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1.
EMBO J ; 43(8): 1445-1483, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38499786

RESUMO

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.


Assuntos
Apoferritinas , Linfócitos T Reguladores , Animais , Humanos , Camundongos , Apoferritinas/genética , Apoferritinas/metabolismo , Linhagem da Célula/genética , Citosina/metabolismo , Fatores de Transcrição Forkhead , Ferro/metabolismo
2.
Clin Chem Lab Med ; 62(6): 1080-1091, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38205643

RESUMO

OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.


Assuntos
Androstenodiona , Sulfato de Desidroepiandrosterona , Testosterona , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Androstenodiona/sangue , Androstenodiona/análise , Calibragem , Sulfato de Desidroepiandrosterona/sangue , Sulfato de Desidroepiandrosterona/análise , Sulfato de Desidroepiandrosterona/normas , Espectrometria de Massa com Cromatografia Líquida/métodos , Espectrometria de Massa com Cromatografia Líquida/normas , Espectrometria de Massas em Tandem/normas , Espectrometria de Massas em Tandem/métodos , Testosterona/sangue , Testosterona/análise , Testosterona/normas
3.
Int J Mol Sci ; 25(10)2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38791102

RESUMO

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder impairing cortisol synthesis due to reduced enzymatic activity. This leads to persistent adrenocortical overstimulation and the accumulation of precursors before the blocked enzymatic step. The predominant form of CAH arises from mutations in CYP21A2, causing 21-hydroxylase deficiency (21-OHD). Despite emerging treatment options for CAH, it is not always possible to physiologically replace cortisol levels and counteract hyperandrogenism. Moreover, there is a notable absence of an effective in vivo model for pre-clinical testing. In this work, we developed an animal model for CAH with the clinically relevant point mutation p.R484Q in the previously humanized CYP21A2 mouse strain. Mutant mice showed hyperplastic adrenals and exhibited reduced levels of corticosterone and 11-deoxycorticosterone and an increase in progesterone. Female mutants presented with higher aldosterone concentrations, but blood pressure remained similar between wildtype and mutant mice in both sexes. Male mutant mice have normal fertility with a typical testicular appearance, whereas female mutants are infertile, exhibit an abnormal ovarian structure, and remain in a consistent diestrus phase. Conclusively, we show that the animal model has the potential to contribute to testing new treatment options and to prevent comorbidities that result from hormone-related derangements and treatment-related side effects in CAH patients.


Assuntos
Hiperplasia Suprarrenal Congênita , Modelos Animais de Doenças , Esteroide 21-Hidroxilase , Animais , Hiperplasia Suprarrenal Congênita/genética , Hiperplasia Suprarrenal Congênita/patologia , Hiperplasia Suprarrenal Congênita/metabolismo , Esteroide 21-Hidroxilase/genética , Esteroide 21-Hidroxilase/metabolismo , Camundongos , Feminino , Masculino , Humanos , Corticosterona/metabolismo , Corticosterona/sangue , Aldosterona/metabolismo , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Mutação , Progesterona/metabolismo
4.
Hepatology ; 75(4): 881-897, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34519101

RESUMO

BACKGROUND AND AIMS: NAFLD is initiated by steatosis and can progress through fibrosis and cirrhosis to HCC. The RNA binding protein human antigen R (HuR) controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte HuR in NAFLD development and progression to fibrosis and HCC. APPROACH AND RESULTS: Hepatocyte-specific, HuR-deficient mice and control HuR-sufficient mice were fed either a normal diet or an NAFLD-inducing diet. Hepatic lipid accumulation, inflammation, fibrosis, and HCC development were studied by histology, flow cytometry, quantitative PCR, and RNA sequencing. The liver lipidome was characterized by lipidomics analysis, and the HuR-RNA interactions in the liver were mapped by RNA immunoprecipitation sequencing. Hepatocyte-specific, HuR-deficient mice displayed spontaneous hepatic steatosis and fibrosis predisposition compared to control HuR-sufficient mice. On an NAFLD-inducing diet, hepatocyte-specific HuR deficiency resulted in exacerbated inflammation, fibrosis, and HCC-like tumor development. A multi-omic approach, including lipidomics, transcriptomics, and RNA immunoprecipitation sequencing revealed that HuR orchestrates a protective network of hepatic-metabolic and lipid homeostasis-maintaining pathways. Consistently, HuR-deficient livers accumulated, already at steady state, a triglyceride signature resembling that of NAFLD livers. Moreover, up-regulation of secreted phosphoprotein 1 expression mediated, at least partially, fibrosis development in hepatocyte-specific HuR deficiency on an NAFLD-inducing diet, as shown by experiments using antibody blockade of osteopontin. CONCLUSIONS: HuR is a gatekeeper of liver homeostasis, preventing NAFLD-related fibrosis and HCC, suggesting that the HuR-dependent network could be exploited therapeutically.


Assuntos
Carcinoma Hepatocelular , Proteína Semelhante a ELAV 1 , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Animais , Carcinoma Hepatocelular/patologia , Proteína Semelhante a ELAV 1/metabolismo , Homeostase , Inflamação/metabolismo , Fígado/patologia , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , RNA , Triglicerídeos/metabolismo
5.
Reproduction ; 166(3): 221-234, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37432973

RESUMO

In brief: Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is expressed by various murine ovarian cells. Morphological and molecular investigations, including a proteomic study of adult Chrna7 knockout (KO) mouse ovaries, reveal the roles of these receptors in the local regulation of the ovary. Abstract: Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is involved in cellular functions ranging from synaptic transmission in neurons to regulation of inflammation, cell growth and metabolism to cell death in other cells. Our qPCR results and other studies indicated that nAChRa7 is expressed in the adult mouse ovary, while in situ hybridization and single-cell sequencing data suggested this expression may be shared by several ovarian cells, including fibroblast-like and steroidogenic stroma cells, macrophages and oocytes of small follicles. To explore a possible involvement of nAChRa7 in ovarian functions, we evaluated ovarian morphology of Chrna7-null mutant adult mice (KO) and wildtype mice (WT; 3 months, metestrus) by performing immunohistochemistry, qPCR studies, measurements of serum progesterone and proteomic analyses. The evaluation of serial sections indicated fewer primordial follicles but similar numbers of primary, secondary and tertiary follicles, as well as corpora lutea in KO and WT mice. Atresia was unchanged. Serum progesterone and mRNA levels of proliferation and most apoptosis markers were not changed, yet two typical macrophage markers were elevated. Furthermore, the proteomes of KO ovaries were significantly altered with 96 proteins increased and 32 decreased in abundance in KOs compared to WTs. Among the elevated proteins were markers for stroma cells. Hence, the lack of nAChRa7 causes changes in small follicle counts and alterations of the ovarian stroma cells. The ovarian phenotype of Chrna7 mutant mice links this channel protein to the local regulation of ovarian cells, including stroma cells.


Assuntos
Ovário , Receptores Nicotínicos , Animais , Feminino , Camundongos , Camundongos Knockout , Ovário/metabolismo , Fenótipo , Progesterona/metabolismo , Proteômica , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
6.
J Neurooncol ; 165(2): 329-342, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37976029

RESUMO

PURPOSE: Primary brain tumors are a leading cause of cancer-related death in children, and medulloblastoma is the most common malignant pediatric brain tumor. The current molecular characterization of medulloblastoma is mainly based on protein-coding genes, while little is known about the involvement of long non-coding RNAs (lncRNAs). This study aimed to elucidate the role of the lncRNA OTX2-AS1 in medulloblastoma. METHODS: Analyses of DNA copy number alterations, methylation profiles, and gene expression data were used to characterize molecular alterations of OTX2-AS1 in medulloblastoma tissue samples. In vitro analyses of medulloblastoma cell models and orthotopic in vivo experiments were carried out for functional characterization of OTX2-AS1. High-throughput drug screening was employed to identify pharmacological inhibitors, while proteomics and metabolomics analyses were performed to address potential mechanisms of drug action. RESULTS: We detected amplification and consecutive overexpression of OTX2 and OTX2-AS1 in a subset of medulloblastomas. In addition, OTX2-AS1 promoter methylation was linked to OTX2-AS1 expression. OTX2-AS1 knockout reduced medulloblastoma cell viability and cell migration in vitro and prolonged survival in the D283 orthotopic medulloblastoma mouse xenograft model. Pharmacological inhibition of BCL-2 suppressed the growth of OTX2-AS1 overexpressing medulloblastoma cells in vitro. CONCLUSIONS: Our study revealed a pro-tumorigenic role of OTX2-AS1 in medulloblastoma and identified BCL-2 inhibition as a potential therapeutic approach to target OTX2-AS1 overexpressing medulloblastoma cells.


Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , RNA Longo não Codificante , Animais , Criança , Humanos , Camundongos , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Regulação Neoplásica da Expressão Gênica , Meduloblastoma/patologia , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Longo não Codificante/genética
7.
Clin Chem Lab Med ; 61(1): 67-77, 2023 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-36288389

RESUMO

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration. METHODS: Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation. RESULTS: Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from -5.6 to 12.3% for corticosterone, -14.6 to 12.4% for 11-deoxycortisol and -4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability. CONCLUSIONS: Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization.


Assuntos
Cortisona , Humanos , Cromatografia Líquida/métodos , Cortodoxona , Corticosterona , Espectrometria de Massas em Tandem/métodos , Reprodutibilidade dos Testes
8.
Clin Chem Lab Med ; 60(12): 1929-1937, 2022 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-35851438

RESUMO

OBJECTIVES: Mass spectrometry-based steroidomics combined with machine learning (ML) provides a potentially powerful approach in endocrine diagnostics, but is hampered by limitations in the conveyance of results and interpretations to clinicians. We address this shortcoming by integration of the two technologies with a laboratory information management systems (LIMS) model. METHODS: The approach involves integration of ML algorithm-derived models with commercially available mathematical programming software and a web-based LIMS prototype. To illustrate clinical utility, the process was applied to plasma steroidomics data from 22 patients tested for primary aldosteronism (PA). RESULTS: Once mass spectrometry data are uploaded into the system, automated processes enable generation of interpretations of steroid profiles from ML models. Generated reports include plasma concentrations of steroids in relation to age- and sex-specific reference intervals along with results of ML models and narrative interpretations that cover probabilities of PA. If PA is predicted, reports include probabilities of unilateral disease and mutations of KCNJ5 known to be associated with successful outcomes of adrenalectomy. Preliminary results, with no overlap in probabilities of disease among four patients with and 18 without PA and correct classification of all four patients with unilateral PA including three of four with KCNJ5 mutations, illustrate potential utility of the approach to guide diagnosis and subtyping of patients with PA. CONCLUSIONS: The outlined process for integrating plasma steroidomics data and ML with LIMS may facilitate improved diagnostic-decision-making when based on higher-dimensional data otherwise difficult to interpret. The approach is relevant to other diagnostic applications involving ML.


Assuntos
Hiperaldosteronismo , Masculino , Feminino , Humanos , Hiperaldosteronismo/diagnóstico , Inteligência Artificial , Esteroides , Espectrometria de Massas , Gestão da Informação , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética
9.
Clin Chem Lab Med ; 60(5): 726-739, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35172417

RESUMO

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. METHODS: Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. RESULTS: Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from -6.6 to 6.9%, -17.2 to 7.8% and -12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation. CONCLUSIONS: Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.


Assuntos
Hidrocortisona , Progesterona , Aldosterona , Calibragem , Cromatografia Líquida/métodos , Humanos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos
10.
Cell Mol Life Sci ; 78(7): 3577-3590, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33464382

RESUMO

Endogenous steroid hormones, especially glucocorticoids and mineralocorticoids, derive from the adrenal cortex, and drastic or sustained changes in their circulatory levels affect multiple organ systems. Although hypoxia signaling in steroidogenesis has been suggested, knowledge on the true impact of the HIFs (Hypoxia-Inducible Factors) in the adrenocortical cells of vertebrates is scant. By creating a unique set of transgenic mouse lines, we reveal a prominent role for HIF1α in the synthesis of virtually all steroids in vivo. Specifically, mice deficient in HIF1α in adrenocortical cells displayed enhanced levels of enzymes responsible for steroidogenesis and a cognate increase in circulatory steroid levels. These changes resulted in cytokine alterations and changes in the profile of circulatory mature hematopoietic cells. Conversely, HIF1α overexpression resulted in the opposite phenotype of insufficient steroid production due to impaired transcription of necessary enzymes. Based on these results, we propose HIF1α to be a vital regulator of steroidogenesis as its modulation in adrenocortical cells dramatically impacts hormone synthesis with systemic consequences. In addition, these mice can have potential clinical significances as they may serve as essential tools to understand the pathophysiology of hormone modulations in a number of diseases associated with metabolic syndrome, auto-immunity or even cancer.


Assuntos
Glândulas Suprarrenais/metabolismo , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Esteroides/biossíntese , Animais , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais
11.
Clin Chem ; 67(8): 1098-1112, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-33993248

RESUMO

BACKGROUND: Plasma-free normetanephrine and metanephrine (metanephrines) are the recommended biomarkers for testing of pheochromocytoma and paraganglioma (PPGL). This study evaluated the status of harmonization of liquid chromatography-tandem mass spectrometry-based measurements of plasma metanephrines and methoxytyramine and clinical interpretation of test results. METHODS: 125 plasma samples from patients tested for PPGLs were analyzed in 12 laboratories. Analytical performance was also assessed from results of a proficiency-testing program. Agreement of test results from different laboratories was assessed by Passing-Bablok regression and Bland-Altman analysis. Agreement in clinical test interpretation based on laboratory specific reference intervals was also examined. RESULTS: Comparisons of analytical test results by regression analysis revealed strong correlations for normetanephrine and metanephrine (R ≥ 0.95) with mean slopes of 1.013 (range 0.975-1.078), and 1.019 (range 0.963-1.081), and intercepts of -0.584 (-53.736 to 54.790) and -3.194 (-17.152 to 5.933), respectively. The mean bias between methods was 1.2% (-11.6% to 16.0%) for metanephrine and 0.1% (-18.0% to 9.5%) for normetanephrine. Measurements of 3-methoxytyramine revealed suboptimal agreement between laboratories with biases ranging from -32.2% to 64.0%. Interrater agreement in test interpretation was >94% for metanephrine and >84% for normetanephrine; improvements in interrater agreement were observed with use of harmonized reference intervals, including age-specific cut-offs for normetanephrine. CONCLUSIONS: Analytical methods for metanephrines are well harmonized between laboratories. However, the 16% disagreement in test interpretation for normetanephrine suggests use of suboptimal method-dependent reference intervals for clinical decision-making for this metabolite. Improved analytical methods and reference interval harmonization are particularly required for 3-methoxytyramine.


Assuntos
Neoplasias das Glândulas Suprarrenais , Metanefrina , Neoplasias das Glândulas Suprarrenais/diagnóstico , Cromatografia Líquida , Dopamina/análogos & derivados , Humanos , Normetanefrina , Espectrometria de Massas em Tandem
12.
Horm Metab Res ; 53(5): 326-334, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33902135

RESUMO

The adrenal gland is important for many physiological and pathophysiological processes, but studies are often restricted by limited availability of sample material. Improved methods for sample preparation are needed to facilitate analyses of multiple classes of adrenal metabolites and macromolecules in a single sample. A procedure was developed for preparation of chromaffin cells, mouse adrenals, and human chromaffin tumors that allows for multi-omics analyses of different metabolites and preservation of native proteins. To evaluate the new procedure, aliquots of samples were also prepared using conventional procedures. Metabolites were analyzed by liquid-chromatography with mass spectrometry or electrochemical detection. Metabolite contents of chromaffin cells and tissues analyzed with the new procedure were similar or even higher than with conventional methods. Catecholamine contents were comparable between both procedures. The TCA cycle metabolites, cis-aconitate, isocitate, and α-ketoglutarate were detected at higher concentrations in cells, while in tumor tissue only isocitrate and potentially fumarate were measured at higher contents. In contrast, in a broad untargeted metabolomics approach, a methanol-based preparation procedure of adrenals led to a 1.3-fold higher number of detected metabolites. The established procedure also allows for simultaneous investigation of adrenal hormones and related enzyme activities as well as proteins within a single sample. This novel multi-omics approach not only minimizes the amount of sample required and overcomes problems associated with tissue heterogeneity, but also provides a more complete picture of adrenal function and intra-adrenal interactions than previously possible.


Assuntos
Glândulas Suprarrenais/química , Glândulas Suprarrenais/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Hormônios/metabolismo , Espectrometria de Massas/métodos , Metabolômica/métodos , Neoplasias das Glândulas Suprarrenais/química , Neoplasias das Glândulas Suprarrenais/metabolismo , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Paraganglioma/química , Paraganglioma/metabolismo , Feocromocitoma/química , Feocromocitoma/metabolismo
13.
Horm Metab Res ; 53(10): 699-704, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34607368

RESUMO

The impact of dietary sodium reduction on mouse models of type 2 diabetes is not well understood. Therefore, we analyzed the effect of a low-salt diet on obesity and parameters of type 2 diabetes in db/db mice. Five-week-old male db/db and lean db/m mice were fed a normal salt (0.19% Na+, NS) or a low-salt diet (<0.03% Na+, LS) for 5 weeks. Body and organ weight and parameters of glucose and insulin tolerance were analyzed. Plasma levels of steroids were determined by liquid chromatography tandem mass spectrometry. Body weight, glucose, and insulin tolerance were not affected by LS. The amount of gonadal adipose tissue showed a trend to be increased by LS whereas liver, pancreas, kidney, heart, and adrenal weight remained unaffected. LS reduced urinary sodium-to-creatinine ratio but did not affect plasma Na+ levels in both genotypes. Plasma and urinary potassium-to-creatinine ratio did not differ in all groups of mice. Aldosterone as a major determinant of changes in dietary sodium remained unaffected by LS in db/db mice as well as further investigated steroid hormones. The present study showed reduced sodium-to-creatinine ratio, but no additional effects of dietary sodium reduction on major metabolic parameters and steroid levels in obese and hyper-glycemic db/db mice.


Assuntos
Diabetes Mellitus Tipo 2/dietoterapia , Dieta Hipossódica , Obesidade/dietoterapia , Animais , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Progressão da Doença , Regulação para Baixo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/metabolismo , Obesidade/patologia , Tamanho do Órgão/efeitos dos fármacos , Cloreto de Sódio na Dieta/administração & dosagem , Cloreto de Sódio na Dieta/farmacologia , Sódio na Dieta/farmacologia
14.
J Pathol ; 251(4): 378-387, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32462735

RESUMO

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours with a hereditary background in over one-third of patients. Mutations in succinate dehydrogenase (SDH) genes increase the risk for PPGLs and several other tumours. Mutations in subunit B (SDHB) in particular are a risk factor for metastatic disease, further highlighting the importance of identifying SDHx mutations for patient management. Genetic variants of unknown significance, where implications for the patient and family members are unclear, are a problem for interpretation. For such cases, reliable methods for evaluating protein functionality are required. Immunohistochemistry for SDHB (SDHB-IHC) is the method of choice but does not assess functionality at the enzymatic level. Liquid chromatography-mass spectrometry-based measurements of metabolite precursors and products of enzymatic reactions provide an alternative method. Here, we compare SDHB-IHC with metabolite profiling in 189 tumours from 187 PPGL patients. Besides evaluating succinate:fumarate ratios (SFRs), machine learning algorithms were developed to establish predictive models for interpreting metabolite data. Metabolite profiling showed higher diagnostic specificity compared to SDHB-IHC (99.2% versus 92.5%, p = 0.021), whereas sensitivity was comparable. Application of machine learning algorithms to metabolite profiles improved predictive ability over that of the SFR, in particular for hard-to-interpret cases of head and neck paragangliomas (AUC 0.9821 versus 0.9613, p = 0.044). Importantly, the combination of metabolite profiling with SDHB-IHC has complementary utility, as SDHB-IHC correctly classified all but one of the false negatives from metabolite profiling strategies, while metabolite profiling correctly classified all but one of the false negatives/positives from SDHB-IHC. From 186 tumours with confirmed status of SDHx variant pathogenicity, the combination of the two methods resulted in 185 correct predictions, highlighting the benefits of both strategies for patient management. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Neoplasias de Cabeça e Pescoço/diagnóstico , Aprendizado de Máquina , Metabolômica , Paraganglioma/diagnóstico por imagem , Feocromocitoma/diagnóstico , Succinato Desidrogenase/genética , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/patologia , Estudos de Coortes , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imuno-Histoquímica , Mutação , Paraganglioma/genética , Paraganglioma/patologia , Feocromocitoma/genética , Feocromocitoma/patologia
15.
Proc Natl Acad Sci U S A ; 115(51): 12997-13002, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30514817

RESUMO

The adrenal gland is a master regulator of the human body during response to stress. This organ shows constant replacement of senescent cells by newly differentiated cells. A high degree of plasticity is critical to sustain homeostasis under different physiological demands. This is achieved in part through proliferation and differentiation of adult adrenal progenitors. Here, we report the isolation and characterization of a Nestin+ population of adrenocortical progenitors located under the adrenal capsule and scattered throughout the cortex. These cells are interconnected with progenitors in the medulla. In vivo lineage tracing revealed that, under basal conditions, this population is noncommitted and slowly migrates centripetally. Under stress, this migration is greatly enhanced, and the cells differentiate into steroidogenic cells. Nestin+ cells cultured in vitro also show multipotency, as they differentiate into mineralocorticoid and glucocorticoid-producing cells, which can be further influenced by the exposure to Angiotensin II, adrenocorticotropic hormone, and the agonist of luteinizing hormone-releasing hormone, triptorelin. Taken together, Nestin+ cells in the adult adrenal cortex exhibit the features of adrenocortical progenitor cells. Our study provides evidence for a role of Nestin+ cells in organ homeostasis and emphasizes their role under stress. This cell population might be a potential source of cell replacement for the treatment of adrenal insufficiency.


Assuntos
Adaptação Fisiológica , Córtex Suprarrenal/citologia , Hormônio Adrenocorticotrópico/metabolismo , Homeostase , Células-Tronco/citologia , Estresse Fisiológico , Córtex Suprarrenal/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Camundongos , Células-Tronco/fisiologia
16.
Pediatr Blood Cancer ; 67(2): e28081, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31724812

RESUMO

BACKGROUND: Neuroblastoma, the most common extracranial solid tumor of childhood, produces catecholamines that are metabolized within tumor cells. Homovanillic acid (HVA) and vanillylmandelic acid (VMA), the end products of catecholamine metabolism, have limited accuracy for testing of the tumors. This study assessed whether metabolites produced in earlier steps of catecholamine metabolism might offer improved diagnostic accuracy over urinary HVA and VMA. PROCEDURE: Plasma concentrations of 3-methoxytyramine, normetanephrine, and metanephrine were measured in two pediatric cohorts: (i) 96 children with confirmed neuroblastoma and (ii) 41 children with signs and symptoms of a catecholamine-producing tumor or other neoplasms and in whom neuroblastoma was excluded. Additional measurements of plasma 3-O-methyldopa and relationships of metabolites to MYCN amplification were examined in patient subgroups. RESULTS: Overall, 94 of the 96 patients with neuroblastoma had concentrations of 3-methoxytyramine or normetanephrine above age-specific upper limits of reference intervals, providing a diagnostic sensitivity of 97.9% that was higher (P < 0.0001) than that of 82.2% for HVA and VMA. One of the two patients with normal plasma results showed an elevation of plasma 3-O-methyldopa. Diagnostic specificities were, respectively, 95.1% and 84.8%. Areas under receiver-operating characteristic curves confirmed the superior diagnostic power of the plasma than the urinary test (0.994 vs 0.945; P = 0.0095). Ratios of plasma 3-methoxytyramine to normetanephrine were 7.2-fold higher (P < 0.0001) for patients who had neuroblastomas with MYCN amplification than without MYCN amplification. CONCLUSIONS: Measurements of plasma 3-methoxytyramine and normetanephrine provide a highly accurate diagnostic test for neuroblastoma and also offer potential for prognostic risk stratification.


Assuntos
Biomarcadores Tumorais/análise , Dopamina/análogos & derivados , Neuroblastoma/diagnóstico , Normetanefrina/análise , Tirosina/análogos & derivados , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Dopamina/análise , Feminino , Seguimentos , Humanos , Lactente , Masculino , Neuroblastoma/sangue , Neuroblastoma/urina , Prognóstico , Estudos Retrospectivos , Tirosina/análise
17.
Clin Chem Lab Med ; 59(2): 353-363, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33001846

RESUMO

Objectives: Plasma free metanephrines are commonly used for diagnosis of pheochromocytoma and paraganglioma (PPGLs), but can also provide other information. This multicenter study prospectively examined whether tumor size, location, and mutations could be predicted by these metabolites. Methods: Predictions of tumor location, size, and mutation type, based on measurements of plasma normetanephrine, metanephrine, and methoxytyramine were made without knowledge of disease in 267 patients subsequently determined to have PPGLs. Results: Predictions of adrenal vs. extra-adrenal locations according to increased plasma concentrations of metanephrine and methoxytyramine were correct in 93 and 97% of the respective 136 and 33 patients in who these predictions were possible. Predicted mean tumor diameters correlated positively (p<0.0001) with measured diameters; predictions agreed well for pheochromocytomas but were overestimated for paragangliomas. Considering only patients with mutations, 51 of the 54 (94%) patients with NF1 or RET mutations were correctly predicted with those mutations according to increased plasma metanephrine, whereas no or minimal increase in metanephrine correctly predicted all 71 patients with either VHL or SDHx mutations; furthermore, among the latter group increases in methoxytyramine correctly predicted SDHx mutations in 93% of the 29 cases for this specific prediction. Conclusions: Extents and patterns of increased plasma O-methylated catecholamine metabolites among patients with PPGLs allow predictions of tumor size, adrenal vs. extra-adrenal locations and general types of mutations. Predictions of tumor location are, however, only possible for patients with clearly increased plasma methoxytyramine or metanephrine. Where possible or clinically relevant the predictions are potentially useful for subsequent clinical decision-making.


Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Biomarcadores Tumorais/sangue , Metanefrina/sangue , Paraganglioma/diagnóstico , Feocromocitoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Dopamina/análogos & derivados , Dopamina/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neurofibromatose 1/genética , Normetanefrina/sangue , Estudos Prospectivos , Proto-Oncogenes/genética , Fatores de Risco
18.
Thorax ; 74(11): 1102-1105, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31467191

RESUMO

The aim of this investigation was to elucidate the effect of CPAP withdrawal on neurometabolic and cardiometabolic markers in patients with obstructive sleep apnoea. We evaluated 70 patients (mean age 61±10 years, 82% men) treated with CPAP in two 2-week, parallel, randomised controlled trials. CPAP withdrawal resulted in elevated 3,4-dihydroxyphenylglycol, norepinephrine and cortisol after 2 weeks of CPAP withdrawal; however, no statistically significant changes of the renin-angiotensin-aldosterone system (RAAS) determinants were documented. In summary, CPAP withdrawal may be more prominently linked to short-term increases in sympathetic activation than hypothalamic-pituitary-adrenal axis or RAAS activation. ClinicalTrials.gov Identifier: NCT02493673 and NCT02050425.


Assuntos
Pressão Positiva Contínua nas Vias Aéreas , Hidrocortisona/sangue , Metoxi-Hidroxifenilglicol/análogos & derivados , Norepinefrina/sangue , Apneia Obstrutiva do Sono/sangue , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Metoxi-Hidroxifenilglicol/sangue , Pessoa de Meia-Idade , Sistema Renina-Angiotensina , Índice de Gravidade de Doença , Apneia Obstrutiva do Sono/terapia , Suspensão de Tratamento
19.
Int J Obes (Lond) ; 43(2): 263-275, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29717268

RESUMO

BACKGROUND: Obesity-associated activation of sympathetic nervous outflow is well documented, whereas involvement of dysregulated adrenomedullary hormonal function in obesity is less clear. This study assessed relationships of sympathoadrenal function with indices of obesity and influences of circulating catecholamines on body mass. METHODS: Anthropometric and clinical data along with plasma and 24-h urine samples were collected from 590 volunteers and 1368 patients tested for phaeochromocytoma and paraganglioma (PPGL), among whom tumours were diagnosed in 210 individuals. RESULTS: Among patients tested for PPGL, those with tumours less often had a body mass index (BMI) above 30 kg/m2 (12 vs. 31%) and more often a BMI under 25 kg/m2 (56 vs. 32%) than those without tumours (P < 0.0001). Urinary outputs of catecholamines in patients with PPGL were negatively related to BMI (r = -0.175, P = 0.0133). Post-operative weight gain (P < 0.0001) after resection of PPGL was positively related to presurgical tumoural catecholamine output (r = 0.257, P = 0.0101). Higher BMI in men and women and percent body fat in women of the volunteer group were associated with lower plasma concentrations and urinary outputs of adrenaline and metanephrine, the former indicating obesity-related reduced adrenaline secretion and the latter obesity-related reduced adrenomedullary adrenaline stores. Daytime activity was associated with substantial increases in urinary adrenaline and noradrenaline excretion, with blunted responses in obese subjects. CONCLUSIONS: The findings in patients with PPGL support an influence of high circulating catecholamines on body weight. Additional associations of adrenomedullary dysfunction with obesity raise the possibility of a permissive influence of the adrenal medulla on the regulation of body weight.


Assuntos
Neoplasias das Glândulas Suprarrenais , Peso Corporal/fisiologia , Catecolaminas , Obesidade , Adolescente , Neoplasias das Glândulas Suprarrenais/complicações , Neoplasias das Glândulas Suprarrenais/epidemiologia , Medula Suprarrenal/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Catecolaminas/sangue , Catecolaminas/urina , Células Cromafins/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/epidemiologia , Feocromocitoma , Estudos Prospectivos , Adulto Jovem
20.
Genet Med ; 21(3): 705-717, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30050099

RESUMO

PURPOSE: Metabolic aberrations have been described in neoplasms with pathogenic variants (PV) in the Krebs cycle genes encoding succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH). In turn, accumulation of oncometabolites succinate, fumarate, and 2-hydroxyglutarate can be employed to identify tumors with those PV . Additionally, such metabolic readouts may aid in genetic variant interpretation and improve diagnostics. METHODS: Using liquid chromatography-mass spectrometry, 395 pheochromocytomas and paragangliomas (PPGLs) from 391 patients were screened for metabolites to indicate Krebs cycle aberrations. Multigene panel sequencing was applied to detect driver PV in cases with indicative metabolite profiles but undetermined genetic drivers. RESULTS: Aberrant Krebs cycle metabolomes identified rare cases of PPGLs with germline PV in FH and somatic PV in IDHx and SDHx, including the first case of a somatic IDH2 PV in PPGL. Metabolomics also reliably identified PPGLs with SDHx loss-of-function (LOF) PV. Therefore we utilized tumor metabolite profiles to further classify variants of unknown significance in SDHx, thereby enabling missense variants associated with SDHx LOF to be distinguished from benign variants. CONCLUSION: We propose incorporation of metabolome data into the diagnostics algorithm in PPGLs to guide genetic testing and variant interpretation and to help identify rare cases with PV in FH and IDHx.


Assuntos
Genômica/métodos , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/genética , Cromatografia Líquida , Feminino , Fumarato Hidratase/genética , Fumarato Hidratase/fisiologia , Humanos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Masculino , Espectrometria de Massas , Metaboloma/genética , Succinato Desidrogenase/genética , Succinato Desidrogenase/fisiologia
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