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BACKGROUND: Absolute plasma hepcidin concentrations measured by various procedures differ substantially, complicating interpretation of results and rendering reference intervals method dependent. We investigated the degree of equivalence achievable by harmonization and the identification of a commutable secondary reference material to accomplish this goal. METHODS: We applied technical procedures to achieve harmonization developed by the Consortium for Harmonization of Clinical Laboratory Results. Eleven plasma hepcidin measurement procedures (5 mass spectrometry based and 6 immunochemical based) quantified native individual plasma samples (n = 32) and native plasma pools (n = 8) to assess analytical performance and current and achievable equivalence. In addition, 8 types of candidate reference materials (3 concentrations each, n = 24) were assessed for their suitability, most notably in terms of commutability, to serve as secondary reference material. RESULTS: Absolute hepcidin values and reproducibility (intrameasurement procedure CVs 2.9%-8.7%) differed substantially between measurement procedures, but all were linear and correlated well. The current equivalence (intermeasurement procedure CV 28.6%) between the methods was mainly attributable to differences in calibration and could thus be improved by harmonization with a common calibrator. Linear regression analysis and standardized residuals showed that a candidate reference material consisting of native lyophilized plasma with cryolyoprotectant was commutable for all measurement procedures. Mathematically simulated harmonization with this calibrator resulted in a maximum achievable equivalence of 7.7%. CONCLUSIONS: The secondary reference material identified in this study has the potential to substantially improve equivalence between hepcidin measurement procedures and contributes to the establishment of a traceability chain that will ultimately allow standardization of hepcidin measurement results.
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Servicios de Laboratorio Clínico/normas , Hepcidinas/sangre , Cooperación Internacional , Humanos , Inmunoquímica , Modelos Lineales , Estándares de ReferenciaRESUMEN
BACKGROUND: Hepcidin is considered the master regulator of iron homoeostasis. Novel hepcidin antagonists have recently been introduced as potential treatment for iron-restricted anaemia. Meanwhile, serum hepcidin has been shown to be positively associated with cardiovascular disease and inversely with acute kidney injury. These properties may lead to contrasting effects, especially in renal transplant recipients (RTR), which are prone to cardiovascular diseases and graft failure. To date, the role of serum hepcidin in RTR is unknown. We, therefore, prospectively determined the association of serum hepcidin with risk of graft failure, cardiovascular mortality and all-cause mortality in RTR. MATERIALS AND METHODS: Serum hepcidin was assessed in an extensively phenotyped RTR cohort by dual-monoclonal sandwich ELISA specific immunoassay. Statistical analyses were performed using univariate linear regression followed by stepwise backward linear regression. Cox proportional hazard regression models were performed to determine prospective associations. RESULTS: We included 561 RTR (age 51 ± 12 years). Mean haemoglobin (Hb) was 8·6 ± 1·0 mM. Median [IQR] serum hepcidin was 7·2 [3·2-13·4] ng/mL. Mean estimated glomerular filtration rate was 47 ± 16 mL/min/1·73 m2 . In univariate Cox regression analyses, serum hepcidin was not associated with risk of graft failure, cardiovascular mortality or all-cause mortality. Notably, after adjustment for high sensitivity C-reactive protein and ferritin, serum hepcidin became negatively associated with all-cause mortality (hazard ratio 0·89; 95% confidence interval 0·80-0·99, P = 0·03). CONCLUSIONS: In this study, we did not find an association between serum hepcidin and outcomes, that is graft failure, cardiovascular mortality or all-cause mortality. Based on our results, it is questionable whether serum hepcidin may be used to predict a beneficial effect of hepcidin antagonists.
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Lesión Renal Aguda/sangre , Enfermedades Cardiovasculares/sangre , Hepcidinas/sangre , Fallo Renal Crónico/cirugía , Trasplante de Riñón , Adulto , Proteína C-Reactiva/metabolismo , Enfermedades Cardiovasculares/mortalidad , Causas de Muerte , Ensayo de Inmunoadsorción Enzimática , Ferritinas/sangre , Humanos , Fallo Renal Crónico/sangre , Modelos Lineales , Persona de Mediana Edad , Mortalidad , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Tasa de SupervivenciaRESUMEN
AIMS/HYPOTHESIS: The central nervous system (CNS) is a major player in the regulation of food intake. The gut hormone glucagon-like peptide-1 (GLP-1) has been proposed to have an important role in this regulation by relaying information about nutritional status to the CNS. We hypothesised that endogenous GLP-1 has effects on CNS reward and satiety circuits. METHODS: This was a randomised, crossover, placebo-controlled intervention study, performed in a university medical centre in the Netherlands. We included patients with type 2 diabetes and healthy lean control subjects. Individuals were eligible if they were 40-65 years. Inclusion criteria for the healthy lean individuals included a BMI <25 kg/m(2) and normoglycaemia. Inclusion criteria for the patients with type 2 diabetes included BMI >26 kg/m(2), HbA1c levels between 42 and 69 mmol/mol (6.0-8.5%) and treatment for diabetes with only oral glucose-lowering agents. We assessed CNS activation, defined as blood oxygen level dependent (BOLD) signal, in response to food pictures in obese patients with type 2 diabetes (n = 20) and healthy lean individuals (n = 20) using functional magnetic resonance imaging (fMRI). fMRI was performed in the fasted state and after meal intake on two occasions, once during infusion of the GLP-1 receptor antagonist exendin 9-39, which was administered to block actions of endogenous GLP-1, and on the other occasion during saline (placebo) infusion. Participants were blinded for the type of infusion. The order of infusion was determined by block randomisation. The primary outcome was the difference in BOLD signal, i.e. in CNS activation, in predefined regions in the CNS in response to viewing food pictures. RESULTS: All patients were included in the analyses. Patients with type 2 diabetes showed increased CNS activation in CNS areas involved in the regulation of feeding (insula, amygdala and orbitofrontal cortex) in response to food pictures compared with lean individuals (p ≤ 0.04). Meal intake reduced activation in the insula in response to food pictures in both groups (p ≤ 0.05), but this was more pronounced in patients with type 2 diabetes. Blocking actions of endogenous GLP-1 significantly prevented meal-induced reductions in bilateral insula activation in response to food pictures in patients with type 2 diabetes (p ≤ 0.03). CONCLUSIONS/INTERPRETATION: Our findings support the hypothesis that endogenous GLP-1 is involved in postprandial satiating effects in the CNS of obese patients with type 2 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT 01363609. Funding The study was funded in part by a grant from Novo Nordisk.
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Diabetes Mellitus Tipo 2/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Periodo Posprandial , Recompensa , Respuesta de Saciedad , Adulto , Anciano , Estudios Cruzados , Diabetes Mellitus Tipo 2/psicología , Femenino , Alimentos , Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/agonistas , Hemoglobina Glucada/análisis , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Obesidad/sangre , Oxígeno/sangre , Fragmentos de Péptidos/efectos adversos , Fragmentos de Péptidos/uso terapéutico , Estimulación LuminosaRESUMEN
Small interfering RNA (siRNA) is gaining momentum as a therapeutic modality with six approved products. Since siRNA has the potential to elicit undesired immune responses in patients, immunogenicity assessment is required during clinical development by regulatory authorities. In this study, anti-siRNA polyclonal antibodies were generated through animal immunization. These cross-reactive polyclonal antibodies recognized mostly the N-acetylgalactosamine (GalNAc) moiety with a small fraction against sequence-independent epitopes. We demonstrate that the polyclonal antibodies can be utilized as immunogenicity assay positive controls for the same class of GalNAc-conjugated siRNAs. In addition, anti-GalNAc mAbs showed desired sensitivity and drug tolerance, supporting their use as alternative surrogate positive controls. These findings can guide positive control selection and immunogenicity assay development for GalNAc-conjugated siRNAs and other oligonucleotide therapeutics.
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Acetilgalactosamina , Oligonucleótidos , Animales , Humanos , ARN Interferente Pequeño/genética , Anticuerpos MonoclonalesRESUMEN
During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.
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Apolipoproteínas A/química , Apolipoproteínas A/metabolismo , Hipertrigliceridemia/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Apolipoproteína A-V , Apolipoproteínas A/genética , Western Blotting , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Liposomas/química , Liposomas/metabolismo , Masculino , Persona de Mediana Edad , Mutagénesis Sitio-Dirigida , Mutación , Adulto JovenRESUMEN
BACKGROUND: Glucose-dependent insulinotropic peptide (GIP) is an incretin peptide secreted by intestinal K cells that stimulates insulin secretion in a glucose-dependent manner. It is secreted as an active, intact 42-amino acid peptide GIP(1-42), which is rapidly degraded by dipeptidyl peptidase 4 to GIP(3-42), which is inactive. There is currently no described monoclonal antibody-based sandwich immunoassay to quantify concentrations of GIP(1-42), the active form of the peptide. METHODS: To create a sandwich ELISA for GIP(1-42), we generated a monoclonal antibody specific for the intact N-terminus of the peptide, which was further optimized to increase its affinity. We used this antibody as a conjugate antibody in a sandwich ELISA and paired it with an anti-total GIP capture monoclonal antibody to create a dual monoclonal sandwich ELISA for GIP(1-42). RESULTS: The sandwich ELISA was highly specific for GIP(1-42) and did not recognize GIP(3-42). The ELISA demonstrated a broad dynamic range and a lower limit of quantification of 5 ng/L. Using the ELISA, we were able to show that GIP(1-42) concentrations in healthy volunteers increased dramatically in the postprandial state compared to the fasting state. GIP(1-42) values were correlated with total GIP values overall; however, there was substantial interindividual variation. CONCLUSIONS: The use of an N-terminal-specific monoclonal antibody in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of GIP(1-42), the active form of the incretin hormone. This ELISA should help to improve our understanding of the role of GIP(1-42) in regulating glucose-dependent insulin secretion.
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Anticuerpos Monoclonales , Polipéptido Inhibidor Gástrico/sangre , Incretinas/sangre , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Polipéptido Inhibidor Gástrico/inmunología , Humanos , Incretinas/inmunología , RatonesRESUMEN
INTRODUCTION: Apolipoprotein (apo) A-II is the second most abundant high-density lipoprotein (HDL) apolipoprotein. We assessed the mechanism involved in the altered postprandial triglyceride-rich lipoprotein metabolism of female human apoA-II-transgenic mice (hapoA-II-Tg mice), which results in up to an 11-fold increase in plasma triglyceride concentration. The relationships between apoA-II, HDL composition, and lipoprotein lipase (LPL) activity were also analyzed in a group of normolipidemic women. METHODS AND RESULTS: Triglyceride-rich lipoprotein catabolism was decreased in hapoA-II-Tg mice compared to control mice. This suggests that hapoA-II, which was mainly associated with HDL during fasting and postprandially, impairs triglyceride-rich lipoprotein lipolysis. HDL isolated from hapoA-II-Tg mice impaired bovine LPL activity. Two-dimensional gel electrophoresis, mass spectrometry, and immunonephelometry identified a marked deficiency in the HDL content of apoA-I, apoC-III, and apoE in these mice. In normolipidemic women, apoA-II concentration was directly correlated with plasma triglyceride and inversely correlated with the HDL-apoC-II+apoE/apoC-III ratio [corrected]. HDL-mediated induction of LPL activity was inversely correlated with apoA-II and directly correlated with the HDL-apoC-II+apoE/apoC-III ratio [corrected]. Purified hapoA-II displaced apoC-II, apoC-III, and apoE from human HDL2. Human HDL3 was, compared to HDL2, enriched in apoA-II but poorer in apoC-II, apoC-III, and apoE. CONCLUSIONS: ApoA-II plays a crucial role in triglyceride catabolism by regulating LPL activity, at least in part, through HDL proteome modulation.
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Apolipoproteína A-II/sangre , Lipólisis , Lipoproteína Lipasa/sangre , Lipoproteínas HDL2/sangre , Lipoproteínas HDL3/sangre , Triglicéridos/sangre , Animales , Apolipoproteína A-I/sangre , Apolipoproteína A-II/genética , Apolipoproteína C-II/sangre , Apolipoproteína C-III/sangre , Apolipoproteínas E/sangre , Biomarcadores/sangre , Grasas de la Dieta/metabolismo , Electroforesis en Gel Bidimensional , Femenino , Humanos , Intestinos/enzimología , Hígado/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nefelometría y Turbidimetría , Periodo Posprandial , Proteoma , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Aim: We present a novel methodology to compare results between distinct immunogenicity assays, performed by two laboratories, for the same biotherapeutic. Materials & methods: Human serum pools from clinical trials were generated to provide representative immunogenicity titers. Pools were evaluated at two laboratories in a blinded fashion to assess the effect of assay format and laboratory change on clinical interpretation of immunogenicity results. Results: The laboratories validated two different assay formats and demonstrated comparable sensitivity and drug tolerance. Overall, the comparisons in assay format and laboratory ensured a comparable ability to detect treatment-emergent antidrug antibodies for a biotherapeutic. Conclusion: We have established an approach, using pooling of patient samples, that allows for the interlaboratory comparisons without creating duplicative results.
Lay abstract Measuring immunogenicity, an immune response to a drug, is important in understanding the benefits and risks associated with a drug. Immunogenicity is measured by specific tests within a laboratory; however, these tests and laboratories may change over time. This paper proposes a method to determine if a change in test and laboratory will impact the interpretation of immunogenicity for a drug. Blood samples from clinical trial patients were combined in order to provide representative samples for the immunogenicity tests. The samples were tested at two laboratories with two tests to measure if any interpretation of immunogenicity results would change due to the different tests and laboratories. Laboratories and tests demonstrated similar and reliable results of the samples. This study has established a method which allows for the comparison of immunogenicity results when tests and/or laboratories are changed.
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Polyethylene glycol (PEG) represents an effective strategy to improve the pharmacokinetic profile of a molecule as it extends the biotherapeutic's half-life, masks immunogenic epitopes or modifies its distribution. The addition of one or multiple PEG moieties, in either linear or branched form, is known to carry the risk of potentially inducing an immunogenic response against PEG. The importance of accurately quantifying anti-PEG antibodies during a clinical study is well recognized and stems from the fact that anti-PEG antibodies have been shown to negatively impact the efficacy of the biotherapeutic that the PEG is coupled to. As a consequence, sponsors are encouraged to develop immunogenicity assays to assess appropriately the presence of anti-drug antibodies (ADA) against the protein component as well as the PEG. However, detection of anti-PEG antibodies is complicated by a number of technical challenges, including the availability of appropriate positive control material. In addition, the fact that some anti-PEG antibodies are known to circulate as low-affinity IgM, drives the need for an assay able to detect low affinity anti-PEG ADA even in the presence of high concentrations of the biotherapeutic. To address this need, we developed and validated an Affinity Capture Elution (ACE)-AGL assay to detect anti-drug and anti-PEG antibodies. In this assay, which we call ACE-AGL, ADA are captured by biotin-PEG-drug, acid eluted and re-captured on a second plate coated with protein AGL. ADA are then detected using Ruthenium-PEG-drug. The new assay format described is highly sensitive to both anti-drug and anti-PEG antibodies and very drug-tolerant. The ACE-AGL assay is easy to perform and has been successfully validated at two separate CROs. We propose the ACE-AGL format as a valid and effective alternative to the currently available assay methods.
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Productos Biológicos/inmunología , Excipientes/química , Inmunoensayo , Inmunoglobulina M/sangre , Polietilenglicoles/química , Proteínas Recombinantes/inmunología , Adulto , Productos Biológicos/química , Ácidos Cólicos/química , Detergentes/química , Composición de Medicamentos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polisorbatos/química , Proteínas Recombinantes/química , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
BACKGROUND: Anemia and vitamin D deficiency are highly prevalent in critical illness, and vitamin D status has been associated with hemoglobin concentrations in epidemiologic studies. We examined the effect of high-dose vitamin D therapy on hemoglobin and hepcidin concentrations in critically ill adults. MATERIALS AND METHODS: Mechanically ventilated critically ill adults (N = 30) enrolled in a pilot double-blind, randomized, placebo-controlled trial of high-dose vitamin D3 (D3 ) were included in this analysis. Participants were randomized to receive placebo, 50,000 IU D3 , or 100,000 IU D3 daily for 5 days (totaling 250,000 IU D3 and 500,000 IU D3 , respectively). Blood was drawn weekly throughout hospitalization for up to 4 weeks. Linear mixed-effects models were used to assess change in hemoglobin and hepcidin concentrations by treatment group over time. RESULTS: At enrollment, >75% of participants in all groups had plasma 25-hydroxyvitamin D (25(OH)D) concentrations <30 ng/mL, and >85% of participants across groups were anemic. In the 500,000-IU D3 group, hemoglobin concentrations increased significantly over time (Pgroup × time = .01) compared with placebo but did not change in the 250,000-IU D3 group (Pgroup × time = 0.59). Hepcidin concentrations decreased acutely in the 500,000-IU D3 group relative to placebo after 1 week (P = .007). Hepcidin did not change significantly in the 250,000-IU D3 group. CONCLUSION: In these critically ill adults, treatment with 500,000 IU D3 was associated with increased hemoglobin concentrations over time and acutely reduced serum hepcidin concentrations. These findings suggest that high-dose vitamin D may improve iron metabolism in critical illness and should be confirmed in larger studies.
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Colecalciferol/uso terapéutico , Cuidados Críticos/métodos , Hemoglobinas/efectos de los fármacos , Respiración Artificial , Vitaminas/uso terapéutico , Anciano , Enfermedad Crítica , Método Doble Ciego , Femenino , Hepcidinas/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Proyectos PilotoRESUMEN
BACKGROUND & AIMS: In vitro studies suggest that vitamin D may reduce hepcidin expression and pro-inflammatory cytokine release from monocytes. However, data assessing the vitamin D-mediated effects on iron recycling in healthy individuals are lacking. We aimed to examine the effect of high-dose vitamin D3 on plasma hepcidin, inflammatory cytokine, and ferritin concentrations in healthy adults. METHODS: This was a pilot, double-blind, placebo-controlled trial in healthy adults (N = 28) randomized to receive a one-time oral dose of 250,000 IU of vitamin D3 or placebo. Between- and within-group differences in plasma hepcidin, pro-inflammatory cytokine [interleukin (IL)-1ß, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1)], and ferritin concentrations at baseline and 1 week were determined using two-sample and paired t-tests, respectively. RESULTS: At baseline, plasma 25-hydroxyvitamin D [25(OH)D], hepcidin, pro-inflammatory cytokine, and ferritin concentrations did not differ between the two groups, and greater than 70% of subjects in both groups were vitamin D deficient (25(OH)D < 20 ng/mL). After 1 week, plasma hepcidin concentrations decreased by 73% from baseline in those who received vitamin D3 (geometric mean ratio [GMR] = 0.27 (95% CI: 0.11-0.62); P = 0.005); there was no significant change in the placebo group (GMR = 0.73 (95% CI: 0.49-1.09); P = 0.11). Plasma cytokine and ferritin concentrations did not change significantly in either group. CONCLUSIONS: High-dose vitamin D3 significantly reduced plasma hepcidin concentrations in healthy adults 1 week post-dosing, without a change in plasma pro-inflammatory cytokine or ferritin concentrations. These data suggest that vitamin D may have a role in regulating iron recycling by acting independently of changes in pro-inflammatory markers.
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Anemia Ferropénica/dietoterapia , Colecalciferol/administración & dosificación , Suplementos Dietéticos , Regulación hacia Abajo , Hepcidinas/sangre , Estado Nutricional , Deficiencia de Vitamina D/dietoterapia , Adulto , Anemia Ferropénica/sangre , Anemia Ferropénica/complicaciones , Anemia Ferropénica/epidemiología , Enfermedades Asintomáticas/epidemiología , Enfermedades Asintomáticas/terapia , Biomarcadores/sangre , Calcifediol/sangre , Colecalciferol/efectos adversos , Colecalciferol/uso terapéutico , Estudios de Cohortes , Citocinas/sangre , Suplementos Dietéticos/efectos adversos , Método Doble Ciego , Femenino , Ferritinas/sangre , Georgia/epidemiología , Humanos , Masculino , Proyectos Piloto , Prevalencia , Deficiencia de Vitamina D/sangre , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/epidemiología , Adulto JovenRESUMEN
OBJECTIVE: New-onset diabetes after transplantation (NODAT) is a major complication in renal transplant recipients (RTRs). Cholesterol metabolism has been linked to diabetes development. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is crucial in LDL receptor regulation. Its association with NODAT is unknown. We prospectively determined the association between serum PCSK9 levels and NODAT development and then with all-cause mortality, cardiovascular mortality, and renal graft failure. RESEARCH DESIGN AND METHODS: In a university setting, nondiabetic RTRs recruited between 2001 and 2003 with a functional graft for ≥1 year were eligible. Serum PCSK9 was measured by ELISA. Cox proportional hazards analysis was used to assess the association of PCSK9 with the development of NODAT, all-cause mortality, cardiovascular mortality, and graft failure. RESULTS: In 453 RTRs (age 51 ± 12 years, 56% male; 6.1 [2.7-11.7] years after transplantation), serum PCSK9 was 107.1 ± 43.4 µg/L. During a median follow-up of 10 years, 70 RTRs developed NODAT, 123 died, and 59 developed graft failure. NODAT occurred more frequently in the upper PCSK9 tertile (23%) versus the lowest two PCSK9 tertiles (12%; P < 0.001). In crude Cox regression analyses, PCSK9 was significantly associated with development of NODAT (hazard ratio 1.34 [95% CI 1.10-1.63]) per SD change (P = 0.004). This association remained independent of adjustment for potential confounders, including statin use. PCSK9 was not associated with all-cause mortality, cardiovascular mortality, or graft failure. CONCLUSIONS: Circulating PCSK9 is associated with NODAT in RTRs. The PCSK9 pathway may contribute to the pathogenesis of NODAT.
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Diabetes Mellitus/sangre , Trasplante de Riñón/efectos adversos , Proproteína Convertasa 9/sangre , Adulto , LDL-Colesterol/sangre , Diabetes Mellitus/etiología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Factores de RiesgoRESUMEN
Growth/differentiation factor 15 (GDF15), also known as MIC-1, is a distant member of the transforming growth factor-ß (TGF-ß) superfamily and has been implicated in various biological functions, including cancer cachexia, renal and heart failure, atherosclerosis and metabolism. A connection between GDF15 and body-weight regulation was initially suggested on the basis of an observation that increasing GDF15 levels in serum correlated with weight loss in individuals with advanced prostate cancer. In animal models, overexpression of GDF15 leads to a lean phenotype, hypophagia and other improvements in metabolic parameters, suggesting that recombinant GDF15 protein could potentially be used in the treatment of obesity and type 2 diabetes. However, the signaling and mechanism of action of GDF15 are poorly understood owing to the absence of a clearly identified cognate receptor. Here we report that GDNF-family receptor α-like (GFRAL), an orphan member of the GFR-α family, is a high-affinity receptor for GDF15. GFRAL binds to GDF15 in vitro and is required for the metabolic actions of GDF15 with respect to body weight and food intake in vivo in mice. Gfral-/- mice were refractory to the effects of recombinant human GDF15 on body-weight, food-intake and glucose parameters. Blocking the interaction between GDF15 and GFRAL with a monoclonal antibody prevented the metabolic effects of GDF15 in rats. Gfral mRNA is highly expressed in the area postrema of mouse, rat and monkey, in accordance with previous reports implicating this region of the brain in the metabolic actions of GDF15 (refs. 4,5,6). Together, our data demonstrate that GFRAL is a receptor for GDF15 that mediates the metabolic effects of GDF15.
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Área Postrema/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor 15 de Diferenciación de Crecimiento/farmacología , Obesidad/metabolismo , Pérdida de Peso/efectos de los fármacos , Animales , Encéfalo/metabolismo , Ingestión de Alimentos/genética , Citometría de Flujo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Células HEK293 , Humanos , Immunoblotting , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Resonancia por Plasmón de Superficie , Pérdida de Peso/genéticaRESUMEN
OBJECTIVE: Hepcidin reduces iron absorption by binding to the intestinal iron transporter ferroportin, thereby causing its degradation. Although short-term administration of testosterone or growth hormone (GH) has been reported to decrease circulating hepcidin levels, little is known about how hepcidin is influenced in human endocrine conditions associated with anemia. RESEARCH DESIGN AND METHODS: We used a sensitive and specific dual-monoclonal antibody sandwich immunoassay to measure hepcidin-25 in patients (a) during initiation of in vitro fertilization when endogenous estrogens were elevated vs. suppressed, (b) with GH deficiency before and after 12 months substitution treatment, (c) with hyperthyroidism before and after normalization, and (d) with hyperprolactinemia before and after six months of treatment with a dopamine agonist. RESULTS: In response to a marked stimulation of endogenous estrogen production, median hepcidin levels decreased from 4.85 to 1.43 ng/mL (p < 0.01). Hyperthyroidism, hyperprolactinemia, or GH substitution to GH-deficient patients did not influence serum hepcidin-25 levels. CONCLUSIONS: In humans, gonadotropin-stimulated endogenous estrogen markedly decreases circulating hepcidin-25 levels. No clear and stable correlation between iron biomarkers and hepcidin-25 was seen before or after treatment of hyperthyroidism, hyperprolactinemia or growth hormone deficiency.
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Anemia/sangre , Estrógenos/fisiología , Hepcidinas/sangre , Adolescente , Adulto , Anciano , Anemia/metabolismo , Proteína C-Reactiva/biosíntesis , Proteína C-Reactiva/metabolismo , Agonistas de Dopamina/química , Femenino , Ferritinas/biosíntesis , Ferritinas/metabolismo , Fertilización In Vitro , Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/metabolismo , Humanos , Hiperprolactinemia/complicaciones , Hipertiroidismo/complicaciones , Inmunoensayo , Hierro/metabolismo , Masculino , Persona de Mediana Edad , Prolactinoma/sangre , Transferrina/biosíntesis , Transferrina/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Increased emphasis on the development of biologics has placed a significant focus on anti-drug antibody (ADA) detection. To address this need, several immunoassay formats have been described for use in characterizing potential immune responses. Two commonly utilized methods include the affinity capture elution (ACE) and bridging formats. While these approaches have been effective in supporting many clinical initiatives, both possess potential disadvantages. Here, we compare these standard methods to a novel format that addresses these noted drawbacks. RESULTS: A novel assay format has been designed to incorporate the benefits of the ACE and bridging methods while overcoming the disadvantages incurred with each approach. The described ACE-Bridge format exhibits excellent sensitivity and precision while providing superior drug tolerance when compared to bridging formats. Further, this assay format is not susceptible to the endogenous target interference that can be an issue in the ACE format. CONCLUSIONS: The ACE-Bridge format provides an often superior option as a screening method to monitor patient ADA responses. This method is unique in its ability to measure ADA in the presence of high circulating endogenous target concentrations (>100 ng/mL) while demonstrating very high drug tolerance.
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Anticuerpos/sangre , Anticuerpos/inmunología , Inmunoensayo/métodos , Proteínas/inmunología , Proteínas/uso terapéutico , HumanosRESUMEN
AIM: Immunogenicity testing of biotherapeutic drugs is a regulatory requirement. Herein, we describe a drug-tolerant assay for detecting neutralizing antibodies against a therapeutic antibody. RESULTS: Excess target of the therapeutic antibody was incorporated into the detection step of an affinity capture elution assay. Signal generated from binding of antidrug antibody (ADA) to the therapeutic antibody was compared with signal from binding of ADA to the therapeutic antibody preincubated with its target. The results demonstrated that the target blocked binding of the therapeutic antibody to neutralizing monkey ADA and to two anti-idiotypic antibodies. CONCLUSION: This highly drug-tolerant novel approach enables the detection of neutralizing antibodies and allows for one basic assay format to achieve complete characterization of ADA responses.
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Anticuerpos Antiidiotipos/sangre , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/sangre , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/inmunología , Biotina/química , Cromatografía de Afinidad , Haplorrinos , Humanos , InmunoensayoRESUMEN
Administration of blosozumab, a humanized monoclonal antibody that binds sclerostin, increases bone formation and bone mineral density (BMD) in postmenopausal women with low BMD. To evaluate the effect of discontinuing blosozumab, we studied women enrolled in a 1-year randomized, placebo-controlled phase 2 trial for an additional year after they completed treatment. Of the 120 women initially enrolled in the study, 106 women completed treatment and continued into follow-up; 88 women completed 1 year of follow-up. At the beginning of follow-up, groups remained balanced for age, race, and body mass index, but lumbar spine and total hip BMD were increased in prior blosozumab groups, reflecting an anabolic treatment effect. At the end of follow-up, 1 year after discontinuing treatment, lumbar spine BMD remained significantly greater than placebo in women initially treated with blosozumab 270 mg every 2 weeks (Q2W) and blosozumab 180 mg Q2W (6.9% and 3.6% above baseline, respectively). Total hip BMD also declined after discontinuation of treatment but at 1 year after treatment remained significantly greater than placebo in women initially treated with blosozumab 270 mg Q2W and blosozumab 180 mg Q2W (3.9% and 2.6% above baseline, respectively). During follow-up, median serum P1NP was not consistently different between the prior blosozumab groups and placebo. A similar pattern was apparent for median serum C-terminal telopeptide of type 1 collagen (CTx) levels, with more variability. Mean serum total sclerostin concentration increased with blosozumab, indicating target engagement, and declined to baseline after discontinuation. There were no adverse events considered related to prior treatment with blosozumab. Anti-drug antibodies generally declined in patients who had detectable levels during prior treatment. These findings support the continued study of blosozumab as an anabolic therapy for treatment of osteoporosis.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Densidad Ósea , Proteínas Morfogenéticas Óseas/sangre , Osteoporosis Posmenopáusica/tratamiento farmacológico , Posmenopausia , Proteínas Adaptadoras Transductoras de Señales , Anciano , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados/sangre , Conservadores de la Densidad Ósea/administración & dosificación , Conservadores de la Densidad Ósea/sangre , Proteínas Morfogenéticas Óseas/inmunología , Resorción Ósea , Método Doble Ciego , Esquema de Medicación , Femenino , Estudios de Seguimiento , Marcadores Genéticos/inmunología , Humanos , Persona de Mediana Edad , Osteogénesis , Esteroides/química , Factores de TiempoRESUMEN
Chronic kidney disease affects 40% of adults aged 65 and older. Anemia of CKD is present in 30% of patients with CKD and is associated with increased cardiovascular risk, decreased quality of life, and increased mortality. Hepcidin-25 (hepcidin), the key iron regulating hormone, prevents iron egress from macrophages and thus prevents normal recycling of the iron needed to support erythropoiesis. Hepcidin levels are increased in adults and children with CKD. Vitamin D insufficiency is highly prevalent in CKD and is associated with erythropoietin hyporesponsiveness. Recently, hepcidin levels were found to be inversely correlated with vitamin D status in CKD. The aim of this study was to investigate the role of vitamin D in the regulation of hepcidin expression in vitro and in vivo. This study reports that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D, is associated with decreased hepcidin and increased ferroportin expression in lipopolysaccharide (LPS) stimulated THP-1 cells. 1,25(OH)2D3 also resulted in a dose-dependent decrease in pro-hepcidin cytokines, IL-6 and IL-1ß, release in vitro. Further, we show that high-dose vitamin D therapy impacts systemic hepcidin levels in subjects with early stage CKD. These data suggest that improvement in vitamin D status is associated with lower systemic concentrations of hepcidin in subjects with CKD. In conclusion, vitamin D regulates the hepcidin-ferroportin axis in macrophages which may facilitate iron egress. Improvement in vitamin D status in patients with CKD may reduce systemic hepcidin levels and may ameliorate anemia of CKD.
RESUMEN
BACKGROUND: FGF21 is involved in glucose and lipid metabolism. Our objective was to develop two novel sandwich immunoassays that measure active and total levels of FGF21 in human plasma. RESULTS: Both immunoassays utilized affinity-optimized monoclonal antibodies generated specifically for either the mid-domain or the intact C-terminus of the respective FGF21 peptides. Total FGF21 levels measured from normal human plasma samples ranged from 42 to 462 pg/ml, while active FGF21 levels ranged from 11 to 399 pg/ml. The data also suggested no significant differences in the concentrations of active FGF21 when measured from pre- and post-prandial samples. CONCLUSION: We have successfully developed sensitive assays to measure active and total FGF21, which show the majority of total FGF21 in plasma is active FGF21.
Asunto(s)
Análisis Químico de la Sangre/métodos , Factores de Crecimiento de Fibroblastos/sangre , Inmunoensayo/métodos , Adolescente , Adulto , Anciano , Calibración , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Accurate measurement of a total protein target (free plus bound) is essential to optimize dose selection for monoclonal antibody drugs. Herein, we describe a novel sandwich immunoassay format in which the biotherapeutic antibody itself serves as the primary detection antibody. A signal is then generated through the addition of a labeled secondary antibody that recognizes the biotherapeutic antibody. The secondary antibody is conjugated with ruthenium to facilitate electrochemiluminescent analysis. RESULTS: Data are presented from the analysis of two protein biomarkers having disparate size and structure; a 4.5 kDa peptide and a 60 kDa protein. In both cases, validated, highly specific assays were developed and shown to be tolerant to elevated levels of the therapeutic monoclonal antibody in question. CONCLUSION: Our novel format allows drug-tolerant measurement of soluble protein biomarkers targeted by monoclonal antibodies when only two independent epitopes for antibody binding are available and one is recognized by the therapeutic antibody.