RESUMO
A major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma, non-alcoholic fatty liver disease (NAFLD) results from excessive liver fat accumulation. Vitamin D (VitD) plays multiple important roles in diverse physiologic processes. Here, we describe the role of VitD in the complex pathogenesis of NAFLD and explore the possible therapeutic role of VitD supplementation in NAFLD therapy. To compare the effect of VitD to other interventions such as low-calorie diet, we induced NAFLD in young adult zebrafish (Danio rerio, AB strain) and monitored the effects of VitD supplementation on the disease course. The zebrafish administered with high-dose VitD (1.25 µg) had significantly reduced liver fat compared to those that received low-dose VitD (0.049 µg) or caloric restriction. Gene expression analysis revealed that VitD downregulated several pathways that may play a role in NAFLD etiology, which affected fatty acid metabolism, vitamins and their cofactors, ethanol oxidation, and glycolysis. The pathway analysis revealed that the cholesterol biosynthesis pathway and the isoprenoid biosynthetic process pathway were significantly upregulated whereas the small molecule catabolic process pathway significantly downregulated following the exposure of NAFLD zebrafish model to high VitD dose. Therefore, our findings suggest the association of novel biochemical pathways with NAFLD and highlight the potential of VitD supplementation to reverse the severity of NAFLD, especially in younger people.
Assuntos
Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Animais , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Vitamina D/farmacologia , Vitamina D/uso terapêutico , Vitamina D/metabolismo , Peixe-Zebra , Dieta Hiperlipídica , Fígado/metabolismo , Vitaminas/metabolismo , Neoplasias Hepáticas/metabolismoRESUMO
Intestinal metaplasia (IM) is an intermediate step in the progression from premalignant to malignant stages of gastric cancer (GC). The Popeye domain containing (POPDC) gene family encodes three transmembrane proteins, POPDC1, POPDC2, and POPDC3, initially described in muscles and later in epithelial and other cells, where they function in cell-cell interaction, and cell migration. POPDC1 and POPDC3 downregulation was described in several tumors, including colon and gastric cancers. We questioned whether IM-to-GC transition involves POPDC gene dysregulation. Gastric endoscopic biopsies of normal, IM, and GC patients were examined for expression levels of POPDC1-3 and several suggested IM biomarkers, using immunohistochemistry and qPCR. Immunostaining indicated lower POPDC1 and POPDC3 labeling in IM compared with normal tissues. Significantly lower POPDC1 and POPDC3 mRNA levels were measured in IM and GC biopsies and in GC-derived cell lines. The reduction in focal IM was smaller than in extensive IM that resembled GC tissues. POPDC1 and POPDC3 transcript levels were highly correlated with each other and inversely correlated with LGR5, OLFM4, CDX2, and several mucin transcripts. The association of POPDC1 and POPDC3 downregulation with IM-to-GC transition implicates a role in tumor suppression and highlights them as potential biomarkers for GC progression and prospective treatment targets.
Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas Musculares/metabolismo , Lesões Pré-Cancerosas/patologia , Idoso , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Sobrevivência Celular/genética , Feminino , Mucosa Gástrica/patologia , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/metabolismo , Metaplasia/patologia , Pessoa de Meia-Idade , Proteínas Musculares/genética , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Estudos Prospectivos , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
Leptin is an adipokine of pleiotropic effects linked to energy metabolism, satiety, the immune response, and cardioprotection. We have recently shown that leptin causally conferred resistance to myocardial infarction-induced damage in transgenic αMUPA mice overexpressing leptin compared to their wild type (WT) ancestral mice FVB/N. Prompted by these findings, we have investigated here if leptin can counteract the inflammatory response triggered after LPS administration in tissues in vivo and in cardiomyocytes in culture. The results have shown that LPS upregulated in vivo and in vitro all genes examined here, both pro-inflammatory and antioxidant, as well as the leptin gene. Pretreating mice with leptin neutralizing antibodies further upregulated the expression of TNFα and IL-1ß in the adipose tissue of both mouse types, and in the αMUPA heart. The antibodies also increased the levels of serum markers for cell toxicity in both mouse types. These results indicate that under LPS, leptin actually reduced the levels of these inflammatory-related parameters. In addition, pretreatment with leptin antibodies reduced the levels of HIF-1α and VEGF mRNAs in the heart, indicating that under LPS leptin increased the levels of these mRNAs. In cardiomyocytes, pretreatment with exogenous leptin prior to LPS reduced the expression of both pro-inflammatory genes, enhanced the expression of the antioxidant genes HO-1, SOD2 and HIF-1α, and lowered ROS staining. In addition, results obtained with leptin antibodies and the SMLA leptin antagonist indicated that endogenous and exogenous leptin can inhibit leptin gene expression. Together, these findings have indicated that under LPS, leptin concomitantly downregulated pro-inflammatory genes, upregulated antioxidant genes, and lowered ROS levels. These results suggest that leptin can counteract inflammation in the heart and adipose tissue by modulating gene expression.
Assuntos
Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Leptina/metabolismo , Miócitos Cardíacos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Metabolismo Energético/efeitos dos fármacos , Inflamação/metabolismo , Leptina/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacosRESUMO
Leptin, an adipocyte-derived satiety hormone, has been previously linked to cardioprotection. We have shown before that leptin conferred resistance to ischemic damage in the heart in long-lived transgenic αMUPA mice overexpressing leptin compared to the wild type (WT) FVB/N control mice. To better understand the contribution of leptin to the ischemic heart, we measured here the expression of genes encoding leptin and ischemia-related proteins in αMUPA and WT mice in the heart vs adipose tissue after MI. In addition, we investigated gene expression in neonatal rat cardiomyocytes under hypoxia in the absence and presence of exogenously added leptin or a leptin antagonist. We used real time RT-PCR and ELISA or Western blot assays to measure, respectively, mRNA and protein levels. The results have shown that circulating leptin levels and mRNA levels of leptin and heme oxygenase-1 (HO-1) in the heart were elevated in both mouse genotypes after 24 h myocardial infarction (MI), reaching higher values in αMUPA mice. In contrast, leptin gene expression in the adipose tissue was significantly increased only in WT mice, but reaching lower levels compared to the heart. Expression of the proinflammatory genes encoding TNFα and IL-1ß was also largely increased after MI in the heart in both mouse types, however reaching considerably lower levels in αMUPA mice indicating a mitigated inflammatory state. In cardiomyocytes, mRNA levels of all aforementioned genes as well as HIF-1α and SOD2 genes were elevated after hypoxia. Pretreatment with exogenous leptin largely reduced the mRNA levels of TNFα and IL-1ß after hypoxia, while enhancing expression of all other genes and reducing ROS levels. Pretreating the cells with a leptin antagonist increased solely the levels of leptin mRNA, suggesting a negative regulation of the hormone on the expression of its own gene. Overall, the results have shown that leptin affects expression of genes in cardiomyocytes under hypoxia in a manner that could mitigate inflammation and oxidative stress, suggesting a similar influence by endogenous leptin in αMUPA mice. Furthermore, leptin is likely to function in the ischemic murine heart more effectively in an autocrine compared to paracrine manner. These results suggest that leptin can reduce ischemic damage by modulating gene expression in the heart.
Assuntos
Biomarcadores/análise , Regulação da Expressão Gênica/efeitos dos fármacos , Leptina/farmacologia , Isquemia Miocárdica/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Miócitos Cardíacos/metabolismo , RatosRESUMO
Previously, we have reported that the active vitamin D metabolite, calcitriol and vitamin D3 (cholecalciferol), both remarkably inhibit hepatitis C virus production. The mechanism by which vitamin D3 exerts its effect is puzzling due to the low levels of calcitriol produced in vitamin D3-treated Huh7.5 cells. In this study, we aimed to explore the mechanism of vitamin D3 anti-hepatitis C virus effect. We show that vitamin D3 activity is not mediated by its metabolic conversion to calcitriol, but may be due to its primary metabolic product 25(OH)D3. This is inferred from the findings that 25(OH)D3 could inhibit hepatitis C virus production in our system, and that adequate concentrations needed to exert this effect are produced in Huh7.5 cells treated with vitamin D3. Using the CRISPR-Cas9 editing technology to knockout the vitamin D receptor, we found that the antiviral activity of vitamin D3 and 25(OH)D3 was not impaired in the vitamin D receptor knockout cells. This result indicates that 25(OH)D3 anti-hepatitis C virus effect is exerted by a vitamin D receptor-independent mode of action. The possibility that vitamin D3 and 25(OH)D3, being 3ß-hydroxysteroids, affect hepatitis C virus production by direct inhibition of the Hedgehog pathway in a vitamin D receptor-independent manner was ruled out. Taken together, this study proposes a novel mode of action for the anti-hepatitis C virus activity of vitamin D3 that is mediated by 25(OH)D3 in a vitamin D receptor-independent mechanism.
Assuntos
Calcifediol/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Receptores de Calcitriol/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Colecalciferol/farmacologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Calcitriol/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Type 2 diabetes mellitus (DM2) leads to cardiomyopathy characterized by cardiomyocyte hypertrophy, followed by mitochondrial dysfunction and interstitial fibrosis, all of which are exacerbated by angiotensin II (AT). SIRT1 and its transcriptional coactivator target PGC-1α (peroxisome proliferator-activated receptor-γ coactivator), and heme oxygenase-1 (HO-1) modulates mitochondrial biogenesis and antioxidant protection. We have previously shown the beneficial effect of caloric restriction (CR) on diabetic cardiomyopathy through intracellular signaling pathways involving the SIRT1-PGC-1α axis. In the current study, we examined the role of HO-1 in diabetic cardiomyopathy in mice subjected to CR. METHODS: Cardiomyopathy was induced in obese diabetic (db/db) mice by AT infusion. Mice were either fed ad libitum or subjected to CR. In an in vitro study, the reactive oxygen species (ROS) level was determined in cardiomyocytes exposed to different glucose levels (7.5-33 mM). We examined the effects of Sn(tin)-mesoporphyrin (SnMP), which is an inhibitor of HO activity, the HO-1 inducer cobalt protoporphyrin (CoPP), and the SIRT1 inhibitor (EX-527) on diabetic cardiomyopathy. RESULTS: Diabetic mice had low levels of HO-1 and elevated levels of the oxidative marker malondialdehyde (MDA). CR attenuated left ventricular hypertrophy (LVH), increased HO-1 levels, and decreased MDA levels. SnMP abolished the protective effects of CR and caused pronounced LVH and cardiac metabolic dysfunction represented by suppressed levels of adiponectin, SIRT1, PPARγ, PGC-1α, and increased MDA. High glucose (33 mM) increased ROS in cultured cardiomyocytes, while SnMP reduced SIRT1, PGC-1α levels, and HO activity. Similarly, SIRT1 inhibition led to a reduction in PGC-1α and HO-1 levels. CoPP increased HO-1 protein levels and activity, SIRT1, and PGC-1α levels, and decreased ROS production, suggesting a positive feedback between SIRT1 and HO-1. CONCLUSION: These results establish a link between SIRT1, PGC-1α, and HO-1 signaling that leads to the attenuation of ROS production and diabetic cardiomyopathy. CoPP mimicked the beneficial effect of CR, while SnMP increased oxidative stress, aggravating cardiac hypertrophy. The data suggest that increasing HO-1 levels constitutes a novel therapeutic approach to protect the diabetic heart. Brief Summary: CR attenuates cardiomyopathy, and increases HO-1, SIRT activity, and PGC-1α protein levels in diabetic mice. High glucose reduces adiponectin, SIRT1, PGC1-1α, and HO-1 levels in cardiomyocytes, resulting in oxidative stress. The pharmacological activation of HO-1 activity mimics the effect of CR, while SnMP increased oxidative stress and cardiac hypertrophy. These data suggest the critical role of HO-1 in protecting the diabetic heart.
Assuntos
Restrição Calórica/métodos , Cardiomiopatias Diabéticas/tratamento farmacológico , Cardiomiopatias Diabéticas/metabolismo , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/uso terapêutico , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Angiotensina II/metabolismo , Animais , Glicemia , Carbazóis/farmacologia , Cardiomegalia/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/complicações , Masculino , Malondialdeído/sangue , Mesoporfirinas/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Protoporfirinas/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/metabolismoRESUMO
Hepatitis B virus (HBV) targets the liver and is a major driver for liver cancer. Clinical data suggest that HBV infection is associated with reduced response to treatment with the multi-kinase inhibitor sorafenib, the first available molecularly targeted anti-hepatocellular carcinoma (HCC) drug. Given that Raf is one of the major targets of sorafenib, we investigated the activation state of the Raf-Mek-Erk pathway in the presence of HBV and in response to sorafenib. Here we show that hepatoma cells with replicating HBV are less susceptible to sorafenib inhibitory effect as compared to cells in which HBV expression is suppressed. However, although HBV replication is associated with increased level of pErk, its blockade only modestly augments sorafenib effect. In contrast, the phosphorylated form of the pro-oncogenic Mitogen-Activated Protein Kinase 14 (pMAPK14), a protein kinase that was recently linked to sorafenib resistance, is induced in sorafenib-treated hepatoma cells in association with HBV X protein expression. Knocking down pMAPK14 results in augmentation of the therapeutic efficacy of sorafenib and largely alleviates resistance to sorafenib in the presence of HBV. Thus, this study suggests that HBV promotes HCC resistance to sorafenib. Combining pMAPK14 inhibitors with sorafenib may be beneficial in patients with HBV-associated HCC.
RESUMO
BACKGROUND: Toll-like receptors (TLRs) are expressed in immune cells and hepatocytes. We examined whether hepatic Toll-like receptor 4 (TLR4) is involved in the acute hepatic injury caused by the administration of lipopolysaccharide (LPS) (septic shock model). METHODS: Wild type (WT), TLR4-deficient and chimera mice underwent myeloablative bone marrow transplantation to dissociate between TLR4 expression in the liver or in the immune-hematopoietic system. Mice were injected with LPS and sacrificed 4 hours later. RESULTS: Compared to TLR4 deficient mice, WT mice challenged with LPS displayed increased serum liver enzymes and hepatic cellular inflammatory infiltrate together with increased serum and hepatic levels of interleukin 1ß (IL-1ß), tumor necrosis factor α (TNFα) ,Up-regulation of hepatic mRNA encoding TLR4, IκB and c-jun expressions. TLR4 mutant mice transplanted with WT bone marrow were more protected than WT chimeric mice bearing TLR4 mutant hemopoietic cells from LPS, as seen by IL-1ß and TNFα levels. We then used hepatocytes (Huh7) and macrophages from monocytic cell lines to detect TLR mRNA expression. Macrophages expressed a significantly higher level of TLR4 mRNA and TLR2 (more than 3000- and 8000-fold respectively) compared with the hepatocyte cell line. LPS administration induced TLR4 activation in a hepatocyte cell line in a dose dependent manner while TLR2 mRNA hardly changed. CONCLUSIONS: These results suggest that TLR4 activation of hepatocytes participate in the immediate response to LPS induced hepatic injury. However, in this response, the contribution of TLR4 on bone marrow derived cells is more significant than those of the hepatocytes. The absence of the TLR4 gene plays a pivotal role in reducing hepatic LPS induced injury.
Assuntos
Medula Óssea/metabolismo , Endotoxemia/metabolismo , Endotoxemia/patologia , Fígado/metabolismo , Fígado/patologia , Receptor 4 Toll-Like/metabolismo , Doença Aguda , Animais , Linhagem Celular Tumoral , Endotoxemia/sangue , Endotoxemia/genética , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/sangue , Estimativa de Kaplan-Meier , Lipopolissacarídeos , Fígado/enzimologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sepse/metabolismo , Sepse/patologia , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/sangueRESUMO
Serodiagnosis of infectious disease is often based on the detection of pathogen-specific antibodies in a patient's blood. For this, mixtures of pathogen-related antigens are used as bait to capture corresponding antibodies in solid phase immunoassays such as enzyme immunoassay (EIA). Western blots provide improved diagnostic power as compared with EIA due to the fact that the mixture of markers in the EIA well is resolved and tested as individual antigens on the Western blot. Hence, confirmation of EIA results is accomplished using the antigen arrays of Western blots. Here we took this approach one step further and tested the attributes of using epitope arrays in a diagnostic platform coined "combinatorial diagnostics." As a case in point, we tested a panel of phage-displayed epitope-based markers in the serodiagnosis of hepatitis C virus (HCV). The repertoire of HCV antigens was deconvoluted into panels of distinct linear and conformational epitopes and tested individually by quantitative EIA. Combinatorial diagnostics proved to be effective for the discrimination between positive and negative sera as well as serotyping of HCV.
Assuntos
Epitopos/imunologia , Hepacivirus/imunologia , Hepatite C/diagnóstico , Técnicas Imunoenzimáticas , Sequência de Aminoácidos , Genótipo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/imunologia , Anticorpos Anti-Hepatite C/sangue , Antígenos da Hepatite C/sangue , Antígenos da Hepatite C/imunologia , Humanos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Análise Serial de ProteínasRESUMO
Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1ß levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaß (NF-κ ß) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1ß, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IκB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-κB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.
Assuntos
Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/metabolismo , Fígado/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Galactosamina/farmacologia , Interleucina-1beta/análise , Interleucina-1beta/sangue , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/sangueRESUMO
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named "zymoxins". These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the "first generation zymoxins" by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases.
Assuntos
Toxinas Bacterianas/administração & dosagem , Hepatite C/tratamento farmacológico , Precursores de Proteínas/uso terapêutico , Antitoxinas/genética , Proteínas de Bactérias , Toxinas Bacterianas/uso terapêutico , Morte Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Sistemas de Liberação de Medicamentos , Endorribonucleases/genética , Proteínas de Escherichia coli/genética , Terapia Genética , Hepacivirus , Humanos , Precursores de Proteínas/administração & dosagem , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismoRESUMO
UNLABELLED: Vitamin D supplementation was reported to improve the probability of achieving a sustained virological response when combined with antiviral treatment against hepatitis C virus (HCV). Our aim was to determine the in vitro potential of vitamin D to inhibit HCV infectious virus production and explore the mechanism(s) of inhibition. Here we show that vitamin D(3) remarkably inhibits HCV production in Huh7.5 hepatoma cells. These cells express CYP27B1, the gene encoding for the enzyme responsible for the synthesis of the vitamin D hormonally active metabolite, calcitriol. Treatment with vitamin D(3) resulted in calcitriol production and induction of calcitriol target gene CYP24A1, indicating that these cells contain the full machinery for vitamin D metabolism and activity. Notably, treatment with calcitriol resulted in HCV inhibition. Collectively, these findings suggest that vitamin D(3) has an antiviral activity which is mediated by its active metabolite. This antiviral activity involves the induction of the interferon signaling pathway, resulting in expression of interferon-ß and the interferon-stimulated gene, MxA. Intriguingly, HCV infection increased calcitriol production by inhibiting CYP24A1 induction, the enzyme responsible for the first step in calcitriol catabolism. Importantly, the combination of vitamin D(3) or calcitriol and interferon-α synergistically inhibited viral production. CONCLUSION: This study demonstrates for the first time a direct antiviral effect of vitamin D in an in vitro infectious virus production system. It proposes an interplay between the hepatic vitamin D endocrine system and HCV, suggesting that vitamin D has a role as a natural antiviral mediator. Importantly, our study implies that vitamin D might have an interferon-sparing effect, thus improving antiviral treatment of HCV-infected patients.
Assuntos
Calcitriol/biossíntese , Colecalciferol/farmacocinética , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Hepatócitos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Antivirais/metabolismo , Antivirais/farmacologia , Calcitriol/metabolismo , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Sinergismo Farmacológico , Hepacivirus/crescimento & desenvolvimento , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Interferon-alfa/farmacologia , Neoplasias Hepáticas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Vitamina D3 24-Hidroxilase , Vitaminas/farmacocinéticaRESUMO
HBV and HCV have major roles in hepatocarcinogenesis. More than 500 million people are infected with hepatitis viruses and, therefore, HCC is highly prevalent, especially in those countries endemic for HBV and HCV. Viral and host factors contribute to the development of HCC. The main viral factors include the circulating load of HBV DNA or HCV RNA and specific genotypes. Various mechanisms are involved in the host-viral interactions that lead to HCC development, among which are genetic instability, self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasiveness. Prevention of HBV by vaccination, as well as antiviral therapy against HBV and for HCV seem able to inhibit the development of HCC.
Assuntos
Carcinoma Hepatocelular/virologia , Hepacivirus/fisiologia , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/patologia , Hepatite C Crônica/patologia , Neoplasias Hepáticas/virologia , Animais , Feminino , Humanos , Neoplasias Hepáticas Experimentais/virologia , MasculinoRESUMO
The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins"). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.
Assuntos
Hepacivirus/metabolismo , Engenharia de Proteínas/métodos , Deleção de Sequência/fisiologia , Toxinas Biológicas/genética , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Toxina Diftérica , Precursores Enzimáticos , Escherichia coli/genética , Hepacivirus/genética , Humanos , Fragmentos de Peptídeos , Estrutura Terciária de Proteína , Ricina , Serina Proteases , Toxinas Biológicas/síntese química , Proteínas não Estruturais Virais/genéticaRESUMO
Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small-molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing.
Assuntos
Hepacivirus , Inibidores de Proteases/farmacologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêutico , Proteínas não Estruturais Virais/imunologia , Anticorpos Antivirais/imunologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepacivirus/imunologia , Hepacivirus/fisiologia , Humanos , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , Plasmídeos , Reação em Cadeia da Polimerase , Poliproteínas/metabolismo , RNA Viral/genética , RNA Polimerase Dependente de RNA , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
BACKGROUND: Increasing evidence suggests that adipose tissue contains mesenchymal stem cells (MSC) that possess the ability to transdifferentiate into other cell types including hepatocytes, similar to bone marrow-derived stem cells. The existence of precommitted cells in the MSC population may explain transdifferentiation. AIMS: Our aim was to identify a population of putative hepatocyte-like precursor cells in human adipose tissue. METHODS: We analysed the 'basal' hepatic potential of undifferentiated, naïve human adipose-derived mesenchymal stem cells (hADMSC). hADMSC were isolated from human adipose tissue and characterized for cell surface markers and for liver-specific gene expression. RESULTS: The isolated undifferentiated naïve hADMSCs expressed MSC surface markers. They also expressed alpha-fetoprotein, CK18, CK19 and HNF4, which are known as early liver expressing genes. Interestingly, the undifferentiated naïve hADMSC were also positive for albumin, G-6-P and alpha-1-antitrypsin (AAT), which are all known to be predominantly expressed in adult liver cells. These cells acquired a hepatocyte-specific phenotype and function upon treatment with a differentiation medium, resulting in the upregulation of albumin, G-6-P and AAT. Moreover, urea production, glycogen storage ability and cellular uptake of indocyanine green, which were absent in the basal state, were evident in the treated cells. CONCLUSIONS: Our findings suggest the presence of cells with hepatocyte-like properties that are isolated from human adipose tissue and that can readily acquire hepatocyte-like functions. Adipose tissue could thus be an exciting alternative means for repopulating the liver after various injuries, and might serve as a source for the transplantation of liver cells.
Assuntos
Tecido Adiposo/citologia , Fígado/metabolismo , Células-Tronco Mesenquimais/química , Albuminas/análise , Antígenos CD34/análise , Antígenos de Superfície/análise , Biomarcadores , Diferenciação Celular , Células Cultivadas , Hepatócitos/citologia , Humanos , Células-Tronco Mesenquimais/citologia , alfa-Fetoproteínas/análiseRESUMO
BACKGROUND/AIMS: Hepatitis C virus infection is a major worldwide health problem, causing chronic hepatitis, cirrhosis and primary liver cancer. In addition to its role in the viral polyprotein-processing, the viral NS3 serine protease has been implicated in interactions with various cell constituents resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for anti-viral drugs thus specific inhibitors of its activities should be important. With the aim of inhibiting NS3 protease activity as a means to inhibit HCV replication we used a novel bacterial genetic screen to isolate NS3-inhibiting peptide aptamers. METHODS: We have isolated and characterized seven NS3-inhibiting peptide aptamers. We investigated the phenotypic changes that SEAP-secreting subgenomic RNA replicons undergo upon intracellular expression of these peptide aptamers, assayed by real-time RT-PCR and inhibition of SEAP secretion by transfected replicon cells. RESULTS AND CONCLUSIONS: The peptide aptamers inhibited NS3 protease activity in vitro with an IC50 in the low micromolar range. Upon transfection, aptamers inhibited the replication of SEAP-secreting genotype 1b subgenomic RNA replicons. Aptamer-based intracellular immunization may emerge as a promising antiviral approach to interfere with the life cycle and pathogenicity of HCV.
Assuntos
Antivirais/farmacologia , Aptâmeros de Peptídeos/farmacologia , Hepacivirus/efeitos dos fármacos , Inibidores de Proteases/farmacologia , RNA Viral/análise , RNA Viral/biossíntese , Replicação Viral/efeitos dos fármacos , Antivirais/isolamento & purificação , Aptâmeros de Peptídeos/isolamento & purificação , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Inibidores de Proteases/isolamento & purificação , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
BACKGROUND: Transmission of hepatitis C virus (HCV) from infected health care workers to patients rarely occurs. In 2003, a cluster of patients with HCV infection was identified at a medical center in Israel. All patients had a common history of various surgical procedures performed during the period 2001-2003. All patients had been anesthetized by an anesthesiologist who was an injection drug user and was infected with genotype 2a HCV. Screening was initiated by the hospital to identify newly infected patients with HCV infection and to determine the source of the iatrogenic HCV infection outbreak using comparative molecular analysis of the HCV E1 and HCV E2 hypervariable regions (HVR1 and HVR2). METHODS: A total of 1200 patients who were anesthetized by the anesthesiologist (the related group) and 873 hospital personnel and patients anesthetized by other anesthetists (the unrelated group) were examined. Serum samples were screened for anti-HCV antibodies, HCV RNA, and genotype. Sequence analysis of HVR1 and HVR2 was performed after reverse-transcriptase polymerase chain reaction. RESULTS: HCV type 2a was found in 33 patients in the related group but in only 1 patient in the unrelated group. The differences between the sequences isolated from the related group serum samples and the sequences isolated from genotype 2a control group serum samples (obtained from 15 patients) were highly statistically significant. The genetic distances from the anesthesiologist sequence were 1.4%-4.4% in the HVR1 and 0%-3% in the HVR2 in the related group serum samples, whereas in the HCV genotype 2a control group serum samples, the genetic distances were 22%-45% and 10%-35%, respectively. CONCLUSIONS: Molecular analysis revealed sequence similarity of HVR1 and HVR2 in the related group, suggesting that the anesthesiologist with chronic HCV infection may have transmitted HCV to 33 patients.
Assuntos
Surtos de Doenças , Hepacivirus/genética , Hepatite C/transmissão , Doença Iatrogênica/epidemiologia , Transmissão de Doença Infecciosa do Profissional para o Paciente , Proteínas do Envelope Viral/genética , Genótipo , Hepacivirus/classificação , Hepacivirus/isolamento & purificação , Hepatite C/epidemiologia , Hepatite C/virologia , Filogenia , Proteínas do Envelope Viral/químicaRESUMO
The hepatitis C virus (HCV) F protein is a recently described, frameshift product of HCV core encoding sequence with unknown biological function. In this study we sought to characterize the prevalence of specific anti-F antibodies in patients with chronic HCV infection and to analyze the anti-F antibody profile before, during, and after antiviral treatment in order to gain a better understanding of the role of F protein in HCV pathogenesis. Serum samples were collected from 44 patients with chronic HCV infection and from 19 healthy controls. Consecutive samples from 27 patients taken before, during, and after treatment with antiviral therapy. The F and the core proteins were cloned from the HCV genome. The recombinant proteins were expressed in Escherichia coli and affinity purified. A sensitive and specific enzyme-linked immunosorbent assay was developed to assess the prevalence of anti-F antibodies. Eighty-nine percent of chronic HCV patients had evidence of anti-F antibodies, and 95% of them had anti-core antibodies. No correlation of anti-F antibodies was found with response to treatment, genotype, or seroconversion. We conclude that the F protein elicits specific antibodies in most individuals chronically infected with HCV with no correlation with response to treatment. Our results confirm the expression of F protein during natural HCV infection.
Assuntos
Antivirais/uso terapêutico , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C Crônica , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Ribavirina/uso terapêutico , Proteínas do Core Viral/imunologia , Portadores de Fármacos , Quimioterapia Combinada , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Genótipo , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Humanos , Interferon alfa-2 , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Viral/genética , Proteínas Recombinantes , Resultado do TratamentoRESUMO
Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable beta-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant beta-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal) plates), while induction of NS3 expression results in loss of beta-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources.