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1.
Hepatology ; 72(3): 829-844, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-31925967

RESUMO

BACKGROUND AND AIMS: To date, conflicting data exist as to whether hepatitis B virus (HBV) has the ability to induce innate immune responses. Here, we investigated cellular changes after the first contact between HBV and primary human hepatocytes (PHH) in vitro and in vivo. APPROACH AND RESULTS: The exposure of PHH to HBV particles resulted in nuclear translocation of NFκB, followed by the expression and secretion of inflammatory cytokines (IL [interleukin] 1B, IL6, and TNF [tumor necrosis factor]). Ultraviolet irradiation of viral particles suppressed HBV infectivity but not the induction of cytokines in PHH, suggesting that the inoculum contains the immune-inducing agent. Purified HBV particles on the whole, which were prepared from HBV DNA-positive and protein-rich fractions after heparin column separation, still had immune-inducing capacity in PHH. The HBV-induced gene expression profile was similar to that induced by toll-like receptor 2 (TLR2) ligand Pam3Cys, but different from those induced by the viral sensors TLR3 or TLR7-9. Treatment of PHH with both HBV particles and Pam3Cys led to phosphorylation of ERK (extracellular signal-regulated kinase), JNK, and p38 mitogen-activated protein kinases as well as NFκB (nuclear factor kappa B). Finally, HBV-induced gene expression could be neutralized by TLR2-specific antibodies. Of note, pretreatment with an HBV entry inhibitor attenuated the TLR2-mediated response to HBV, suggesting a receptor binding-related mechanism. In liver-humanized uPA/severe combined immunodeficient (SCID)/beige mice challenged with HBV in vivo, immune induction could only marginally be seen. CONCLUSIONS: PHHs are able to sense HBV particles through TLR2, leading to an activation of anti-HBV immune responses in vitro. These findings challenge the previously described stealth properties of HBV.


Assuntos
Vírus da Hepatite B/imunologia , Hepatite B , Hepatócitos , Receptor 2 Toll-Like/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Hepatite B/imunologia , Hepatite B/metabolismo , Hepatócitos/imunologia , Hepatócitos/metabolismo , Humanos , Imunidade Inata , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Lipoproteínas/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , NF-kappa B/metabolismo , Fosforilação , Transcriptoma , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
Int J Cancer ; 141(2): 354-363, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28419472

RESUMO

Previous studies have shown that hepatocellular carcinoma (HCC) develops more frequently in hepatitis B virus surface antigen (HBsAg)-transgenic mice (Alb/HBs) than in wild-type (WT) mice. However, the mechanism of this HCC model has not been well documented. Toll-like receptor 4 (Tlr4) signaling probably links innate immunity and HCC progression. This study was designed to investigate the role of innate immunity in hepatocarcinogenesis in Alb/HBs mice. Immunohistochemical analysis of liver specimens from Alb/HBs mice (16 per group) showed that the oncogenes Bmi1 (16/16, 100%) and Dkk1 (13/16, 81.25%) were highly expressed in Alb/HBs mice, whereas the other oncogenes evaluated were expressed in smaller percentages of mice (Afp, 9/16, 56.2%; Ctnnb1, 5/16, 31.3%; Epcam, 0/16; 0%). Comparable results were obtained by quantitative PCR analysis. Hepatic gene expression of Tlr2, Tlr4, Il6 and Tnf was additionally elevated in Alb/HBs mice. Stimulation of primary murine hepatocytes with cell culture-derived HBV particles or LPS increased the expression of oncogenes (Bmi1, Dkk1) and inflammatory factors (Tnf, Il6, Tlr4). Proliferation and colony formation of hepatoma cells were enhanced by treatment with HBV and LPS and were impaired by the suppression of Bmi1 and Dkk1 by small interfering RNAs. Substantial induction of BMI1 and DKK1 was found in liver biopsy samples from patients with HBV-related HCC but not in HCC samples without HBV infection background. These findings suggest that innate immunity may link inflammation and tumor progression during chronic HBV infection, involving the oncogenes BMI1 and DKK1.


Assuntos
Carcinoma Hepatocelular/virologia , Antígenos de Superfície da Hepatite B/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas/virologia , Complexo Repressor Polycomb 1/metabolismo , Regulação para Cima , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Antígenos de Superfície da Hepatite B/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular/genética , Lipopolissacarídeos/farmacologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Transgênicos , Complexo Repressor Polycomb 1/genética , Vírion/patogenicidade
3.
Anesthesiology ; 121(2): 372-82, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24758774

RESUMO

BACKGROUND: Phosphodiesterase 2A (PDE2A) is an evolutionarily conserved enzyme that catalyzes the degradation of the cyclic nucleotides, cyclic adenosine monophosphate, and/or cyclic guanosine monophosphate. Recent studies reported the expression of PDE2A in the dorsal horn of the spinal cord, pointing to a potential contribution to the processing of pain. However, the functions of PDE2A in spinal pain processing in vivo remained elusive. METHODS: Immunohistochemistry, laser microdissection, and quantitative real-time reverse transcription polymerase chain reaction experiments were performed to characterize the localization and regulation of PDE2A protein and messenger RNA in the mouse spinal cord. Effects of the selective PDE2A inhibitor, BAY 60-7550 (Cayman Chemical, Ann Arbor, MI), in animal models of inflammatory pain (n = 6 to 10), neuropathic pain (n = 5 to 6), and after intrathecal injection of cyclic nucleotides (n = 6 to 8) were examined. Also, cyclic adenosine monophosphate and cyclic guanosine monophosphate levels in spinal cord tissues were measured by liquid chromatography tandem mass spectrometry. RESULTS: The authors here demonstrate that PDE2A is distinctly expressed in neurons of the superficial dorsal horn of the spinal cord, and that its spinal expression is upregulated in response to hind paw inflammation. Administration of the selective PDE2A inhibitor, BAY 60-7550, increased the nociceptive behavior of mice in animal models of inflammatory pain. Moreover, BAY 60-7550 increased the pain hypersensitivity induced by intrathecal delivery of cyclic adenosine monophosphate, but not of cyclic guanosine monophosphate, and it increased the cyclic adenosine monophosphate levels in spinal cord tissues. CONCLUSION: Our findings indicate that PDE2A contributes to the processing of inflammatory pain in the spinal cord.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/fisiologia , Inflamação/enzimologia , Inflamação/fisiopatologia , Dor/enzimologia , Dor/fisiopatologia , Medula Espinal/enzimologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/biossíntese , Hipersensibilidade a Drogas/fisiopatologia , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Imuno-Histoquímica , Inflamação/complicações , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microdissecção , Neuralgia/enzimologia , Neuralgia/fisiopatologia , Neuralgia/psicologia , Dor/etiologia , Medição da Dor , Inibidores de Fosfodiesterase/administração & dosagem , Inibidores de Fosfodiesterase/farmacologia , Células do Corno Posterior/enzimologia , Reação em Cadeia da Polimerase em Tempo Real , Triazinas/administração & dosagem , Triazinas/farmacologia , Regulação para Cima/genética , Regulação para Cima/fisiologia , Zimosan
4.
Int Immunol ; 26(1): 35-46, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24065781

RESUMO

OBJECTIVES: The therapeutic application of small interfering RNAs (siRNAs) is limited by the induction of severe off-target effects, especially in the liver. Therefore, we assessed the potential of differently modified siRNAs to induce the hepatic innate immune system in vitro and in vivo. METHODS: Primary isolated liver cells were transfected with siRNAs against apolipoprotein B1 (APOB1), luciferase (LUC) or galactosidase (GAL). For in vivo use, siRNAs were formulated in lipid nanoparticles (LNPs) and administered intravenously to C57BL/6 mice. Liver tissue was collected 6-48 h after injection and knock-down efficiency or immune responses were determined by quantitative reverse-transcription-linked PCR. RESULTS: Unmodified GAL siRNA transiently induced the expression of TNF-α, IL-6, IL-10, IFN-ß and IFN-sensitive gene 15 in vivo, whereas a formulation of 2'-O-methylated-LUC siRNA had no such effects. Formulation of unmodified APOB1-specific siRNA suppressed APOB1 mRNA levels by ~80% in the liver 48h after application. The results were paralleled in vitro, where transfection of liver cells with unmodified siRNAs, but not with chemically modified siRNAs, led to cell-type-specific induction of immune genes. These immune responses were not observed in MYD88-deficient mice or in chloroquine-treated cells in vitro. CONCLUSIONS: Our data indicate that siRNAs activate endosomal Toll-like receptors in different liver-derived cell types to various degrees, in vitro. LNP-formulated siRNA selectively leads to hepatic knock-down of target genes in vivo. Here, off-target immune responses are restricted to non-parenchymal liver cells. However, 2'-O-methyl modifications of siRNA largely avoid immune-stimulatory effects, which is a crucial prerequisite for the development of safe and efficient RNA-interference-based therapeutics.


Assuntos
Hepatócitos/imunologia , Sistema Imunitário/imunologia , Imunidade Inata/imunologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/imunologia , Receptores Toll-Like/imunologia , Animais , Imunidade Inata/genética , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Fígado , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Interferente Pequeno/genética , Receptores Toll-Like/genética , Transfecção/métodos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
5.
Antiviral Res ; 100(3): 654-61, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24416772

RESUMO

In previous studies we identified the interferon stimulated gene 15 (ISG15) as a pro-viral host factor in the pathogenesis of hepatitis C virus (HCV) infection. However, the functional link between ISG15 and the HCV replication cycle is not well understood. Aim of the present study was to functionally analyze the role of ISG15 and to identify possible HCV promoting effector molecules. Isg15 suppression was investigated in the murine subgenomic HCV replicon (MH1) transfected with Isg15-specific siRNA and in C57BL/6 mice intravenously injected with lipid nanoparticles (LNP)-formulated siRNA. Interestingly, the LNP-formulated siRNA led to hepatocyte-specific knockdown of Isg15 in vivo, which mediated a hypo-responsiveness to endogenous and exogenous interferon. A label free proteome analysis accompanied by western blot and quantitative RT-PCR techniques led to identification of five candidate proteins (Heterogeneous nuclear ribonucleoprotein A3 (HnrnpA3), Heterogeneous nuclear ribonucleoprotein K (HnrnpK), Hydroxymethylglutaryl-CoA synthase (Hmgcs1), Isocitrate dehydrogenase cytoplasmic (Idh1) and Thioredoxin domain-containing protein 5 (Txndc5)) that are either involved in lipid metabolism or belong to the family of Heterogeneous nuclear ribonucleoprotein (Hnrnp). All candidate proteins are likely to be associated with the HCV replication complex. Furthermore treatment with HnrnpK-specific siRNA directly suppressed HCV replication in vitro. Taken together these data suggest that targeting Isg15 may represent an attractive novel therapeutic option for the treatment of chronic HCV infection.


Assuntos
Citocinas/fisiologia , Hepacivirus/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular Tumoral , Citocinas/antagonistas & inibidores , Citocinas/genética , Hepatócitos/metabolismo , Hepatócitos/virologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Hidroximetilglutaril-CoA Sintase/fisiologia , Isocitrato Desidrogenase/fisiologia , Neoplasias Hepáticas Experimentais/patologia , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas , Proteoma , Interferência de RNA , RNA Interferente Pequeno/genética , Ribonucleoproteínas/fisiologia , Tiorredoxinas/fisiologia , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/genética , Ubiquitinas/fisiologia
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