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1.
Ann Rheum Dis ; 79(9): 1218-1226, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32561607

RESUMO

OBJECTIVES: The analysis of annotated transcripts from genome-wide expression studies may help to understand the pathogenesis of complex diseases, such as systemic sclerosis (SSc). We performed a whole blood (WB) transcriptome analysis on RNA collected in the context of the European PRECISESADS project, aiming at characterising the pathways that differentiate SSc from controls and that are reproducible in geographically diverse populations. METHODS: Samples from 162 patients and 252 controls were collected in RNA stabilisers. Cases and controls were divided into a discovery (n=79+163; Southern Europe) and validation cohort (n=83+89; Central-Western Europe). RNA sequencing was performed by an Illumina assay. Functional annotations of Reactome pathways were performed with the Functional Analysis of Individual Microarray Expression (FAIME) algorithm. In parallel, immunophenotyping of 28 circulating cell populations was performed. We tested the presence of differentially expressed genes/pathways and the correlation between absolute cell counts and RNA transcripts/FAIME scores in regression models. Results significant in both populations were considered as replicated. RESULTS: Overall, 15 224 genes and 1277 functional pathways were available; of these, 99 and 225 were significant in both sets. Among replicated pathways, we found a deregulation in type-I interferon, Toll-like receptor cascade, tumour suppressor p53 protein function, platelet degranulation and activation. RNA transcripts or FAIME scores were jointly correlated with cell subtypes with strong geographical differences; neutrophils were the major determinant of gene expression in SSc-WB samples. CONCLUSIONS: We discovered a set of differentially expressed genes/pathways validated in two independent sets of patients with SSc, highlighting a number of deregulated processes that have relevance for the pathogenesis of autoimmunity and SSc.


Assuntos
Autoimunidade/genética , Escleroderma Sistêmico/genética , Transdução de Sinais/genética , Transcriptoma/genética , Adulto , Idoso , Estudos de Coortes , Europa (Continente) , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Imunofenotipagem , Interferon Tipo I/sangue , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Análise de Sequência de RNA , Receptores Toll-Like/sangue
2.
Ann Rheum Dis ; 77(12): 1782-1789, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30065042

RESUMO

OBJECTIVES: Chronic renal impairment remains a feared complication of lupus nephritis (LN). The present work aimed at identifying mechanisms and markers of disease severity in renal tissue samples from patients with LN. METHODS: We performed high-throughput transcriptomic studies (Illumina HumanHT-12 v4 Expression BeadChip) on archived kidney biopsies from 32 patients with LN and eight controls (pretransplant donors). Histological staging (glomerular and tubular scores) and immunohistochemistry experiments were performed on the same and on a replication set of 37 LN kidney biopsy samples. RESULTS: A group of LN samples was identified by unsupervised clustering studies based on their gene expression features, that is, the overexpression of transcripts involved in antigen presentation, T and B cell activation. These samples were characterised by a significantly lower estimated glomerular filtration rate (eGFR) at the time of biopsy (T0) compared with the other systemic lupus erythematosus samples. Yet, apparent disease duration at T0, double-stranded DNA antibody titres at T0 and other relevant characteristics (serum C3, proteinuria, histological scores, numbers of previous flares) were not different between groups.Immunohistochemistry studies confirmed the association between interstitial infiltration by adaptive immune effectors and decreased renal function in the same and in a replication group of LN kidney biopsies. This was associated with transcriptomic, histological and immunohistochemical evidence of renal tubular cell involvement. CONCLUSION: Interstitial infiltration of LN kidney biopsies by adaptive immune effectors is associated with impaired renal tubular cell function and decreased eGFR. These results open new perspectives in evaluating and treating patients with LN, focusing on intrarenal mechanisms of immune cell activation.


Assuntos
Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Adulto , Feminino , Humanos , Túbulos Renais/patologia , Masculino , Insuficiência Renal/imunologia , Insuficiência Renal/patologia , Transcriptoma
3.
Lab Invest ; 96(9): 972-80, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27428080

RESUMO

Notch signaling pathways have recently been implicated in the pathogenesis of metabolic diseases. However, the role of hepatic Notch signaling in glucose and lipid metabolism remains unclear and needs further investigation as it might be a candidate therapeutic target in metabolic diseases such as nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). We used hepatocyte-specific Notch1 knockout (KO) mice and liver biopsies from NASH and NAFLD patients to analyze the role of Notch1 in glucose and lipid metabolism. Hepatocyte-specific Notch1 KO mice were fed with a high fat diet (HFD) or a regular diet (RD). We assessed the metabolic phenotype, glucose and insulin tolerance tests, and liver histology. Hepatic mRNA expression was profiled by Affymetrix Mouse Gene arrays and validated by quantitative reverse transcription PCR (qPCR). Akt phosphorylation was visualized by immunoblotting. Gene expression was analyzed in liver biopsies from NASH, NAFLD, and control patients by qPCR. We found that Notch1 KO mice had elevated fasting glucose. Gene expression analysis showed an upregulation of glucose-6-phosphatase, involved in the final step of gluconeogenesis and glucose release from glycogenolysis, and perilipin-5, a regulator of hepatic lipid accumulation. When fed with an HFD KO mice developed overt diabetes and hepatic steatosis. Akt was highly phosphorylated in KO animals and the Foxo1 target gene expression was altered. Accordingly, a reduction in Notch1 and increase in glucose-6-phosphatase and perilipin-5 expression was observed in liver biopsies from NAFLD/NASH compared with controls. Notch1 is a regulator of hepatic glucose and lipid homeostasis. Hepatic impairment of Notch1 expression may be involved in the pathogenesis of human NAFLD/NASH.


Assuntos
Diabetes Mellitus/genética , Fígado Gorduroso/genética , Predisposição Genética para Doença/genética , Glucose-6-Fosfatase/genética , Perilipina-1/genética , Receptor Notch1/genética , Animais , Diabetes Mellitus/etiologia , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/etiologia , Perfilação da Expressão Gênica/métodos , Hepatócitos/metabolismo , Humanos , Immunoblotting , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
4.
Hepatology ; 62(5): 1497-510, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26173433

RESUMO

UNLABELLED: The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature. CONCLUSIONS: These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins.


Assuntos
Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/patologia , Proteínas de Ligação a DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Neovascularização Patológica/etiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Apoptose , Neoplasias dos Ductos Biliares/irrigação sanguínea , Neoplasias dos Ductos Biliares/tratamento farmacológico , Proteínas de Ciclo Celular , Proliferação de Células , Colangiocarcinoma/irrigação sanguínea , Colangiocarcinoma/tratamento farmacológico , Proteínas Contráteis/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Oncogenes , Fatores de Transcrição de Domínio TEA
5.
Lab Invest ; 95(3): 351-62, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25418579

RESUMO

Hepatic angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited as animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aims of this study were to: (1) establish and characterize a cell line derived from this murine AS, (2) identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and (3) generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and Von Willebrand factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors, confirming the tumorigenic potential of these cells. In summary, we established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, supporting further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing.


Assuntos
Modelos Animais de Doenças , Hemangiossarcoma/patologia , Neoplasias Hepáticas/patologia , Fígado/patologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hemangiossarcoma/genética , Hemangiossarcoma/metabolismo , Imuno-Histoquímica , Fígado/metabolismo , Fígado/ultraestrutura , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Microscopia Eletrônica , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Compostos de Fenilureia/farmacologia , Receptor Notch1/deficiência , Receptor Notch1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sorafenibe , Carga Tumoral/genética
6.
Hepatology ; 59(6): 2121-30, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24122862

RESUMO

UNLABELLED: Approximately 50% of patients with chronic hepatitis C (CHC) have ongoing expression of interferon stimulated genes (ISGs) in the liver. It is unclear why this endogenous antiviral response is inefficient in eradicating the infection. Several viral escape strategies have been identified in vitro, including inhibition of interferon (IFN) induction and ISG messenger RNA (mRNA) translation. The in vivo relevance of these mechanisms is unknown, because reliable methods to identify hepatitis C virus (HCV)-infected cells in human liver are lacking. We developed a highly sensitive in situ hybridization (ISH) system capable of HCV RNA and ISG mRNA detection in human liver biopsies and applied it to study the interaction of HCV with the endogenous IFN system. We simultaneously monitored HCV RNA and ISG mRNA using HCV isolate- and ISG mRNA-specific probes in liver biopsy sections from 18 CHC patients. The signals were quantified at the single-cell resolution in a series of random high-power fields. The proportion of infected hepatocytes ranged from 1%-54% and correlated with viral load, but not with HCV genotype or ISG expression. Infected cells occurred in clusters, pointing to cell-to-cell spread as the predominant mode of HCV transmission. ISG mRNAs were readily detected in HCV-infected cells, challenging previously proposed mechanisms of viral interference with the immune system. Conversely, infected cells and neighboring cells showed increased ISG mRNA levels, demonstrating that the stimulus driving ISG expression originates from HCV-infected hepatocytes. CONCLUSION: HCV infection in human hepatocytes during CHC does not efficiently interfere with IFN induction, IFN signaling, or transcription of ISG mRNA.


Assuntos
Regulação Viral da Expressão Gênica , Hepatite C/virologia , Interferons/fisiologia , Fígado/virologia , Hepatite C/genética , Hepatite C/metabolismo , Hepatócitos/virologia , Humanos , Hibridização In Situ , Fígado/metabolismo , RNA Viral/genética , Carga Viral/genética
7.
Hepatology ; 59(3): 911-23, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24114970

RESUMO

UNLABELLED: Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related death. Despite the advances in diagnosis and management of HCC, the biology of this tumor remains poorly understood. Recent evidence highlighted long noncoding RNAs (lncRNAs) as crucial determinants of HCC development. In this study we report the lncRNA HOXA transcript at the distal tip (HOTTIP) as significantly up-regulated in HCC specimens. The HOTTIP gene is located in physical contiguity with HOXA13 and directly controls the HOXA locus gene expression by way of interaction with the WDR5/MLL complex. HOX genes encode transcription factors regulating embryonic development and cell fate. We previously described HOX genes deregulation to be involved in hepatocarcinogenesis. Indeed, we observed the marked up-regulation of HOXA13 in HCC. Here, by correlating clinicopathological and expression data, we demonstrate that the levels of HOTTIP and HOXA13 are associated with HCC patients' clinical progression and predict disease outcome. In contrast to the majority of similar studies, our data were obtained from snap-frozen needle HCC biopsies (n=52) matched with their nonneoplastic counterparts collected from patients who had not yet received any HCC-tailored therapeutic treatments at the time of biopsy. In addition, taking advantage of gain and loss of function experiments in liver cancer-derived cell lines (HuH-6 and HuH-7), we uncover a novel bidirectional regulatory loop between HOTTIP/HOXA13. CONCLUSION: Our study highlights the key role of HOTTIP and HOXA13 in HCC development by associating their expression with metastasis and survival in HCC patients, provides novel insights on the function of lncRNA-driven hepatocarcinogenesis, and paves the way for further investigation about the possible role of HOTTIP as a predictive biomarker of HCC.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Homeodomínio/genética , Neoplasias Hepáticas/genética , RNA Longo não Codificante/genética , Idoso , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/secundário , Linhagem Celular Transformada , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Gradação de Tumores , Valor Preditivo dos Testes , RNA Interferente Pequeno/genética
8.
Liver Int ; 35(7): 1824-32, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25533046

RESUMO

BACKGROUND & AIMS: Differences in intrahepatic gene expression patterns may be associated with therapy response in peginterferon-treated chronic hepatitis B (CHB) patients. METHODS: We employed gene expression profiling in baseline liver biopsies of 40 CHB patients (19 HBeAg-positive; 21 HBeAg-negative) treated with peginterferon and adefovir for 48 weeks, and compared expression patterns of combined responders (HBeAg loss, HBV-DNA <2000 IU/ml, alanine aminotransferase normalization after 1 year of treatment-free follow-up) with non-responders. Genes identified by transcriptome analysis in 15 biopsies were confirmed in 25 additional biopsies by RT-qPCR. RESULTS: Transcriptome analysis demonstrated significant differences in expression of 41 genes between responders and non-responders. In responders, pathway analysis showed specific upregulation of genes related to the immune response, including chemotaxis and antigen processing and presentation. Genes upregulated in responders exhibited strongest similarity with a set of genes induced in livers of chimpanzees with acute Hepatitis B infection. Differential expression was confirmed for eight selected genes. A 2-gene subset (HLA-DPB1, SERPIN-E1) was found to predict response most accurately. Incorporation of these genes in a multivariable model with HBeAg status, HBV genotype and baseline HBsAg level correctly classified 90% of all patients, in which HLA-DPB1 and SERPIN-E1 were independent predictors of response. CONCLUSION: We identified an intrahepatic transcriptional signature associated with enhanced immune activation which predicts therapy response. These novel associations could lead to better understanding of responsiveness to peginterferon in CHB patients, and may assist in selecting possible responders to interferon-based treatment.


Assuntos
Antivirais/uso terapêutico , Perfilação da Expressão Gênica/métodos , Hepatite B Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Fígado/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Polietilenoglicóis/uso terapêutico , Adenina/análogos & derivados , Adenina/uso terapêutico , Adulto , Biópsia , Distribuição de Qui-Quadrado , Quimioterapia Combinada , Feminino , Marcadores Genéticos , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/genética , Hepatite B Crônica/imunologia , Humanos , Fígado/imunologia , Fígado/metabolismo , Fígado/virologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Organofosfonatos/uso terapêutico , Seleção de Pacientes , Medicina de Precisão , Valor Preditivo dos Testes , Proteínas Recombinantes/uso terapêutico , Reprodutibilidade dos Testes , Fatores de Tempo , Resultado do Tratamento
9.
Carcinogenesis ; 35(1): 114-22, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23901063

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most HCCs develop in cirrhotic livers. Alcoholic liver disease, chronic hepatitis B and chronic hepatitis C are the most common underlying liver diseases. Hepatitis C virus (HCV)-specific mechanisms that contribute to HCC are presently unknown. Transgenic expression of HCV proteins in the mouse liver induces an overexpression of the protein phosphatase 2A catalytic subunit (PP2Ac). We have previously reported that HCV-induced PP2Ac overexpression modulates histone methylation and acetylation and inhibits DNA damage repair. In this study, we analyze tumor formation and gene expression using HCV transgenic mice that overexpress PP2Ac and liver tissues from patients with HCC. We demonstrate that PP2Ac overexpression interferes with p53-induced apoptosis. Injection of the carcinogen, diethylnitrosamine, induced significantly more and larger liver tumors in HCV transgenic mice that overexpress PP2Ac compared with control mice. In human liver biopsies from patients with HCC, PP2Ac expression was significantly higher in HCC tissue compared with non-tumorous liver tissue from the same patients. Our findings demonstrate an important role of PP2Ac overexpression in liver carcinogenesis and provide insights into the molecular pathogenesis of HCV-induced HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteína Fosfatase 2/metabolismo , Animais , Biópsia , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/enzimologia , Dietilnitrosamina/toxicidade , Modelos Animais de Doenças , Etoposídeo/análogos & derivados , Etoposídeo/farmacologia , Regulação Enzimológica da Expressão Gênica , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite B Crônica/enzimologia , Hepatite B Crônica/patologia , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Compostos Organofosforados/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
10.
J Hepatol ; 58(4): 743-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23207140

RESUMO

BACKGROUND & AIMS: The toll-like receptor 9 (TLR9) agonist IMO-2125 is currently evaluated in clinical trials for chronic hepatitis C therapy. The aim of this study was to investigate the in vivo mode of action of a closely related compound, referred to as immunomodulatory oligonucleotide (IMO). METHODS: We analyzed the Jak-STAT pathway activation and induction of interferon-stimulated genes in the liver of wild type, interferon-α/ß receptor-deficient and interferon-γ-deficient mice, after administration of IMO. RESULTS: IMO induced a prolonged activation of the Jak-STAT pathway and upregulation of interferon-stimulated genes in the mouse liver. Contrary to the response observed after interferon-α injection, the signalling induced by IMO was not abrogated following repeated administration. At early time points after IMO injection, STAT1 phosphorylation and interferon-stimulated gene induction required a functional interferon-α/ß receptor, whereas at the later time points, the activation was type I interferon-independent. Microarray analysis revealed that IMO induced a broad transcriptional response in the mouse liver. This included upregulation of cytokine and chemokine genes responsible for recruitment of IFN-γ producers, such as T cells and natural killer cells. Interferon-γ-deficient mice showed a transient response to IMO, demonstrating the central role of interferon-γ in sustained activation of Jak-STAT pathway by IMO. CONCLUSIONS: The bimodal kinetics of response to IMO in the mouse liver are driven by the sequential endogenous production of type I and II interferons. The lack of refractoriness to IMO, combined with the long-lasting induction of interferon-stimulated genes, reveals a favourable pharmacodynamics profile of this novel TLR9 agonist for the treatment of chronic viral hepatitis.


Assuntos
Interferon Tipo I/biossíntese , Interferon gama/biossíntese , Fígado/efeitos dos fármacos , Fígado/imunologia , Receptor Toll-Like 9/agonistas , Animais , Quimiocinas/genética , Citocinas/genética , Fatores Imunológicos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligodesoxirribonucleotídeos/farmacologia , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
11.
Gastroenterology ; 143(3): 777-786.e6, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22677194

RESUMO

BACKGROUND & AIMS: Approximately 50% of patients with chronic hepatitis C (CHC) have a sustained virologic response to treatment with pegylated interferon (pegIFN)-α and ribavirin. Nonresponse to treatment is associated with constitutively increased expression of IFN-stimulated genes (ISGs) in the liver. Treatment of patients with acute hepatitis C (AHC) is more effective, with sustained virologic response rates greater than 90%. We investigated mechanisms of the different responses of patients with CHC and AHC to pegIFN-α therapy. METHODS: We analyzed IFN signaling and ISG expression in liver samples from patients with AHC, patients with CHC, and individuals without hepatitis C (controls) using microarray, immunohistochemical, and protein analyses. Findings were compared with those from primary human hepatocytes stimulated with IFN-α or IFN-γ, as reference sets. RESULTS: Expression levels of hundreds of genes, primarily those regulated by IFN-γ, were altered in liver samples from patients with AHC compared with controls. Expression of IFN-γ-stimulated genes was induced in liver samples from patients with AHC, whereas expression of IFN-α-stimulated genes was induced in samples from patients with CHC. In an expression analysis of negative regulators of IFN-α signaling, we did not observe differences in expression of suppresor of cytokine signaling 1 or SOCS3 between liver samples from patients with AHC and those with CHC. However, USP18 (another negative regulator of IFN-α signaling), was up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in AHC. CONCLUSIONS: Differences in expression of ISGs might account for the greater response of patients with AHC, compared with those with CHC, to treatment with pegIFN-α and ribavirin. Specifically, USP18 is up-regulated in liver samples of patients with CHC that did not respond to therapy, but not in patients with AHC.


Assuntos
Antivirais/uso terapêutico , Endopeptidases/genética , Hepatite C Crônica/tratamento farmacológico , Hepatite C/tratamento farmacológico , Interferon-alfa/uso terapêutico , Interferon gama/metabolismo , Fígado/efeitos dos fármacos , Polietilenoglicóis/uso terapêutico , Ribavirina/uso terapêutico , Doença Aguda , Adolescente , Adulto , Biópsia , Western Blotting , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Farmacorresistência Viral/genética , Quimioterapia Combinada , Endopeptidases/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Hepacivirus/patogenicidade , Hepatite C/diagnóstico , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/genética , Hepatite C Crônica/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Proteínas Recombinantes/uso terapêutico , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Suíça , Fatores de Tempo , Transcrição Gênica , Resultado do Tratamento , Ubiquitina Tiolesterase
12.
Hepatology ; 53(4): 1154-63, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21480323

RESUMO

UNLABELLED: Therapy of chronic hepatitis C with pegylated interferon α (pegIFN-α) and ribavirin achieves sustained virological responses in approximately half of the patients. Nonresponse to treatment is associated with constitutively increased expression of IFN-stimulated genes in the liver already before therapy. This activation of the endogenous IFN system could prevent cells from responding to therapeutically injected (peg)IFN-α, because prolonged stimulation of cells with IFN-α induces desensitization of the IFN signal transduction pathway. Whether all types of IFNs induce refractoriness in the liver is presently unknown. We therefore treated mice with multiple injections and different combinations of IFN-α, IFN-ß, IFN-γ, and IFN-λ. Pretreatment of mice with IFN-α, IFN-ß, and IFN-λ induced a strong expression of the negative regulator ubiquitin-specific peptidase 18 in the liver and gut. As a result, IFN-α signaling was significantly reduced when mice where reinjected 16 hours after the first injection. Surprisingly, both IFN-ß and IFN-λ could activate the Janus kinase-signal transducer and activator of transcription (STAT) pathway and the expression of IFN-stimulated genes despite high levels of ubiquitin-specific peptidase 18. IFN-λ treatment of human liver biopsies ex vivo resulted in strong and maintained phosphorylation of STAT1, whereas IFN-α-induced STAT1 activation was transient. CONCLUSION: Contrary to the action of IFN-α, IFN-ß, and IFN-λ signaling in the liver does not become refractory during repeated stimulation of the IFN signal transduction pathway. The sustained efficacy of IFN-ß and IFN-λ could be an important advantage for the treatment patients who are nonresponders to pegIFN-α, through a preactivated endogenous IFN system.


Assuntos
Citocinas/fisiologia , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Interferon gama/farmacologia , Fígado/efeitos dos fármacos , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Endopeptidases/metabolismo , Hepatite C Crônica/fisiopatologia , Humanos , Indutores de Interferon/farmacologia , Intestino Delgado/efeitos dos fármacos , Janus Quinases/metabolismo , Fígado/fisiologia , Masculino , Camundongos , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina Tiolesterase
13.
Cytokine ; 59(3): 460-6, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22749235

RESUMO

Hepatitis C virus is a global health concern, estimated to infect 2-3% of the world's population. Inter-individual differences in the course of infection and response to therapy, highlighted by recent genomewide association studies, point to the crucial role of the host immune system in the efficient control of infection. Ongoing progress in the studies of the role of innate immunity during hepatitis C virus infection has improved our understanding of the intricacies of the host-virus interactions. In this review, we summarize and discuss the current knowledge concerning interferon signaling in the liver during acute and chronic hepatitis C virus infection and its implications for the outcome of interferon-α-based antiviral therapies.


Assuntos
Hepacivirus/fisiologia , Hepatite C/imunologia , Interferons/metabolismo , Fígado/imunologia , Fígado/virologia , Transdução de Sinais/imunologia , Hepatite C/terapia , Humanos , Fígado/patologia , Resultado do Tratamento
14.
J Transl Autoimmun ; 4: 100090, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33817614

RESUMO

High amount of polyclonal free light chains (FLC) are reported in systemic autoimmune diseases (SAD) and we took advantage of the PRECISESADS study to better characterize them. Serum FLC levels were explored in 1979 patients with SAD (RA, SLE, SjS, Scl, APS, UCTD, MCTD) and 614 healthy controls. Information regarding clinical parameters, disease activity, medications, autoantibodies (Ab) and the interferon α and/or γ scores were recorded. Among SAD patients, 28.4% had raised total FLC (from 12% in RA to 30% in SLE and APS) with a normal kappa/lambda ratio. Total FLC levels were significantly higher in SAD with inflammation, active disease in SLE and SjS, and an impaired pulmonary functional capacity in SSc, while independent from kidney impairment, infection, cancer and treatment. Total FLC concentrations were positively correlated among the 10/17 (58.8%) autoantibodies (Ab) tested with anti-RNA binding protein Ab (SSB, SSA-52/60 kDa, Sm, U1-RNP), anti-dsDNA/nucleosome Ab, rheumatoid factor and negatively correlated with complement fractions C3/C4. Finally, examination of interferon (IFN) expression as a potential driver of FLC overexpression was tested showing an elevated level of total FLC among patients with a high IFNα and IFNγ Kirou's score, a strong IFN modular score, and the detection in the sera of B-cell IFN dependent factors, such as TNF-R1/TNFRSF1A and CXCL10/IP10. In conclusion, an elevated level of FLC, in association with a strong IFN signature, defines a subgroup of SAD patients, including those without renal affectation, characterized by increased disease activity, autoreactivity, and complement reduction.

15.
Arthritis Rheumatol ; 73(7): 1288-1300, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33455083

RESUMO

OBJECTIVE: To identify the genetic variants that affect gene expression (expression quantitative trait loci [eQTLs]) in systemic sclerosis (SSc) and to investigate their role in the pathogenesis of the disease. METHODS: We performed an eQTL analysis using whole-blood sequencing data from 333 SSc patients and 524 controls and integrated them with SSc genome-wide association study (GWAS) data. We integrated our findings from expression modeling, differential expression analysis, and transcription factor binding site enrichment with key clinical features of SSc. RESULTS: We detected 49,123 validated cis-eQTLs from 4,539 SSc-associated single-nucleotide polymorphisms (SNPs) (PGWAS < 10-5 ). A total of 1,436 genes were within 1 Mb of the 4,539 SSc-associated SNPs. Of those 1,436 genes, 565 were detected as having ≥1 eQTL with an SSc-associated SNP. We developed a strategy to prioritize disease-associated genes based on their expression variance explained by SSc eQTLs (r2 > 0.05). As a result, 233 candidates were identified, 134 (58%) of them associated with hallmarks of SSc and 105 (45%) of them differentially expressed in the blood cells, skin, or lung tissue of SSc patients. Transcription factor binding site analysis revealed enriched motifs of 24 transcription factors (5%) among SSc eQTLs, 5 of which were found to be differentially regulated in the blood cells (ELF1 and MGA), skin (KLF4 and ID4), and lungs (TBX4) of SSc patients. Ten candidate genes (4%) can be targeted by approved medications for immune-mediated diseases, of which only 3 have been tested in clinical trials in patients with SSc. CONCLUSION: The findings of the present study indicate a new layer to the molecular complexity of SSc, contributing to a better understanding of the pathogenesis of the disease.


Assuntos
Regulação da Expressão Gênica/genética , Escleroderma Sistêmico/genética , Adulto , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Estudos de Associação Genética , Humanos , Proteínas Inibidoras de Diferenciação/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Proteínas com Domínio T/genética , Fatores de Transcrição/genética
16.
Sci Rep ; 11(1): 23292, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34857786

RESUMO

Primary Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by lymphocytic infiltration and damage of exocrine salivary and lacrimal glands. The etiology of SS is complex with environmental triggers and genetic factors involved. By conducting an integrated multi-omics study, we confirmed a vast coordinated hypomethylation and overexpression effects in IFN-related genes, what is known as the IFN signature. Stratified and conditional analyses suggest a strong interaction between SS-associated HLA genetic variation and the presence of Anti-Ro/SSA autoantibodies in driving the IFN epigenetic signature and determining SS. We report a novel epigenetic signature characterized by increased DNA methylation levels in a large number of genes enriched in pathways such as collagen metabolism and extracellular matrix organization. We identified potential new genetic variants associated with SS that might mediate their risk by altering DNA methylation or gene expression patterns, as well as disease-interacting genetic variants that exhibit regulatory function only in the SS population. Our study sheds new light on the interaction between genetics, autoantibody profiles, DNA methylation and gene expression in SS, and contributes to elucidate the genetic architecture of gene regulation in an autoimmune population.


Assuntos
Autoanticorpos , Epigenômica , Regulação da Expressão Gênica/genética , Expressão Gênica/genética , Variação Genética , Antígenos HLA/genética , Interferons/genética , Síndrome de Sjogren/genética , Síndrome de Sjogren/imunologia , Metilação de DNA/genética , Feminino , Humanos , Masculino , Síndrome de Sjogren/etiologia
17.
Nat Commun ; 12(1): 3523, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112769

RESUMO

There is currently no approved treatment for primary Sjögren's syndrome, a disease that primarily affects adult women. The difficulty in developing effective therapies is -in part- because of the heterogeneity in the clinical manifestation and pathophysiology of the disease. Finding common molecular signatures among patient subgroups could improve our understanding of disease etiology, and facilitate the development of targeted therapeutics. Here, we report, in a cross-sectional cohort, a molecular classification scheme for Sjögren's syndrome patients based on the multi-omic profiling of whole blood samples from a European cohort of over 300 patients, and a similar number of age and gender-matched healthy volunteers. Using transcriptomic, genomic, epigenetic, cytokine expression and flow cytometry data, combined with clinical parameters, we identify four groups of patients with distinct patterns of immune dysregulation. The biomarkers we identify can be used by machine learning classifiers to sort future patients into subgroups, allowing the re-evaluation of response to treatments in clinical trials.


Assuntos
Citocinas/sangue , Metilação de DNA , Interferons/sangue , Proteoma/metabolismo , Síndrome de Sjogren/imunologia , Transcriptoma/genética , Adulto , Autoanticorpos/sangue , Biomarcadores/sangue , Quimiocinas/análise , Quimiocinas/genética , Quimiocinas/metabolismo , Estudos de Coortes , Biologia Computacional , Simulação por Computador , Estudos Transversais , Citocinas/análise , Citocinas/genética , Metilação de DNA/genética , Bases de Dados Genéticas , Bases de Dados de Proteínas , Feminino , Citometria de Fluxo , Estudo de Associação Genômica Ampla , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interferons/genética , Masculino , Pessoa de Meia-Idade , Família Multigênica , Polimorfismo de Nucleotídeo Único , Proteoma/genética , RNA-Seq , Síndrome de Sjogren/sangue , Síndrome de Sjogren/genética , Síndrome de Sjogren/fisiopatologia
18.
Arthritis Rheumatol ; 73(6): 1073-1085, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33497037

RESUMO

OBJECTIVE: Clinical heterogeneity, a hallmark of systemic autoimmune diseases, impedes early diagnosis and effective treatment, issues that may be addressed if patients could be classified into groups defined by molecular pattern. This study was undertaken to identify molecular clusters for reclassifying systemic autoimmune diseases independently of clinical diagnosis. METHODS: Unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data on 955 patients with 7 systemic autoimmune diseases and 267 healthy controls was undertaken. In addition, an inception cohort was prospectively followed up for 6 or 14 months to validate the results and analyze whether or not cluster assignment changed over time. RESULTS: Four clusters were identified and validated. Three were pathologic, representing "inflammatory," "lymphoid," and "interferon" patterns. Each included all diagnoses and was defined by genetic, clinical, serologic, and cellular features. A fourth cluster with no specific molecular pattern was associated with low disease activity and included healthy controls. A longitudinal and independent inception cohort showed a relapse-remission pattern, where patients remained in their pathologic cluster, moving only to the healthy one, thus showing that the molecular clusters remained stable over time and that single pathogenic molecular signatures characterized each individual patient. CONCLUSION: Patients with systemic autoimmune diseases can be jointly stratified into 3 stable disease clusters with specific molecular patterns differentiating different molecular disease mechanisms. These results have important implications for future clinical trials and the study of nonresponse to therapy, marking a paradigm shift in our view of systemic autoimmune diseases.


Assuntos
Doenças Autoimunes/classificação , Doenças Autoimunes/genética , Epigenoma , Perfilação da Expressão Gênica , Adulto , Idoso , Síndrome Antifosfolipídica/genética , Síndrome Antifosfolipídica/imunologia , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Doenças Autoimunes/imunologia , Estudos de Casos e Controles , Análise por Conglomerados , Estudos Transversais , Epigenômica , Feminino , Humanos , Inflamação/imunologia , Interferons/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , Doença Mista do Tecido Conjuntivo/genética , Doença Mista do Tecido Conjuntivo/imunologia , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/imunologia , Síndrome de Sjogren/genética , Síndrome de Sjogren/imunologia , Doenças do Tecido Conjuntivo Indiferenciado/genética , Doenças do Tecido Conjuntivo Indiferenciado/imunologia
19.
Arthritis Res Ther ; 22(1): 147, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32552896

RESUMO

BACKGROUND: Approximately 50% of systemic lupus erythematosus (SLE) patients develop nephritis, which is among the most severe and frequent complications of the disease and a leading cause of morbidity and mortality. Despite intensive research, there are still no reliable lupus nephritis (LN) markers in clinical use that can assess renal damage and activity with a high sensitivity and specificity. To this end, the aim of this study was to identify new clinically relevant tissue-specific protein biomarkers and possible underlying molecular mechanisms associated with renal involvement in SLE, using mass spectrometry (MS)-based proteomics. METHODS: Kidneys were harvested from female triple congenic B6.NZMsle1/sle2/sle3 lupus mice model, and the respective sex- and age-matched C57BL/6 control mice at 12, 24 and 36 weeks of age, representing pre-symptomatic, established and end-stage LN, respectively. Proteins were extracted from kidneys, purified, reduced, alkylated and digested by trypsin. Purified peptides were separated by liquid chromatography and analysed by high-resolution MS. Data were processed by the Progenesis QIp software, and functional annotation analysis was performed using DAVID bioinformatics resources. Immunofluorescence and multiple reaction monitoring (MRM) MS methods were used to confirm prospective biomarkers in SLE mouse strains as well as human serum samples. RESULTS: Proteomic profiling of kidney tissues from SLE and control mice resulted in the identification of more than 3800 unique proteins. Pathway analysis revealed a number of dysregulated molecular pathways that may be mechanistically involved in renal pathology, including phagosome and proximal tubule bicarbonate reclamation pathways. Proteomic analysis supported by human transcriptomic data and pathway analysis revealed Coronin-1A, Ubiquitin-like protein ISG15, and Rho GDP-dissociation inhibitor 2, as potential LN biomarkers. These results were further validated in other SLE mouse strains using MRM-MS. Most importantly, experiments in humans showed that measurement of Coronin-1A in human sera using MRM-MS can segregate LN patients from SLE patients without nephritis with a high sensitivity (100%) and specificity (100%). CONCLUSIONS: These preliminary findings suggest that serum Coronin-1A may serve as a promising non-invasive biomarker for LN and, upon validation in larger cohorts, may be employed in the future as a screening test for renal disease in SLE patients.


Assuntos
Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Proteínas dos Microfilamentos/metabolismo , Animais , Biomarcadores , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteômica
20.
J Pathol Clin Res ; 2(2): 80-92, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27499918

RESUMO

Molecular classification of hepatocellular carcinomas (HCC) could guide patient stratification for personalized therapies targeting subclass-specific cancer 'driver pathways'. Currently, there are several transcriptome-based molecular classifications of HCC with different subclass numbers, ranging from two to six. They were established using resected tumours that introduce a selection bias towards patients without liver cirrhosis and with early stage HCCs. We generated and analyzed gene expression data from paired HCC and non-cancerous liver tissue biopsies from 60 patients as well as five normal liver samples. Unbiased consensus clustering of HCC biopsy profiles identified 3 robust classes. Class membership correlated with survival, tumour size and with Edmondson and Barcelona Clinical Liver Cancer (BCLC) stage. When focusing only on the gene expression of the HCC biopsies, we could validate previously reported classifications of HCC based on expression patterns of signature genes. However, the subclass-specific gene expression patterns were no longer preserved when the fold-change relative to the normal tissue was used. The majority of genes believed to be subclass-specific turned out to be cancer-related genes differentially regulated in all HCC patients, with quantitative rather than qualitative differences between the molecular subclasses. With the exception of a subset of samples with a definitive ß-catenin gene signature, biological pathway analysis could not identify class-specific pathways reflecting the activation of distinct oncogenic programs. In conclusion, we have found that gene expression profiling of HCC biopsies has limited potential to direct therapies that target specific driver pathways, but can identify subgroups of patients with different prognosis.

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