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1.
PLoS One ; 14(3): e0212670, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30913212

RESUMO

Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of-function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens.

2.
Anal Biochem ; 568: 41-50, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30605634

RESUMO

Apelin, the endogenous ligand for the APJ receptor, has generated interest due to its beneficial effects on the cardiovascular system. Synthesized as a 77 amino acid preproprotein, apelin is post-translationally cleaved to a series of shorter peptides. Though (Pyr)1apelin-13 represents the major circulating form in plasma, it is highly susceptible to proteolytic degradation and has an extremely short half-life, making it challenging to quantify. Literature reports of apelin levels in rodents have historically been determined with commercial ELISA kits which suffer from a lack of selectivity, recognizing a range of active and inactive isoforms of apelin peptide. (Pyr)1apelin-13 has demonstrated beneficial hemodynamic effects in humans, and we wished to evaluate if similar effects could be measured in pre-clinical models. Despite development of a highly selective LC/MS/MS method, in rodent studies where (Pyr)1apelin-13 was administered exogenously the peptide was not detectable until a detailed stabilization protocol was implemented during blood collection. Further, the inherent high clearance of (Pyr)1apelin-13 required an extended release delivery system to enable chronic dosing. The ability to deliver sustained doses and stabilize (Pyr)1apelin-13 in plasma allowed us to demonstrate for the first time the link between systemic concentration of apelin and its pharmacological effects in animal models.

3.
J Pharmacol Exp Ther ; 2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30361237

RESUMO

Plasma pyridoxic acid (PDA) and homovanillic acid (HVA) were recently identified as novel endogenous biomarkers of OAT1/3 function in monkeys. Consequently, this clinical study assessed the dynamic changes and utility of plasma PDA and HVA as an initial evaluation of OAT1/3 inhibition in early-phase drug development. The study was designed as a single dose randomized, three-phase, crossover study; 14 Indian healthy volunteers received probenecid (PROB) (1,000 mg orally) alone, furosemide (FSM) (40 mg orally) alone, or FSM 1 h after receiving PROB (40 mg and 1,000 mg orally) on Days 1, 8, and 15, respectively. PDA and HVA plasma concentrations remained stable over time in the prestudy and FSM groups. Administration of PROB significantly increased AUC of PDA by 3.1-fold (dosed alone; p < 0.05), and 3.2-fold (coadministered with FSM; p < 0.01), as compared with the prestudy and FSM groups, respectively. The corresponding increase in HVA AUC was 1.8-fold (p > 0.05) and 2.1-fold (p < 0.05), respectively. The increases in PDA AUC are similar to those in FSM AUC whereas those of HVA are smaller (3.1-3.2 and 1.8-2.1 versus 3.3, respectively). PDA and HVA CLR were decreased by PROB to smaller extents compared to FSM (0.35-0.37 and 0.67-0.73 versus 0.23, respectively). These data demonstrate that plasma PDA is a promising endogenous biomarker for OAT1/3 function and its plasma exposure respond in a similar fashion to FSM upon OAT1/3 inhibition by PROB. The magnitude and variability of response in PDA AUC and CLR between subjects is more favourable relative to HVA.

4.
Bioanalysis ; 10(9): 633-644, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29749254

RESUMO

AIM: Coproporphyrins (CP-I and CP-III) have been identified as possible biomarkers to predict human hepatic organic anion-transporting polypeptides-mediated-drug-interactions for a new drug entering clinical development. RESULTS: The method is applicable to quantify plasma CP-I and CP-III within 0.078-15.0 nM. The results identify and address a number of challenges encountered with porphyrin assays such as photodegradation and interferences. To overcome interferences from ubiquitous porphyrins, a surrogate matrix was used to prepare calibration standards. Quality controls were prepared in plasma and surrogate matrix to ensure parallelism between surrogate matrix and plasma. CONCLUSION: A robust UHPLC-MS/MS assay was developed and validated for CP-I and CP-III in plasma, and is currently applied to clinical studies to confirm suitability of Coproporphyrins as a potential substitute for drug-drug interaction study.


Assuntos
Biomarcadores Farmacológicos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Coproporfirinas/sangue , Transportadores de Ânions Orgânicos/metabolismo , Espectrometria de Massas em Tandem/métodos , Biomarcadores Farmacológicos/química , Coproporfirinas/química , Desenho de Drogas , Interações de Medicamentos , Humanos , Transportadores de Ânions Orgânicos/química , Rifampina/sangue , Rifampina/química , Rosuvastatina Cálcica/sangue , Rosuvastatina Cálcica/química
5.
Drug Metab Dispos ; 46(2): 178-188, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29162614

RESUMO

Perturbation of organic anion transporter (OAT) 1- and OAT3-mediated transport can alter the exposure, efficacy, and safety of drugs. Although there have been reports of the endogenous biomarkers for OAT1/3, none of these have all of the characteristics required for a clinical useful biomarker. Cynomolgus monkeys were treated with intravenous probenecid (PROB) at a dose of 40 mg/kg in this study. As expected, PROB increased the area under the plasma concentration-time curve (AUC) of coadministered furosemide, a known substrate of OAT1 and OAT3, by 4.1-fold, consistent with the values reported in humans (3.1- to 3.7-fold). Of the 233 plasma metabolites analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics method, 29 metabolites, including pyridoxic acid (PDA) and homovanillic acid (HVA), were significantly increased after either 1 or 3 hours in plasma from the monkeys pretreated with PROB compared with the treated animals. The plasma of animals was then subjected to targeted LC-MS/MS analysis, which confirmed that the PDA and HVA AUCs increased by approximately 2- to 3-fold by PROB pretreatments. PROB also increased the plasma concentrations of hexadecanedioic acid (HDA) and tetradecanedioic acid (TDA), although the increases were not statistically significant. Moreover, transporter profiling assessed using stable cell lines constitutively expressing transporters demonstrated that PDA and HVA are substrates for human OAT1, OAT3, OAT2 (HVA), and OAT4 (PDA), but not OCT2, MATE1, MATE2K, OATP1B1, OATP1B3, and sodium taurocholate cotransporting polypeptide. Collectively, these findings suggest that PDA and HVA might serve as blood-based endogenous probes of cynomolgus monkey OAT1 and OAT3, and investigation of PDA and HVA as circulating endogenous biomarkers of human OAT1 and OAT3 function is warranted.


Assuntos
Biomarcadores/sangue , Ácido Homovanílico/sangue , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Ácido Piridóxico/sangue , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Células HEK293 , Humanos , Macaca fascicularis , Metabolômica/métodos , Probenecid/metabolismo
7.
Bioanalysis ; 9(20): 1573-1588, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29072496

RESUMO

AIM: IP-10 is a protein target for the treatment of Crohn's disease. Inhibition of IP-10 by anti-IP-10 mAbs neutralizes its various biological activities. The measurement of free IP-10 suppression as a target engagement biomarker is required for the assessment of drug effect on the target. RESULTS: The development of highly sensitive immunoaffinity-LC-MS/MS assays for quantifying free and total IP-10 in cynomolgus monkey serum is reported for the first time. This paper details strategies for maximizing assay sensitivity by selecting digestion routes, and optimizing immunocapture to achieve full recovery and minimal matrix effect. For the free IP-10 assay, bioanalytical strategies have been established to minimize drug/ligand dissociation. CONCLUSION: The assays have been implemented for target engagement measurement, pharmacokinetic-pharmacodynamic correlation, and human dose projections.


Assuntos
Biomarcadores/sangue , Quimiocina CXCL10/sangue , Cromatografia de Afinidade , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Cromatografia Líquida de Alta Pressão , Humanos , Ligantes , Macaca fascicularis , Peptídeos/química , Peptídeos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
8.
Anal Chem ; 89(9): 5115-5123, 2017 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-28383906

RESUMO

We demonstrate a novel strategy using affinity extraction (AE) LC-MS to directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay. The assay measures total drug and target concentration at the site of therapeutic action, as well as the amount of target bound to drug. The case study presented applies the strategy to measure drug engagement of a membrane bound receptor (CD40) that is critical to immune regulation in colon biopsies collected from monkey dosed with an anti-CD40 antibody. Unlike other techniques that measure receptor occupancy, such as flow cytometry, this technique does not rely on viable cells allowing measurement of frozen samples in a remote setting from the clinic.

9.
Obesity (Silver Spring) ; 25(6): 1069-1076, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28452429

RESUMO

OBJECTIVE: Characteristic pathological changes define the progression of steatosis to nonalcoholic steatohepatitis (NASH) and are correlated to metabolic pathways. A common rodent model of NASH is the methionine and choline deficient (MCD) diet. The objective of this study was to perform full metabolomic analyses on liver samples to determine which pathways are altered most pronouncedly in this condition in humans, and to compare these changes to rodent models of nonalcoholic fatty liver disease (NAFLD). METHODS: A principal component analysis for all 91 metabolites measured indicated that metabolome perturbation is greater and less varied for humans than for rodents. RESULTS: Metabolome changes in human and rat NAFLD were greatest for the amino acid and bile acid metabolite families (e.g., asparagine, citrulline, gamma-aminobutyric acid, lysine); although, in many cases, the trends were reversed when compared between species (cholic acid, betaine). CONCLUSIONS: Overall, these results indicate that metabolites of specific pathways may be useful biomarkers for NASH progression, although these markers may not correspond to rodent NASH models. The MCD model may be useful when studying certain end points of NASH; however, the metabolomics results indicate important differences between humans and rodents in the biochemical pathogenesis of the disease.


Assuntos
Metabolômica/métodos , Obesidade/complicações , Animais , Dieta , Progressão da Doença , Humanos , Fígado/patologia , Masculino , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ratos , Ratos Sprague-Dawley
10.
Cell Metab ; 24(2): 223-33, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27508871

RESUMO

The development of LXR agonists for the treatment of coronary artery disease has been challenged by undesirable properties in animal models. Here we show the effects of an LXR agonist on lipid and lipoprotein metabolism and neutrophils in human subjects. BMS-852927, a novel LXRß-selective compound, had favorable profiles in animal models with a wide therapeutic index in cynomolgus monkeys and mice. In healthy subjects and hypercholesterolemic patients, reverse cholesterol transport pathways were induced similarly to that in animal models. However, increased plasma and hepatic TG, plasma LDL-C, apoB, apoE, and CETP and decreased circulating neutrophils were also evident. Furthermore, similar increases in LDL-C were observed in normocholesterolemic subjects and statin-treated patients. The primate model markedly underestimated human lipogenic responses and did not predict human neutrophil effects. These studies demonstrate both beneficial and adverse LXR agonist clinical responses and emphasize the importance of further translational research in this area.


Assuntos
Movimento Celular , Imidazóis/efeitos adversos , Imidazóis/farmacologia , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Receptores X do Fígado/agonistas , Neutrófilos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Tecido Adiposo/metabolismo , Adolescente , Adulto , Animais , Movimento Celular/efeitos dos fármacos , Colesterol/sangue , Colesterol/metabolismo , Voluntários Saudáveis , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Imidazóis/uso terapêutico , Contagem de Leucócitos , Lipoproteínas/sangue , Macaca fascicularis , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Sistema Fagocitário Mononuclear/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Triglicerídeos/sangue , Adulto Jovem
11.
J Pharmacol Exp Ther ; 358(3): 397-404, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27317801

RESUMO

In the present study, an open-label, three-treatment, three-period clinical study of rosuvastatin (RSV) and rifampicin (RIF) when administered alone and in combination was conducted in 12 male healthy subjects to determine if coproporphyrin I (CP-I) and coproporphyrin III (CP-III) could serve as clinical biomarkers for organic anion transporting polypeptide 1B1 (OATP1B1) and 1B3 that belong to the solute carrier organic anion gene subfamily. Genotyping of the human OATP1B1 gene was performed in all 12 subjects and confirmed absence of OATP1B1*5 and OATP1B1*15 mutations. Average plasma concentrations of CP-I and CP-III prior to drug administration were 0.91 ± 0.21 and 0.15 ± 0.04 nM, respectively, with minimum fluctuation over the three periods. CP-I was passively eliminated, whereas CP-III was actively secreted from urine. Administration of RSV caused no significant changes in the plasma and urinary profiles of CP-I and CP-III. RIF markedly increased the maximum plasma concentration (Cmax) of CP-I and CP-III by 5.7- and 5.4-fold (RIF) or 5.7- and 6.5-fold (RIF+RSV), respectively, as compared with the predose values. The area under the plasma concentration curves from time 0 to 24 h (AUC0-24h) of CP-I and CP-III with RIF and RSV increased by 4.0- and 3.3-fold, respectively, when compared with RSV alone. In agreement with this finding, Cmax and AUC0-24h of RSV increased by 13.2- and 5.0-fold, respectively, when RIF was coadministered. Collectively, we conclude that CP-I and CP-III in plasma and urine can be appropriate endogenous biomarkers specifically and reliably reflecting OATP inhibition, and thus the measurement of these molecules can serve as a useful tool to assess OATP drug-drug interaction liabilities in early clinical studies.


Assuntos
Coproporfirinas/sangue , Coproporfirinas/urina , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Rifampina/farmacologia , Rosuvastatina Cálcica/farmacologia , Adulto , Biomarcadores/sangue , Biomarcadores/urina , Interações de Medicamentos , Humanos , Masculino , Pessoa de Meia-Idade , Rifampina/farmacocinética , Rosuvastatina Cálcica/farmacocinética , Adulto Jovem
12.
Gut Microbes ; 7(4): 313-322, 2016 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-27355107

RESUMO

We previously reported quantitation of gut microbiota in a panel of 89 different inbred strains of mice, and we now examine the question of sex differences in microbiota composition. When the total population of 689 mice was examined together, several taxa exhibited significant differences in abundance between sexes but a larger number of differences were observed at the single strain level, suggesting that sex differences can be obscured by host genetics and environmental factors. We also examined a subset of mice on chow and high fat diets and observed sex-by-diet interactions. We further investigated the sex differences using gonadectomized and hormone treated mice from 3 different inbred strains. Principal coordinate analysis with unweighted UniFrac distances revealed very clear effects of gonadectomy and hormone replacement on microbiota composition in all 3 strains. Moreover, bile acid analyses showed gender-specific differences as well as effects of gonodectomy, providing one possible mechanism mediating sex differences in microbiota composition.


Assuntos
Microbioma Gastrointestinal , Trato Gastrointestinal/microbiologia , Hormônios/metabolismo , Camundongos/microbiologia , Animais , Ácidos e Sais Biliares/metabolismo , Comportamento Alimentar , Feminino , Masculino , Camundongos/fisiologia , Fatores Sexuais
13.
Anal Biochem ; 503: 71-8, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27033006

RESUMO

The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC-MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC-MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time.


Assuntos
Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/metabolismo , Marcação por Isótopo , Metabolômica , Animais , Ácidos e Sais Biliares/química , Cromatografia Líquida , Humanos , Espectrometria de Massas , Estrutura Molecular , Ratos
14.
Amino Acids ; 47(3): 603-15, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25534430

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is a globally widespread disease of increasing clinical significance. The pathological progression of the disease from simple steatosis to nonalcoholic steatohepatitis (NASH) has been well defined, however, the contribution of altered branched chain amino acid metabolomic profiles to the progression of NAFLD is not known. The three BCAAs: leucine, isoleucine and valine are known to mediate activation of several important hepatic metabolic signaling pathways ranging from insulin signaling to glucose regulation. The purpose of this study is to profile changes in hepatic BCAA metabolite levels with transcriptomic changes in the progression of human NAFLD to discover novel mechanisms of disease progression. Metabolomic and transcriptomic data sets representing the spectrum of human NAFLD (normal, steatosis, NASH fatty, and NASH not fatty livers) were utilized for this study. During the transition from steatosis to NASH, increases in the levels of leucine (127% of normal), isoleucine (139%), and valine (147%) were observed. Carnitine metabolites also exhibited significantly elevated profiles in NASH fatty and NASH not fatty samples and included propionyl, hexanoyl, lauryl, acetyl and butyryl carnitine. Amino acid and BCAA metabolism gene sets were significantly enriched among downregulated genes during NASH. These cumulative alterations in BCAA metabolite and amino acid metabolism gene profiles represent adaptive physiological responses to disease-induced hepatic stress in NASH patients.


Assuntos
Isoleucina/metabolismo , Leucina/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Valina/metabolismo , Carnitina/genética , Carnitina/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Isoleucina/genética , Leucina/genética , Masculino , Metabolômica , Hepatopatia Gordurosa não Alcoólica/genética , Transdução de Sinais/genética , Valina/genética
15.
J Lipid Res ; 55(8): 1784-96, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24872406

RESUMO

Lysophosphatidic acids (LPAs) are biologically active signaling molecules involved in the regulation of many cellular processes and have been implicated as potential mediators of fibroblast recruitment to the pulmonary airspace, pointing to possible involvement of LPA in the pathology of pulmonary fibrosis. LPAs have been measured in various biological matrices and many challenges involved with their analyses have been documented. However, little published information is available describing LPA levels in human bronchoalveolar lavage fluid (BALF). We therefore conducted detailed investigations into the effects of extensive sample handling and sample preparation conditions on LPA levels in human BALF. Further, targeted lipid profiling of human BALF and plasma identified the most abundant lysophospholipids likely to interfere with LPA measurements. We present the findings from these investigations, highlighting the importance of well-controlled sample handling for the accurate quantitation of LPA. Further, we show that chromatographic separation of individual LPA species from their corresponding lysophospholipid species is critical to avoid reporting artificially elevated levels. The optimized sample preparation and LC/MS/MS method was qualified using a stable isotope-labeled LPA as a surrogate calibrant and used to determine LPA levels in human BALF and plasma from a Phase 0 clinical study comparing idiopathic pulmonary fibrosis patients to healthy controls.


Assuntos
Líquido da Lavagem Broncoalveolar/química , Fibrose Pulmonar Idiopática/metabolismo , Lisofosfolipídeos/metabolismo , Cromatografia Líquida/métodos , Feminino , Humanos , Masculino , Espectrometria de Massas/métodos
16.
Dig Dis Sci ; 59(2): 365-74, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24048683

RESUMO

BACKGROUND: The worldwide prevalences of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are estimated to range from 30 to 40 % and 5-17 %, respectively. Hepatocellular carcinoma (HCC) is primarily caused by hepatitis B infection, but retrospective data suggest that 4-29 % of NASH cases will progress to HCC. Currently the connection between NASH and HCC is unclear. AIMS: The purpose of this study was to identify changes in expression of HCC-related genes and metabolite profiles in NAFLD progression. METHODS: Transcriptomic and metabolomic datasets from human liver tissue representing NAFLD progression (normal, steatosis, NASH) were utilized and compared to published data for HCC. RESULTS: Genes involved in Wnt signaling were downregulated in NASH but have been reported to be upregulated in HCC. Extracellular matrix/angiogenesis genes were upregulated in NASH, similar to reports in HCC. Iron homeostasis is known to be perturbed in HCC and we observed downregulation of genes in this pathway. In the metabolomics analysis of hepatic NAFLD samples, several changes were opposite to what has been reported in plasma of HCC patients (lysine, phenylalanine, citrulline, creatine, creatinine, glycodeoxycholic acid, inosine, and alpha-ketoglutarate). In contrast, multiple acyl-lyso-phosphatidylcholine metabolites were downregulated in NASH livers, consistent with observations in HCC patient plasma. CONCLUSIONS: These data indicate an overlap in the pathogenesis of NAFLD and HCC where several classes of HCC related genes and metabolites are altered in NAFLD. Importantly, Wnt signaling and several metabolites are different, thus implicating these genes and metabolites as mediators in the transition from NASH to HCC.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Análise por Conglomerados , Bases de Dados Genéticas , Fígado Gorduroso/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Metabolômica , Hepatopatia Gordurosa não Alcoólica , Transdução de Sinais/genética
17.
Bioanalysis ; 5(10): 1195-210, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23721443

RESUMO

BACKGROUND: The application of high-resolution LC-MS metabolomics for drug candidate toxicity screening reflects phenotypic changes of an organism caused by induced chemical interferences. Its success depends not only on the ability to translate the acquired analytical information into biological knowledge, but also on the timely delivery of the results to aid the decision making process in drug discovery and development. Recent improvements in analytical instrumentation have resulted in the ability to acquire extremely information-rich datasets. These new data collection abilities have shifted the bottleneck in the timeline of metabolomic studies to the data analysis step. RESULTS: This paper describes our approach to expedient data analysis of nontargeted high-resolution LC-MS profiles of biological samples. The workflow is illustrated with the example of metabolomics study of time-dependent fasting in male rats. The results from measurement of 220 endogenous metabolites in urine samples illustrate significant biochemical changes induced by fasting. CONCLUSION: The developed software enables the reporting of relative quantities of annotated components while maintaining practical turnaround times. Each component annotation in the report is validated using both calculated isotopic peaks patterns and experimentally determined retention time data on standards.


Assuntos
Biomarcadores/urina , Cromatografia Líquida , Mineração de Dados , Jejum/urina , Espectrometria de Massas , Metabolômica , Software , Animais , Ritmo Circadiano , Hipuratos/urina , Masculino , Ratos
18.
J Lipid Res ; 54(9): 2400-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23614904

RESUMO

Proprotein convertase subtilisin-kexin-9 (PCSK9) inhibition markedly augments the LDL lowering action of statins. The combination is being evaluated for long-term effects on atherosclerotic disease outcomes. However, effects of combined treatment on hepatic cholesterol and bile acid metabolism have not yet been reported. To study this, PCSK9-Y119X mutant (knockout) and wild-type mice were treated with or without atorvastatin for 12 weeks. Atorvastatin progressively lowered plasma LDL in each group, but no differences in liver cholesterol, cholesterol ester, or total bile acid concentrations, or in plasma total bile acid levels were seen. In contrast, atorvastatin increased fecal total bile acids (≈ 2-fold, P < 0.01) and cholesterol concentrations (≈ 3-fold, P < 0.01) versus controls for both PCSK9-Y119X and wild-type mice. All 14 individual bile acids resolved by LC-MS, including primary, secondary, and conjugated species, reflected similar increases. Expression of key liver bile acid synthesis genes CYP7A1 and CYP8B1 were ≈ 2.5-fold higher with atorvastatin in both strains, but mRNA for liver bile acid export and reuptake transporters and conjugating enzymes were not unaffected. The data suggest that hepatocyte cholesterol and bile acid homeostasis is maintained with combined PCSK9 and HMG-CoA reductase inhibition through efficient liver enzymatic conversion of LDL-derived cholesterol into bile acids and excretion of both, with undisturbed enterohepatic recycling.


Assuntos
Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Pró-Proteína Convertases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Colesterol/sangue , Interações de Medicamentos , Fezes/química , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Pró-Proteína Convertase 9 , Serina Endopeptidases
19.
PLoS One ; 8(2): e53192, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383297

RESUMO

BACKGROUND: Chronic glucocorticoid excess has been linked to increased atherosclerosis and general cardiovascular risk in humans. The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) increases active glucocorticoid levels within tissues by catalyzing the conversion of cortisone to cortisol. Pharmacological inhibition of 11ßHSD1 has been shown to reduce atherosclerosis in murine models. However, the cellular and molecular details for this effect have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: To examine the role of 11ßHSD1 in atherogenesis, 11ßHSD1 knockout mice were created on the pro-atherogenic apoE⁻/⁻ background. Following 14 weeks of Western diet, aortic cholesterol levels were reduced 50% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. 11ßHSD1⁺/⁺/apoE⁻/⁻ mice without changes in plasma cholesterol. Aortic 7-ketocholesterol content was reduced 40% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. In the aortic root, plaque size, necrotic core area and macrophage content were reduced ∼30% in 11ßHSD1⁻/⁻/apoE⁻/⁻mice. Bone marrow transplantation from 11ßHSD1⁻/⁻/apoE⁻/⁻ mice into apoE⁻/⁻ recipients reduced plaque area 39-46% in the thoracic aorta. In vivo foam cell formation was evaluated in thioglycollate-elicited peritoneal macrophages from 11ßHSD1⁺/⁺/apoE⁻/⁻ and 11ßHSD1⁻/⁻/apoE⁻/⁻ mice fed a Western diet for ∼5 weeks. Foam cell cholesterol levels were reduced 48% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. Microarray profiling of peritoneal macrophages revealed differential expression of genes involved in inflammation, stress response and energy metabolism. Several toll-like receptors (TLRs) were downregulated in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice including TLR 1, 3 and 4. Cytokine release from 11ßHSD1⁻/⁻/apoE⁻/⁻-derived peritoneal foam cells was attenuated following challenge with oxidized LDL. CONCLUSIONS: These findings suggest that 11ßHSD1 inhibition may have the potential to limit plaque development at the vessel wall and regulate foam cell formation independent of changes in plasma lipids. The diminished cytokine response to oxidized LDL stimulation is consistent with the reduction in TLR expression and suggests involvement of 11ßHSD1 in modulating binding of pro-atherogenic TLR ligands.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Células Espumosas/metabolismo , Glucocorticoides/metabolismo , Análise de Variância , Animais , Aterosclerose/prevenção & controle , Pressão Sanguínea , Transplante de Medula Óssea , Colesterol/metabolismo , Dieta Aterogênica , Cetocolesteróis/metabolismo , Lipídeos/sangue , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Toll-Like/metabolismo
20.
Toxicol Appl Pharmacol ; 268(2): 132-40, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23391614

RESUMO

Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the 'classical' (neutral) and 'alternative' (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH.


Assuntos
Ácidos e Sais Biliares/metabolismo , Fígado Gorduroso/metabolismo , Fígado/efeitos dos fármacos , Ácidos e Sais Biliares/análise , Ácidos e Sais Biliares/genética , Ácidos e Sais Biliares/toxicidade , Análise por Conglomerados , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Metabolômica , Hepatopatia Gordurosa não Alcoólica
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