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In response to global challenges in resource supply, many industries are adopting the principles of the Circular Economy (CE) to improve their resource acquisition strategies. This paper introduces an innovative approach to address the environmental impact of waste Glass Fiber Reinforced-Polymer (GFRP) pipes and panels by repurposing them to manufacture structural components for new bicycle and pedestrian bridges. The study covers the entire process, including conceptualization, analysis, design, and testing of a deck system, with a focus on the manufacturing process for a 7-m-long prototype bridge. The study shows promising results in the concept of a sandwich structure utilizing discarded GFRP pipes and panels, which has the flexibility to account for variabilities in dimensions of incoming products while still meeting mechanical requirements. The LCA analysis shows that the transportation of materials is the governing contributing factor. It was concluded that further development of this concept should be accompanied by a business model that considers the importance of the contributions from the whole value chain.
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Polímeros , Polímeros/química , Reciclaje , Peatones , Transportes , Vidrio/químicaRESUMEN
BACKGROUND: Self-sampling of dried blood spots (DBS) offers new routes to gather valuable health-related information from the general population. Yet, the utility of using deep proteome profiling from home-sampled DBS to obtain clinically relevant insights about SARS-CoV-2 infections remains largely unexplored. METHODS: Our study involved 228 individuals from the general Swedish population who used a volumetric DBS sampling device and completed questionnaires at home during spring 2020 and summer 2021. Using multi-analyte COVID-19 serology, we stratified the donors by their response phenotypes, divided them into three study sets, and analyzed 276 proteins by proximity extension assays (PEA). After normalizing the data to account for variances in layman-collected samples, we investigated the association of DBS proteomes with serology and self-reported information. RESULTS: Our three studies display highly consistent variance of protein levels and share associations of proteins with sex (e.g., MMP3) and age (e.g., GDF-15). Studying seropositive (IgG+) and seronegative (IgG-) donors from the first pandemic wave reveals a network of proteins reflecting immunity, inflammation, coagulation, and stress response. A comparison of the early-infection phase (IgM+IgG-) with the post-infection phase (IgM-IgG+) indicates several proteins from the respiratory system. In DBS from the later pandemic wave, we find that levels of a virus receptor on B-cells differ between seropositive (IgG+) and seronegative (IgG-) donors. CONCLUSIONS: Proteome analysis of volumetric self-sampled DBS facilitates precise analysis of clinically relevant proteins, including those secreted into the circulation or found on blood cells, augmenting previous COVID-19 reports with clinical blood collections. Our population surveys support the usefulness of DBS, underscoring the role of timing the sample collection to complement clinical and precision health monitoring initiatives.
The COVID-19 pandemic has posed multiple challenges to healthcare systems. A significant gap that remains is a lack of understanding of the impact of SARS-CoV-2 on individuals who did not seek or require hospitalization. To address this, we distribute self-sampling devices to random citizens, aiming to analyze how blood protein levels are affected in people who have had COVID-19 but had no or mild symptoms. Conducting multiple molecular measurements in dried blood, our study confirms clinically known markers and their relationship to infection stages, even if the donors themselves collect the sample. Our work highlights the potential of combining self-sampling with laboratory methods to provide useful information on human health. This convenient patient-centric sampling approach may potentially be useful when studying other diseases.
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Protein biomarkers in biological fluids represent an important resource for improving the clinical management of diseases. Current proteomics technologies are capable of performing high-throughput and multiplex profiling in different types of fluids, often leading to the shortlisting of tens of candidate biomarkers per study. However, before reaching any clinical setting, these discoveries require thorough validation and an assay that would be suitable for routine analyses. In the path from biomarker discovery to validation, the performance of the assay implemented for the intended protein quantification is extremely critical toward achieving reliable and reproducible results. Development of robust sandwich immunoassays for individual candidates is challenging and labor and resource intensive, and multiplies when evaluating a panel of interesting candidates at the same time. Here we describe a versatile pipeline that facilitates the systematic and parallel development of multiple sandwich immunoassays using a bead-based technology.
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Líquidos Corporales/química , Ensayo de Inmunoadsorción Enzimática , Proteoma/análisis , Proteómica , Biomarcadores/análisis , HumanosRESUMEN
Serological testing is essential to curb the consequences of the COVID-19 pandemic. However, most assays are still limited to single analytes and samples collected within healthcare. Thus, we establish a multianalyte and multiplexed approach to reliably profile IgG and IgM levels against several versions of SARS-CoV-2 proteins (S, RBD, N) in home-sampled dried blood spots (DBS). We analyse DBS collected during spring of 2020 from 878 random and undiagnosed individuals from the population in Stockholm, Sweden, and use classification approaches to estimate an accumulated seroprevalence of 12.5% (95% CI: 10.3%-14.7%). This includes 5.4% of the samples being IgG+IgM+ against several SARS-CoV-2 proteins, as well as 2.1% being IgG-IgM+ and 5.0% being IgG+IgM- for the virus' S protein. Subjects classified as IgG+ for several SARS-CoV-2 proteins report influenza-like symptoms more frequently than those being IgG+ for only the S protein (OR = 6.1; p < 0.001). Among all seropositive cases, 30% are asymptomatic. Our strategy enables an accurate individual-level and multiplexed assessment of antibodies in home-sampled blood, assisting our understanding about the undiagnosed seroprevalence and diversity of the immune response against the coronavirus.
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Recolección de Muestras de Sangre/métodos , Prueba Serológica para COVID-19/métodos , COVID-19/inmunología , Inmunidad Humoral , Adulto , Anciano , Anticuerpos Antivirales/inmunología , COVID-19/etiología , Pruebas con Sangre Seca , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , SARS-CoV-2/inmunología , Suecia , Adulto JovenRESUMEN
In the era towards precision medicine, we here present the individual specific autoantibody signatures of 193 healthy individuals. The self-reactive IgG signatures are stable over time in a way that each individual profile is recognized in longitudinal sampling. The IgG autoantibody reactivity towards an antigen array comprising 335 protein fragments, representing 204 human proteins with potential relevance to autoimmune disorders, was measured in longitudinal plasma samples from 193 healthy individuals. This analysis resulted in unique autoantibody barcodes for each individual that were maintained over one year's time. The reactivity profiles, or signatures, are person specific in regards to the number of reactivities and antigen specificity. Two independent data sets were consistent in that each healthy individual displayed reactivity towards 0-16 antigens, with a median of six. Subsequently, four selected individuals were profiled on in-house produced high-density protein arrays containing 23,000 protein fragments representing 14,000 unique protein coding genes. Based on a unique, broad and deep longitudinal profiling of autoantibody reactivities, our results demonstrate a unique autoreactive profile in each analyzed healthy individual. The need and interest for broad-ranged and high-resolution molecular profiling of healthy individuals is rising. We have here generated and assessed an initial perspective on the global distribution of the self-reactive IgG repertoire in healthy individuals, by investigating 193 well-characterized healthy individuals. Highlights A unique longitudinal profiling of autoantibody repertoires in healthy individuals Autoantibody profiles are highly individual and stable over time All individuals display IgG binding to human protein fragments The specificity of disease associated autoantigens needs to be thoroughly characterized The identification of a small set of highly reactive autoantigens Importance of stringent antigen and sample specific cut-offs for defining reactivity.
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Especificidad de Anticuerpos , Autoanticuerpos , Autoantígenos , Inmunoglobulina G , Anciano , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Autoantígenos/sangre , Autoantígenos/inmunología , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana EdadRESUMEN
With the increasing availability of collections of antibodies, their evaluation in terms of binding selectivity becomes an important but challenging task. Planar antigen microarrays are very suitable tools to address this task and provide a powerful proteomics platform for the characterization of the binding selectivity of antibodies toward thousands of antigens in parallel. In this chapter, we describe our in-house developed procedures for the generation of high-density planar antigen microarrays with over 21,000 features. We also provide the details of the assay protocol, which we routinely use for the assessment of binding selectivity of the polyclonal antibodies generated within the Human Protein Atlas.
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Especificidad de Anticuerpos/inmunología , Antígenos/inmunología , Análisis por Matrices de Proteínas/métodos , Proteómica/métodos , Anticuerpos/inmunología , Antígenos/genética , Humanos , Unión Proteica/inmunologíaRESUMEN
PURPOSE: Affinity proteomic approaches by antibody bead arrays enable multiplexed analysis of proteins in body fluids. In the presented study, we investigated blood plasma within osteoporosis to discovery differential protein profiles and to propose novel biomarkers candidates for subsequent studies. EXPERIMENTAL DESIGN: Starting with 4608 antibodies and plasma samples from 22 women for an untargeted screening, a set of 72 proteins were suggested for further analysis. Complementing these with targets from literature and other studies, a targeted bead array of 180 antibodies was built to profile for 92 proteins in plasma samples of 180 women from two independent population-based studies. RESULTS: Differential profiles between osteoporosis patients and matched controls were discovered for 12 proteins in at least one of the two study sets. Among these targets, the levels of autocrine motility factor receptor (AMFR) were concordantly lower in plasma of female osteoporosis patients. Subsequently, verification of anti-AMFR antibody selectivity was conducted using high-density peptide and protein arrays, and Western blotting. CONCLUSIONS AND CLINICAL RELEVANCE: Further validation in additional study sets will be needed to determine the clinical value of the observed decrease in AMFR plasma levels in osteoporosis patients, but AMFR may aid our understanding of disease mechanisms and could support existing tools for diagnosis and monitoring of patient mobility within osteoporosis.
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Osteoporosis/diagnóstico , Proteómica/métodos , Receptores del Factor Autocrino de Motilidad/genética , Anciano , Secuencia de Aminoácidos , Anticuerpos/química , Especificidad de Anticuerpos , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Expresión Génica , Humanos , Persona de Mediana Edad , Osteoporosis/sangre , Osteoporosis/genética , Análisis por Matrices de Proteínas , Receptores del Factor Autocrino de Motilidad/sangreRESUMEN
High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions.
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Autoanticuerpos/sangre , Análisis por Matrices de Proteínas/métodos , Secuencia de Aminoácidos , Autoantígenos/genética , Autoantígenos/inmunología , Autoinmunidad , Biotecnología , Ensayos Analíticos de Alto Rendimiento , Humanos , Esclerosis Múltiple Crónica Progresiva/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Biblioteca de Péptidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
RATIONALE: There is a need to further characterize the antibody repertoire in relation to sarcoidosis and potentially related autoantigens. OBJECTIVES: We investigated bronchoalveolar lavage (BAL) and serum samples from patients with sarcoidosis and healthy and diseased control subjects to discover sarcoidosis-associated autoantigens. METHODS: Antigen microarrays built on 3,072 protein fragments were used to screen for IgG reactivity in 73 BAL samples from subjects with sarcoidosis, subjects with asthma, and healthy subjects. A set of 131 targets were selected for subsequent verification on suspension bead arrays using 272 additional BAL samples and 141 paired sera. Reactivity to four antigens was furthermore analyzed in 22 unprocessed BAL samples from patients with fibrosis and 269 plasma samples from patients diagnosed with myositis. MEASUREMENTS AND MAIN RESULTS: Reactivity toward zinc finger protein 688 and mitochondrial ribosomal protein L43 were discovered with higher frequencies in patients with sarcoidosis, for mitochondrial ribosomal protein L43 especially in patients with non-Löfgren syndrome. Increased reactivity toward nuclear receptor coactivator 2 was also observed in patients with non-Löfgren syndrome as compared with patients with Löfgren syndrome. The antigen representing adenosine diphosphate-ribosylation factor GTPase activating protein 1 revealed high reactivity frequency in all sample groups but with significantly higher level of IgG reactivities in patients with sarcoidosis. CONCLUSIONS: Autoantigen reactivity was present in most BAL and serum samples analyzed, and the results revealed high interindividual heterogeneity, with most of the reactivities observed in single individuals only. Four proteins are here proposed as sarcoidosis-associated autoimmune targets and of interest for further validation in independent cohorts.
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Autoantígenos/análisis , Sarcoidosis Pulmonar/diagnóstico , Sarcoidosis Pulmonar/inmunología , Adolescente , Adulto , Anciano , Biomarcadores/análisis , Líquido del Lavado Bronquioalveolar/química , Proteínas Portadoras/análisis , Proteínas Portadoras/sangre , Proteínas Portadoras/inmunología , Femenino , Proteínas Activadoras de GTPasa/análisis , Proteínas Activadoras de GTPasa/inmunología , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/análisis , Proteínas Mitocondriales/sangre , Proteínas Mitocondriales/inmunología , Coactivador 2 del Receptor Nuclear/análisis , Coactivador 2 del Receptor Nuclear/inmunología , Análisis por Matrices de Proteínas , Proteómica , Proteínas Ribosómicas/análisis , Proteínas Ribosómicas/sangre , Proteínas Ribosómicas/inmunología , Sarcoidosis Pulmonar/sangre , Adulto Joven , Dedos de Zinc/inmunologíaRESUMEN
A series of 6-substituted 3-(pyrrolidin-1-ylmethyl)chromen-2-ones (coumarins) have been synthesized and their inhibitory activity to human monoamine oxidase A (MAO A) and B (MAO B) determined. Incorporation of a basic amino function in the C3 position together with substitution at the C6 position produced novel coumarin compounds with selectivity for the MAO A subtype. Substitution in the C6 position with small hydrophilic groups such as hydroxy (19, IC50 = 1.46 µM) or amino (18, IC50 = 3.77 µM) gave the most potent and selective compounds for MAO A. These compounds also showed excellent aqueous solubility properties. Compound 18 [6-amino-3-(pyrrolidin-1-ylmethyl)chromen-2-one] administrated in vivo induced in rat brain a neurotransmitter metabolite profile typical of MAO A inhibition: decreased 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) but increased 3-methoxytyramine (3-MT) levels.
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Cromonas/síntesis química , Inhibidores de la Monoaminooxidasa/síntesis química , Monoaminooxidasa/metabolismo , Animales , Sitios de Unión , Cromonas/química , Cromonas/farmacología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Humanos , Microdiálisis , Modelos Moleculares , Estructura Molecular , Inhibidores de la Monoaminooxidasa/química , Inhibidores de la Monoaminooxidasa/farmacología , Neurotransmisores/metabolismo , Unión Proteica , Ratas , Solubilidad , Relación Estructura-ActividadRESUMEN
To further investigate the structure-activity relationship (SAR) of the 5-hydroxytryptamine type 6 (5-HT6) receptor agonist 5-chloro-2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (EMD386088, 6), a series of 2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles were synthesized, and in vitro affinity to, and functional activity at 5-HT6 receptors was tested. We focused on substituents made at the indole N(1)-, 2- and 5-positions and these were found to not only influence the affinity at 5-HT6 receptors but also the intrinsic activity leading to antagonists, partial agonists and full agonists. In order for a compound to demonstrate potent 5-HT6 receptor agonist properties, the indole N(1) should be unsubstituted, an alkyl group such as 2-methyl is needed and finally halogen substituents in the indole 5-position (fluoro, chloro or, bromo) were essential requirements. However, the introduction of a benzenesulfonyl group at N(1)-position switched the full agonist 6 to be a 5-HT6 receptor antagonist (30). A few compounds within the 2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles were also screened for off-targets and generally they displayed low affinity for other 5-HT subtypes and serotonin transporter protein (SERT).
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Indoles/farmacología , Piridinas/farmacología , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Animales , Unión Competitiva , Línea Celular , Células HEK293 , Células HeLa , Humanos , Enlace de Hidrógeno , Indoles/síntesis química , Indoles/química , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Receptores de Serotonina/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Agonistas de Receptores de Serotonina/síntesis química , Agonistas de Receptores de Serotonina/química , Relación Estructura-Actividad , TransfecciónRESUMEN
OBJECTIVE: 11ß-Hydroxysteroid dehydrogenase type I (11ßHSD1) regenerates active cortisol from inert cortisone in adipose tissue. Elevated adipose tissue 11ßHSD1 activity is observed in obese humans and rodents, where it is linked to obesity and its metabolic consequences. Menopause is also associated with increased abdominal fat accumulation, suggesting that estrogen is also important in adipose tissue metabolism. The purpose of this current study was to establish whether estrogen signaling through estrogen receptor α (ER-α) and estrogen receptor ß (ER-ß) could influence 11ßHSD1 in premenopausal and postmenopausal adipose tissues. METHODS: Nineteen premenopausal (aged 26 ± 5 y; body mass index, 23.6 ± 1.6 kg/m) and 23 postmenopausal (aged 63 ± 4 y; body mass index, 23.4 ± 1.9 kg/m) healthy women were studied. Subcutaneous adipose tissue biopsies and fasting venous blood samples were taken. Body composition was measured by bioelectrical impedance analysis. Human Simpson-Golabi-Behmel syndrome adipocyte cells were treated with ER-α- and ER-ß-specific agonists for 24 hours. Basic anthropometric data, serum 17ß-estradiol and progesterone concentrations, ER-α and ER-ß messenger RNA (mRNA) levels, and 11ßHSD1 mRNA, protein, and activity levels were assessed. RESULTS: ER-ß and 11ßHSD1, but not ER-α, mRNAs were significantly increased in adipose tissue from postmenopausal women compared with premenopausal women. ER-ß had a significant positive correlation with the mRNA level of 11ßHSD1 in adipose tissue from premenopausal and postmenopausal women. This association between ER-ß and 11ßHSD1 was greatest in adipose tissue from postmenopausal women. In human Simpson-Golabi-Behmel syndrome adipocytes, diarylpropiolnitrile, a selective ER-ß agonist, increased 11ßHSD1 mRNA, protein, and activity levels. CONCLUSIONS: We conclude that, in adipose tissue, ER-ß-mediated estrogen signaling can up-regulate 11ßHSD1 and that this may be of particular importance in postmenopausal women.
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11-beta-Hidroxiesteroide Deshidrogenasas/genética , 11-beta-Hidroxiesteroide Deshidrogenasas/metabolismo , Tejido Adiposo/química , Tejido Adiposo/enzimología , Receptor beta de Estrógeno/análisis , Posmenopausia/metabolismo , Adulto , Composición Corporal , Estradiol/sangre , Receptor alfa de Estrógeno/análisis , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/fisiología , Estrógenos/fisiología , Femenino , Humanos , Persona de Mediana Edad , Premenopausia/metabolismo , Progesterona/sangre , ARN Mensajero/análisis , Transducción de Señal/fisiología , Regulación hacia ArribaRESUMEN
A series of 1-propyl-4-arylpiperidines were synthesized and their effects on the dopaminergic and serotonergic systems tested in vivo and in vitro. Scaffold jumping among five- and six-membered bicyclic aryl rings attached to the piperidine ring had a marked impact on these effects. Potent and selective dopamine D(2) receptor antagonists were generated from 3-indoles, 3-benzoisoxazoles, 3-benzimidazol-2-one, and 3-benzothiophenes. In contrast, 3-benzofuran was a potent and selective inhibitor of monoamine oxidase (MAO) A. The effects of the synthesized compounds on 3,4-dihydroxyphenylacetic acid (DOPAC) levels correlated very well with their affinity for dopamine D(2) receptors and MAO A. In the 4-arylpiperidine series, the most promising compound for development was the 6-chloro-3-(1-propyl-4-piperidyl)-1H-benzimidazol-2-one (19), which displayed typical dopamine D(2) receptor antagonist properties in vivo but produced only a partial reduction on spontaneous locomotor activity. This indicates that the compound may have a lower propensity to induce parkinsonism in patients.
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Encéfalo/efectos de los fármacos , Dopaminérgicos/farmacología , Antagonistas de los Receptores de Dopamina D2 , Dopamina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Serotonina/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Locomoción/efectos de los fármacos , Estructura Molecular , Trastornos Psicóticos/tratamiento farmacológico , Ratas , Relación Estructura-ActividadRESUMEN
OBJECTIVE: The menopausal transition is characterized by increased body fat accumulation, including redistribution from peripheral to central fat depots. This distribution is associated with an increased risk of type 2 diabetes and cardiovascular disease that are linked to low-grade inflammation. We determined whether postmenopausal women have higher levels of inflammatory markers, compared with premenopausal women. We also wanted to determine whether these markers are reduced by stable weight loss in obese women. DESIGN AND METHODS: Anthropometric data, blood samples and subcutaneous adipose tissue biopsies were collected from normal weight premenopausal and postmenopausal women and obese women before and 2 years after gastric bypass (GBP) surgery. Serum protein levels and adipose tissue gene expression of inflammatory markers were investigated. RESULTS: IL-8 expression in adipose tissue and circulating levels were higher in postmenopausal vs premenopausal women. IL-8 expression was associated with waist circumference, independent of menopausal status. IL-6 expression and serum levels of monocyte chemoattractant protein (MCP)-1 were higher in postmenopausal vs premenopausal women. Two years after GBP surgery, adipose expression of IL-8, tumour necrosis factor-α and MCP-1 decreased significantly. Serum insulin levels were associated with inflammation-related gene expression before GBP surgery, but these associations disappeared after surgery. CONCLUSION: Postmenopausal women have an increased inflammatory response in the subcutaneous fat and circulation. Inflammatory markers in adipose tissue decreased significantly after surgery-induced weight loss. This effect may be beneficial for metabolic control and reduced cardiovascular risk after weight loss.
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Tejido Adiposo/metabolismo , Interleucina-8/metabolismo , Obesidad/metabolismo , Adulto , Anciano , Proteína C-Reactiva/metabolismo , Quimiocina CCL2/metabolismo , Femenino , Humanos , Interleucina-6/metabolismo , Persona de Mediana Edad , Posmenopausia/metabolismo , Premenopausia/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
BACKGROUND AND AIMS: The role of 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) in the pathogenesis of obesity has been elucidated in humans and in various rodent models. Obesity is accompanied by disturbances in glucocorticoid metabolism, circulating adipokine levels, and fatty acid (FA) reesterification. This study was undertaken to evaluate glucocorticoid metabolism in sc fat before and after weight loss and to explore putative associations between 11beta-HSD1 and leptin, adiponectin, and FA recycling. SUBJECTS AND METHODS: Twenty-seven obese (mean body mass index 44.4 + or - 4.4 kg/m(2)) women underwent gastric bypass surgery. Subcutaneous fat biopsies were collected before and 2 yr after surgery. The expression of 11beta-HSD1, leptin, adiponectin, and phosphoenolpyruvate carboxykinase (PEPCK) mRNA was evaluated with real-time PCR. Serum leptin and adiponectin protein levels were estimated by ELISA. RESULTS: Two years after gastric bypass surgery, significant reductions were observed in the mean body mass index (from 44.4 to 30.8 kg/m(2)) and mean waist circumference (from 121.9 to 90.6 cm). After weight loss, 11beta-HSD1 mRNA expression decreased 4-fold (P < 0.001). Both leptin and adiponectin mRNA expression decreased, with concomitantly decreased circulating leptin and increased circulating adiponectin levels. PEPCK mRNA expression did not change. CONCLUSION: Weight loss after gastric bypass surgery was followed by metabolically favorable changes in insulin sensitivity, circulating leptin and adiponectin, and peripheral glucocorticoid metabolism. A significant reduction in 11beta-HSD1 expression was observed in sc adipose tissue after weight loss. This suggests that up-regulation of 11beta-HSD1 is a consequence, rather than a cause, of obesity.
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11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Derivación Gástrica , Obesidad/cirugía , Grasa Subcutánea/metabolismo , Pérdida de Peso/fisiología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Adipoquinas/genética , Adipoquinas/metabolismo , Antropometría , Índice de Masa Corporal , Ensayo de Inmunoadsorción Enzimática , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Femenino , Humanos , Resistencia a la Insulina/fisiología , Leptina/genética , Leptina/metabolismo , Obesidad/genética , Obesidad/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
With age and menopause there is a shift in adipose distribution from gluteo-femoral to abdominal depots in women. Associated with this redistribution of fat are increased risks of type 2 diabetes and cardiovascular disease. Glucocorticoids influence body composition, and 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) which converts inert cortisone to active cortisol is a putative key mediator of metabolic complications in obesity. Increased 11betaHSD1 in adipose tissue may contribute to postmenopausal central obesity. We hypothesized that tissue-specific 11betaHSD1 gene expression and activity are up-regulated in the older, postmenopausal women compared to young, premenopausal women. Twenty-three pre- and 23 postmenopausal, healthy, normal weight women were recruited. The participants underwent a urine collection, a subcutaneous adipose tissue biopsy and the hepatic 11betaHSD1 activity was estimated by the serum cortisol response after an oral dose of cortisone. Urinary (5alpha-tetrahydrocortisol+5beta-tetrahydrocortisol)/tetrahydrocortisone ratios were higher in postmenopausal women versus premenopausal women in luteal phase (P<0.05), indicating an increased whole-body 11betaHSD1 activity. Postmenopausal women had higher 11betaHSD1 gene expression in subcutaneous fat (P<0.05). Hepatic first pass conversion of oral cortisone to cortisol was also increased in postmenopausal women versus premenopausal women in follicular phase of the menstrual cycle (P<0.01, at 30 min post cortisone ingestion), suggesting higher hepatic 11betaHSD1 activity. In conclusion, our results indicate that postmenopausal normal weight women have increased 11betaHSD1 activity in adipose tissue and liver. This may contribute to metabolic dysfunctions with menopause and ageing in women.
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11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Peso Corporal/fisiología , Especificidad de Órganos/fisiología , Posmenopausia/fisiología , Tejido Adiposo/enzimología , Corticoesteroides/sangre , Corticoesteroides/orina , Adulto , Aromatasa/genética , Aromatasa/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Lípidos/sangre , Hígado/enzimología , Persona de Mediana Edad , Posmenopausia/sangre , Posmenopausia/orina , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tejido Subcutáneo/enzimologíaRESUMEN
The glucocorticoid activating enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) is of major interest in obesity-related morbidity. Alterations in tissue-specific cortisol levels may influence lipogenetic and gluco/glyceroneogenetic pathways in fat and liver. We analyzed the expression and activity of 11betaHSD1 as well as the expression of phosphoenolpyruvate carboxykinase (PEPCK), sterol regulatory element binding protein (SREBP), and fatty acid synthase (FAS) in adipose and liver and investigated putative associations between 11betaHSD1 and energy metabolism genes. A total of 33 obese women (mean BMI 44.6) undergoing gastric bypass surgery were enrolled. Subcutaneous adipose tissue (SAT), omental fat (omental adipose tissue (OmAT)), and liver biopsies were collected during the surgery. 11betaHSD1 gene expression was higher in SAT vs. OmAT (P = 0.013), whereas the activity was higher in OmAT (P = 0.009). The SAT 11betaHSD1 correlated with waist circumference (P = 0.045) and was an independent predictor for the OmAT area in a linear regression model. Energy metabolism genes had AT depot-specific expression; higher leptin and SREBP in SAT than OmAT, but higher PEPCK in OmAT than SAT. The expression of 11betaHSD1 correlated with PEPCK in both AT depots (P = 0.05 for SAT and P = 0.0001 for OmAT). Hepatic 11betaHSD1 activity correlated negatively with abdominal adipose area (P = 0.002) and expression positively with PEPCK (P = 0.003). In human obesity, glucocorticoid regeneration in the SAT is associated with central fat accumulation indicating that the importance of this specific fat depot is underestimated. Central fat accumulation is negatively associated with hepatic 11betaHSD1 activity. A disturbance in peripheral glucocorticoid metabolism is associated with changes in genes involved in fatty acid (FA) recycling in adipose tissue (AT).
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Ácidos Grasos/metabolismo , Glucocorticoides/metabolismo , Grasa Intraabdominal/enzimología , Obesidad/metabolismo , Grasa Subcutánea Abdominal/enzimología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Adiposidad , Adulto , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Femenino , Humanos , Leptina/genética , Leptina/metabolismo , Hígado/enzimología , Persona de Mediana Edad , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Circunferencia de la CinturaRESUMEN
CONTEXT: Hypothalamic-pituitary-adrenal (HPA) axis dysregulation may underlie disorders including obesity, depression, cognitive decline, and the metabolic syndrome. Conventional tests of HPA axis negative feedback rely on glucocorticoid receptor (GR) agonists such as dexamethasone but do not test feedback by endogenous cortisol, potentially mediated by both GR and mineralocorticoid receptors (MR). OBJECTIVE: The objective of the study was to use a combination of GR (RU38486, mifepristone) and MR (spironolactone) antagonists to explore the poorly understood activation of the HPA axis that occurs in obesity. DESIGN: This was a double-blind, placebo-controlled, randomized, crossover study. SETTING: The study was conducted at a clinical research facility. PARTICIPANTS: Participants included 15 lean (body mass index 22.0 +/- 1.6 kg/m(2)) and 16 overweight/obese (body mass index 30.1 +/- 3.5 kg/m(2)) men. INTERVENTION: Subjects attended on four occasions for blood and saliva sampling every 30 min between 1800 and 2200 h. At 1100 and 1600 h before visits, subjects took 200 mg spironolactone, 400 mg RU38486, 200 mg spironolactone + 400 mg RU38486, or placebo orally. MAIN OUTCOME MEASURES: Serum cortisol levels after drug or placebo were measured. RESULTS: Cortisol levels did not differ between lean and obese after placebo. Spironolactone and RU38486 alone had modest effects, increasing cortisol by less than 50% in both groups. However, combined spironolactone plus RU38486 elevated cortisol concentrations substantially, more so in lean than obese men [2.9- (0.3) vs. 2.2 (0.3)-fold elevation, P = 0.002]. CONCLUSIONS: Combined receptor antagonist stimulation of the HPA axis reveals redundancy of MR and GR in negative feedback in humans. Obese men have impaired responses to combined receptor antagonist stimulation, suggesting impaired negative feedback by endogenous cortisol. Such an approach may be useful to dissect abnormal HPA axis control in neuropsychiatric and other disorders.
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Hidrocortisona/sangre , Sistema Hipotálamo-Hipofisario/fisiología , Mifepristona/uso terapéutico , Obesidad/tratamiento farmacológico , Sistema Hipófiso-Suprarrenal/fisiología , Espironolactona/uso terapéutico , Adulto , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Estudios Cruzados , Método Doble Ciego , Retroalimentación , Femenino , Antagonistas de Hormonas/uso terapéutico , Humanos , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Masculino , Persona de Mediana Edad , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , Obesidad/sangre , Obesidad/fisiopatología , Sobrepeso/sangre , Sobrepeso/tratamiento farmacológico , Sobrepeso/fisiopatología , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Placebos , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/fisiología , Receptores de Mineralocorticoides/efectos de los fármacos , Receptores de Mineralocorticoides/fisiología , Relación Cintura-CaderaRESUMEN
A sensitive liquid chromatography-mass spectrometric method was validated for the quantification of RU38486 (mifepristone) in human and murine plasma. The analyte and internal standard (alfaxolone) were extracted by liquid-liquid extraction with diethyl ether, resolved on a C18 column using gradient elution with methanol and ammonium acetate and detected after positive electrospray ionization (m/z 430-->372; m/z 333-->297, respectively). Quantification was linear over the range 0.5-500ng (r(2)>0.997), precise and accurate (intra-assay RSD< or =7.2%, RME< or =8.2%; inter-assay RSD< or =15.7% RME< or =10.2%). The limit of quantification (LOQ) was 50pg injected on column, permitting reproducible analysis of RU38486 in small volumes of plasma.