128) undergoing first-time, single-organ orthotopic liver transplantation (OLT) without known iron overload disorders at 2 academic hospitals in Boston from August 2009 to November 2012. Cox regression compared the associations between different iron markers and the development of first infection at least 1 week after OLT; 47 (37%) patients developed a primary outcome of infection at least 1 week after OLT and 1 patient died. After adjusting for perioperative bleeding complications, number of hospital days, and hepatic artery thrombosis, changes in iron markers were associated with the development of infection post-OLT including increasing ferritin (hazard ratio [HR], 1.51; 95% confidence interval [CI], 1.12-2.05), rising ferritin slope (HR, 1.10; 95% CI, 1.03-1.17), and increasing hepcidin (HR, 1.43; 95% CI, 1.05-1.93). A decreasing iron (HR, 1.76; 95% CI, 1.20-2.57) and a decreasing iron slope (HR, 4.21; 95% CI, 2.51-7.06) were also associated with subsequent infections. In conclusion, hepcidin and other serum iron markers and their slope patterns or their combination are associated with infection in vulnerable patient populations. Liver Transplantation 23 1541-1552 2017 AASLD.

Effects of subcutaneous and intravenous golimumab on inflammatory biomarkers in patients with rheumatoid arthritis: results of a phase 1, randomized, open-label trial.

OBJECTIVE: To evaluate the effects of the anti-TNF-α monoclonal antibody golimumab, administered by s.c. injection or i.v. infusion, on markers of inflammation in patients with RA. METHODS: In this phase 1, open-label study, patients with active RA were randomized to receive s.c. golimumab 100 mg at baseline and every 4 weeks through week 20 (n = 33; group 1) or i.v. golimumab 2 mg/kg at baseline and week 12 (n = 16; group 2). Serum levels of CRP, IL-6, serum amyloid A (SAA), TNF receptor II (TNFRII), MMP-3, hyaluronic acid, haptoglobin, ferritin and haemoglobin and serum/urine hepcidin were measured at various time points. Associations between the biomarkers were assessed with Spearman's correlations. RESULTS: In both groups 1 and 2, decreases in mean serum levels of CRP, IL-6, SAA, TNFRII, MMP-3, haptoglobin, ferritin and hepcidin, and mean urine levels of hepcidin occurred within 1 week and were sustained through week 8. Decreases in concentrations of serum CRP, IL-6, SAA, MMP-3, hepcidin, ferritin and haptoglobin and urine hepcidin were maintained through week 24 in group 1, but began to reverse after week 8 in group 2. Among all patients, decreases in serum hepcidin correlated significantly with decreases in serum CRP and ferritin. CONCLUSION: Decreases in serum and urine concentrations of markers of inflammation occurred as early as 24 h after treatment with golimumab, and most of these improvements were sustained through week 24 in group 1.

Circulating non-transferrin-bound iron after oral administration of supplemental and fortification doses of iron to healthy women: a randomized study.

BACKGROUND: After the oral administration of iron, the production of circulating non-transferrin-bound iron may contribute to an increased risk of illness in malaria-endemic areas that lack effective medical services. OBJECTIVE: In healthy women with a range of body iron stores, we aimed to determine effects on the production of circulating non-transferrin-bound iron resulting from the oral administration of 1) a supplemental dose of iron (60 mg) with water, 2) a supplemental dose of iron (60 mg) with a standard test meal, and 3) a fortification dose of iron (6 mg) with a standard test meal. DESIGN: With the use of serum ferritin as the indicator, healthy women with replete iron stores (ferritin concentration >25 µg/L; n = 16) and reduced iron stores (ferritin concentration ≤25 µg/L; n = 16) were enrolled in a prospective, randomized, crossover study. After the oral administration of aqueous solutions of ferrous sulfate isotopically labeled with 54Fe, 57Fe, or 58Fe, blood samples were collected for 8 h, and iron absorption was estimated by erythrocyte incorporation at 14 d. RESULTS: At 4 h, serum non-transferrin-bound iron reached peaks with geometric mean (95% CI) concentrations of 0.81 µmol/L (0.56, 1.1 µmol/L) for 60 mg Fe with water and 0.26 µmol/L (0.15, 0.38 µmol/L) for 60 mg Fe with food but was at assay limits of detection (0.1 µmol Fe/L) for 6 mg Fe with food. For the 60 mg Fe without food, the area under the curve over 8 h for serum non-transferrin-bound iron was positively correlated with the amount of iron absorbed (R = 0.49, P < 0.01) and negatively correlated with serum ferritin (R = -0.39, P < 0.05). CONCLUSIONS: In healthy women, the production of circulating non-transferrin-bound iron is determined by the rate and amount of iron absorbed. The highest concentrations of non-transferrin-bound iron resulted from the administration of supplemental doses of iron without food. Little or no circulating non-transferrin-bound iron resulted from the consumption of a meal with a fortification dose of iron.

Streptococcus iniae phosphoglucomutase is a virulence factor and a target for vaccine development.

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 DeltaPGM mutant cells reaching sizes approximately 3-fold larger than those of the wild type (WT). The DeltaPGM 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 DeltaPGM 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 DeltaPGM mutant. In analysis of the aborted infectious process, we found that DeltaPGM 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 DeltaPGM 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 DeltaPGM mutant is able to stimulate a protective immune response and may have value as a live attenuated vaccine for aquaculture.

Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections.

Bass hepcidin was purified from the gill of hybrid striped bass (Morone chrysops x Morone saxatilis) based on antimicrobial activity against Escherichia coli. This 21-amino acid peptide has 8 cysteines engaged in 4 disulfide bonds and is very similar to human hepcidin, an antimicrobial peptide with iron regulatory properties. To gain insight into potential role(s) of bass hepcidin in innate immunity in fish, we synthesized the peptide, characterized its antimicrobial activities in vitro, determined its solution structure by NMR, and quantified hepatic gene expression in vivo following infection of bass with the fish pathogens, Streptococcus iniae or Aeromonas salmonicida. Its structure is very similar to that of human hepcidin, including the presence of an antiparallel beta-sheet, a conserved disulfide-bonding pattern, and a rare vicinal disulfide bond. Synthetic bass hepcidin was active in vitro against Gram-negative pathogens and fungi but showed no activity against key Gram-positive pathogens and a single yeast strain tested. Hepcidin was non-hemolytic at microbicidal concentrations and had lower specific activity than moronecidin, a broad spectrum, amphipathic, alpha-helical, antimicrobial peptide constitutively expressed in bass gill tissue. Good synergism between the bacterial killing activities of hepcidin and moronecidin was observed in vitro. Hepcidin gene expression in bass liver increased significantly within hours of infection with Gram-positive (S. iniae) or Gram-negative (A. salmonicida) pathogens and was 4-5 orders of magnitude above base-line 24-48 h post-infection. Our results suggest that hepcidin plays a key role in the antimicrobial defenses of bass and that its functions are potentially conserved between fish and human.

Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge.

We report the isolation of a novel antimicrobial peptide, bass hepcidin, from the gill of hybrid striped bass, white bass (Morone chrysops) x 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-kappaB, 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.

Early reversal of pediatric-neonatal septic shock by community physicians is associated with improved outcome.

OBJECTIVE: Experimental and clinical studies of septic shock support the concept that early resuscitation with fluid and inotropic therapies improves survival in a time-dependent manner. The new American College of Critical Care Medicine-Pediatric Advanced Life Support (ACCM-PALS) Guidelines for hemodynamic support of newborns and children in septic shock recommend this therapeutic approach. The objective of this study was to determine whether early septic shock reversal and use of resuscitation practice consistent with the new ACCM-PALS Guidelines by community physicians is associated with improved outcome. METHODS: A 9-year (January 1993-December 2001) retrospective cohort study was conducted of 91 infants and children who presented to local community hospitals with septic shock and required transport to Children's Hospital of Pittsburgh. Shock reversal (defined by return of normal systolic blood pressure and capillary refill time), resuscitation practice concurrence with ACCM-PALS Guidelines, and hospital mortality were measured. RESULTS: Overall, 26 (29%) patients died. Community physicians successfully achieved shock reversal in 24 (26%) patients at a median time of 75 minutes (when the transport team arrived at the patient's bedside), which was associated with 96% survival and >9-fold increased odds of survival (9.49 [1.07-83.89]). Each additional hour of persistent shock was associated with >2-fold increased odds of mortality (2.29 [1.19-4.44]). Nonsurvivors, compared with survivors, were treated with more inotropic therapies (dopamine/dobutamine [42% vs 20%] and epinephrine/norepinephrine [42% vs 6%]) but not increased fluid therapy (median volume; 32.9 mL/kg vs 20.0 mL/kg). Resuscitation practice was consistent with ACCM-PALS Guidelines in only 27 (30%) patients; however, when practice was in agreement with guideline recommendations, a lower mortality was observed (8% vs 38%). CONCLUSIONS: Early recognition and aggressive resuscitation of pediatric-neonatal septic shock by community physicians can save lives. Educational programs that promote ACCM-PALS recommended rapid, stepwise escalations in fluid as well as inotropic therapies may have value in improving outcomes in these children.

Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass.

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 alpha-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 alpha-helical, broad spectrum antimicrobial peptide isolated from the skin and gills of hybrid striped bass.