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1.
Oncogene ; 33(29): 3820-9, 2014 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-23975421

RESUMO

Helicobacter pylori infection induces chronic inflammation that contributes to gastric tumorigenesis. Tumor necrosis factor (TNF-α) is a proinflammatory cytokine, and polymorphism in the TNF-α gene increases the risk of gastric cancer. We herein investigated the role of TNF-α in gastric tumorigenesis using Gan mouse model, which recapitulates human gastric cancer development. We crossed Gan mice with TNF-α (Tnf) or TNF-α receptor TNFR1 (Tnfrsf1a) knockout mice to generate Tnf-/- Gan and Tnfrsf1a-/- Gan mice, respectively, and examined their tumor phenotypes. Notably, both Tnf-/- Gan mice and Tnfrsf1a-/- Gan mice showed similar, significant suppression of gastric tumor growth compared with control Tnf+/+ or Tnfrsf1a+/+ Gan mice. These results indicate that TNF-α signaling through TNFR1 is important for gastric tumor development. Bone marrow (BM) transplantation experiments showed that TNF-α expressed by BM-derived cells (BMDCs) stimulates the TNFR1 on BMDCs by an autocrine or paracrine manner, which is important for gastric tumor promotion. Moreover, the microarray analysis and colony formation assay indicated that NADPH oxidase organizer 1 (Noxo1) and Gna14 are induced in tumor epithelial cells in a TNF-α-dependent manner, and have an important role in tumorigenicity and tumor-initiating cell property of gastric cancer cells. Accordingly, it is possible that the activation of TNF-α/TNFR1 signaling in the tumor microenvironment promotes gastric tumor development through induction of Noxo1 and Gna14, which contribute to maintaining the tumor cells in an undifferentiated state. The present results indicate that targeting the TNF-α/TNFR1 pathway may be an effective preventive or therapeutic strategy for gastric cancer.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Transformação Celular Neoplásica/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Humanos , Receptores de Hialuronatos/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/imunologia , Fator de Necrose Tumoral alfa/genética
2.
Cancer Gene Ther ; 19(5): 312-9, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22402625

RESUMO

Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system combined with monocyte chemoattractant protein-1 (MCP-1) provides significant antitumor efficacy. The current study was designed to evaluate the antitumor immunity of a newly developed membrane-bound form of MCP-1 (mMCP-1) in an immunocompetent mouse model of hepatocellular carcinoma (HCC). A recombinant adenovirus vector (rAd) harboring the human MCP-1 gene and the membrane-spanning domain of the CX3CL1 gene was used. Large amounts of MCP-1 protein were expressed and accumulated on the tumor cell surface. The growth of subcutaneous tumors was markedly suppressed when tumors were treated with mMCP-1, as compared with soluble MCP-1, in combination with the HSV-tk/GCV system (P<0.01). The numbers of Mac-1-, CD4- and CD8a-positive cells were significantly higher in tumor tissues (P<0.05), and tumor necrosis factor (TNF) mRNA expression levels with mMCP-1 were almost five-fold higher than those with soluble MCP-1. These results indicate that the delivery of the mMCP-1 gene greatly enhanced antitumor effects following the apoptotic stimuli by promoting the recruitment and activation of macrophages and T lymphocytes, suggesting a novel strategy of immune-based gene therapy in the treatment of patients with HCC.


Assuntos
Quimiocina CCL2/genética , Genes Transgênicos Suicidas , Terapia Genética/métodos , Neoplasias Hepáticas Experimentais/terapia , Animais , Linhagem Celular Tumoral , Quimiocina CCL2/biossíntese , Quimiocina CCL2/metabolismo , Quimiocina CX3CL1/genética , Modelos Animais de Doenças , Feminino , Ganciclovir/farmacocinética , Ganciclovir/farmacologia , Herpes Simples/enzimologia , Herpes Simples/genética , Humanos , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Timidina Quinase/biossíntese , Timidina Quinase/genética , Timidina Quinase/metabolismo
3.
Clin Exp Immunol ; 163(2): 165-77, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21087443

RESUMO

Despite curative locoregional treatments for hepatocellular carcinoma (HCC), tumour recurrence rates remain high. The current study was designed to assess the safety and bioactivity of infusion of dendritic cells (DCs) stimulated with OK432, a streptococcus-derived anti-cancer immunotherapeutic agent, into tumour tissues following transcatheter hepatic arterial embolization (TAE) treatment in patients with HCC. DCs were derived from peripheral blood monocytes of patients with hepatitis C virus-related cirrhosis and HCC in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor and stimulated with 0·1 KE/ml OK432 for 2 days. Thirteen patients were administered with 5 × 106 of DCs through arterial catheter during the procedures of TAE treatment on day 7. The immunomodulatory effects and clinical responses were evaluated in comparison with a group of 22 historical controls treated with TAE but without DC transfer. OK432 stimulation of immature DCs promoted their maturation towards cells with activated phenotypes, high expression of a homing receptor, fairly well-preserved phagocytic capacity, greatly enhanced cytokine production and effective tumoricidal activity. Administration of OK432-stimulated DCs to patients was found to be feasible and safe. Kaplan-Meier analysis revealed prolonged recurrence-free survival of patients treated in this manner compared with the historical controls (P = 0·046, log-rank test). The bioactivity of the transferred DCs was reflected in higher serum concentrations of the cytokines IL-9, IL-15 and tumour necrosis factor-α and the chemokines CCL4 and CCL11. Collectively, this study suggests that a DC-based, active immunotherapeutic strategy in combination with locoregional treatments exerts beneficial anti-tumour effects against liver cancer.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/terapia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/transplante , Embolização Terapêutica , Imunoterapia Ativa/métodos , Neoplasias Hepáticas/terapia , Picibanil/farmacologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/virologia , Terapia Combinada , Citocinas/sangue , Citocinas/imunologia , Intervalo Livre de Doença , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Hepatite C/imunologia , Humanos , Interleucina-4/farmacologia , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Recidiva Local de Neoplasia/terapia , Radiografia
4.
Oncogene ; 29(15): 2228-37, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20101231

RESUMO

Pim-3, a proto-oncogene with serine/threonine kinase activity, was enhanced in hepatocellular carcinoma (HCC) tissues. To address the roles of Pim-3 in HCC development, we prepared transgenic mice that express human Pim-3 selectively in liver. The mice were born at a Mendelian ratio, were fertile and did not exhibit any apparent pathological changes in the liver until 1 year after birth. Pim-3-transgenic mouse-derived hepatocytes exhibited accelerated cell cycle progression. The administration of a potent hepatocarcinogen, diethylnitrosamine (DEN), induced accelerated proliferation of liver cells in Pim-3 transgenic mice in the early phase, compared with that observed for wild-type mice. Treatment with DEN induced lipid droplet accumulation with increased proliferating cell numbers 6 months after the treatment. Eventually, wild-type mice developed HCC with a frequency of 40% until 10 month after the treatment. Lipid accumulation was accelerated in Pim-3 transgenic mice with higher proliferating cell numbers, compared with that observed for wild-type mice. Pim-3 transgenic mice developed HCC with a higher incidence (80%) and a heavier burden, together with enhanced intratumoral CD31-positive vascular areas, compared with that observed for wild-type mice. These observations indicate that Pim-3 alone cannot cause, but can accelerate HCC development when induced by a hepatocarcinogen, such as DEN.


Assuntos
Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , Fígado/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transgenes/genética , Albuminas/genética , Animais , Carcinoma Hepatocelular/genética , Proliferação de Células , Humanos , Fígado/patologia , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Proto-Oncogene Mas
5.
J Physiol Pharmacol ; 60 Suppl 7: 101-6, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20388952

RESUMO

UNLABELLED: Inducible nitric oxide synthase (iNOS) and interleukin-8 (IL-8) mediate gastric inflammation. Nitric oxide (NO) produced by iNOS may activate oxidant-sensitive transcription factors. There are the binding sites for NF-kappaB, AP-1, and C/EBP (CCAAT/enhancer binding protein) in the promoter regions of IL-8 gene. The present study aims to investigate whether NO donors, SIN-1 and NOC-18, activate oxidant-sensitive transcription factors NF-kappaB and AP-1 as well as C/EBP to induce IL-8 expression in gastric epithelial AGS cells. Gastric epithelial AGS cells were treated with NO donors, SIN-1 and NOC-18. mRNA expression and protein level of IL-8 in the medium were determined. Nitrite level in the medium and DNA binding activities of NF-kappaB, AP-1, and C/EBP were assessed. NO donors induced the increase in the levels of IL-8 and nitrite in the medium as well as mRNA expression of IL-8 in AGS cells time-dependently. The induction of IL-8 by NO donors was accompanied with the activation of NF-kappaB and AP-1 but not C/EBP in AGS cells. CONCLUSION: Large amount of NO, which may be produced by iNOS, may induce the activation of NF-kappaB and AP-1 and the expression of IL-8 in gastric epithelial cells.


Assuntos
Mucosa Gástrica/efeitos dos fármacos , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/fisiologia , Fator de Transcrição AP-1/metabolismo , Antiulcerosos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Células Epiteliais , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Interleucina-8/genética , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Óxido Nítrico/análise , Doadores de Óxido Nítrico/farmacologia , Compostos Nitrosos/farmacologia , RNA Mensageiro/metabolismo , Fatores de Tempo
6.
Int J Mol Med ; 19(2): 335-40, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17203209

RESUMO

Interaction between proteases and protease-activated receptor (PAR) 2 has been proposed to mediate inflammatory and immune response in the gastrointestinal tract. Recently, increase in interleukin (IL)-8 in the esophageal mucosa has been associated with the pathogenesis of esophagitis induced by reflux of gastric acids, bile acids or trypsin. The aims of the present study were to determine PAR2 expression in normal human esophageal epithelial cells (HEEC) and to evaluate the mediation of IL-8 production by trypsin-PAR2 interaction in HEEC. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis revealed that PAR2 mRNA and protein were constitutively expressed in HEEC without upregulation by the stimulation with tumor necrosis factor alpha or trypsin. IL-8 was produced in a dose-dependent fashion when cells were stimulated with a PAR2 agonist such as trypsin or SLIGKV-amide. Blocking antibody to PAR2, camostat mesilate (a trypsin inhibitor), p-38 mitogen-activated protein kinase (MAPK) inhibitors or ERK1/2 inhibitors reduced IL-8 production from trypsin-stimulated HEEC. Mutation of the NFkappaB-, AP-1- and NF-IL-6-binding site on the IL-8 gene promoter abrogated the induction of luciferase activities stimulated with trypsin by 100, 80 and 50%, respectively. These results indicate that PAR2 activation in HEEC by trypsin induces NFkappaB- and AP-1-dependent IL-8 production in association with activation of p38 MAPK and ERK1/2, suggesting that esophageal inflammation may be induced by PAR2 activation via reflux of trypsin.


Assuntos
Células Epiteliais/metabolismo , Esôfago/metabolismo , Interleucina-8/biossíntese , Receptor PAR-2/metabolismo , Anticorpos/imunologia , Linhagem Celular , Genes Reporter/genética , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor PAR-2/genética , Receptor PAR-2/imunologia , Tripsina/metabolismo
7.
Clin Exp Immunol ; 147(2): 296-305, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17223971

RESUMO

The curative treatments for hepatocellular carcinoma (HCC), including surgical resection and radiofrequency ablation (RFA), do not prevent tumour recurrence effectively. Dendritic cell (DC)-based immunotherapies are believed to contribute to the eradication of the residual and recurrent tumour cells. The current study was designed to assess the safety and bioactivity of DC infusion into tumour tissues following transcatheter hepatic arterial embolization (TAE) for patients with cirrhosis and HCC. Peripheral blood mononuclear cells (PBMCs) were differentiated into phenotypically confirmed DCs. Ten patients were administered autologous DCs through an arterial catheter during TAE treatment. Shortly thereafter, some HCC nodules were treated additionally to achieve the curative local therapeutic effects. There was no clinical or serological evidence of adverse events, including hepatic failure or autoimmune responses in any patients, in addition to those due to TAE. Following the infusion of (111)Indium-labelled DCs, DCs were detectable inside and around the HCC nodules for up to 17 days, and were associated with lymphocyte and monocyte infiltration. Interestingly, T lymphocyte responses were induced against peptides derived from the tumour antigens, Her-2/neu, MRP3, hTERT and AFP, 4 weeks after the infusion in some patients. The cumulative survival rates were not significantly changed by this strategy. These results demonstrate that transcatheter arterial DC infusion into tumour tissues following TAE treatment is feasible and safe for patients with cirrhosis and HCC. Furthermore, the antigen-non-specific, immature DC infusion may induce immune responses to unprimed tumour antigens, providing a plausible strategy to enhance tumour immunity.


Assuntos
Carcinoma Hepatocelular/terapia , Células Dendríticas/transplante , Embolização Terapêutica/métodos , Neoplasias Hepáticas/terapia , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/imunologia , Terapia Combinada , Células Dendríticas/imunologia , Intervalo Livre de Doença , Feminino , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Cirrose Hepática/complicações , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/imunologia , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento
8.
Cancer Gene Ther ; 13(4): 357-66, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16224495

RESUMO

Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system is a well-characterized tool for cancer gene therapy; however, it does not yet exhibit sufficient efficacy to cure patients of malignancies. We have reported that adenovirally delivered monocyte chemoattractant protein (MCP)-1 augmented the antitumor effects of the HSV-tk/GCV system in an athymic nude mouse model. The current study, which uses an immunocompetent mouse model of colon cancer, was designed to evaluate the antitumor effects of MCP-1 gene delivery in conjunction with this suicide gene therapy system. Subcutaneous tumor foci were directly transduced with both recombinant adenoviruses (rAds) expressing an HSV-tk gene and either of the MCP-1, CD80 and LacZ genes, followed by GCV administration. The growth of tumors was markedly suppressed by codelivery of HSV-tk and MCP-1 genes, which was exclusively associated with the recruitment of monocytes/macrophages, T helper 1 (Th1) cytokine gene expression and cytotoxic activity of the splenocytes. Furthermore, the antitumor effects were more efficient than that obtained by the combination of HSV-tk and CD80 genes. These results suggest an immunomodulatory effect of MCP-1 in the context of suicide gene therapy of colon cancer via orchestration of innate and acquired immune responses.


Assuntos
Antivirais/uso terapêutico , Quimiocina CCL2/genética , Neoplasias do Colo/terapia , Ganciclovir/uso terapêutico , Terapia Genética , Timidina Quinase/genética , Adenoviridae/genética , Animais , Antígeno B7-1/biossíntese , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL2/biossíntese , Quimiocina CCL2/imunologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Citocinas/genética , Citocinas/metabolismo , Vetores Genéticos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Transplante de Neoplasias , Simplexvirus/metabolismo , Timidina Quinase/biossíntese
9.
Clin Exp Allergy ; 34(8): 1321-8, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15298576

RESUMO

BACKGROUND: Airway inflammation and remodelling are characteristic features of chronic asthma. OBJECTIVE: To elucidate the role of interleukin (IL)-6 in airway responses to chronic antigen exposure. METHODS: We compared airway inflammation, subepithelial collagen deposition, cytokine mRNA expression, and airway responsiveness between IL-6-deficient and wild-type (WT) mice following sensitization and repeated exposure to ovalbumin (OVA) three times a week for 8 weeks. RESULTS: The repeated exposure to OVA induced infiltration of eosinophils, neutrophils, and lymphocytes into the airway, and caused thickening of the basement membrane and subepithelial fibrosis. IL-6-deficient mice exhibited more pronounced infiltration of these cells, a thinner basement membrane, and decreased subepithelial fibrosis, compared with WT mice. The repeated OVA exposure increased expression of IL-4, IL-13, eotaxin, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-beta1 mRNA in WT mice. Among these factors, expression of IL-13 and MCP-1 mRNA was further enhanced in IL-6-deficient mice, compared with WT mice. However, both WT and IL-6-deficient mice exhibited similar levels of airway responsiveness to increasing doses of methacholine, even after repeated exposure to OVA. CONCLUSION: These results suggest that IL-6 has dual roles in the chronic phase of asthma: down-regulation of inflammatory cell infiltration and enhancement of airway remodelling.


Assuntos
Alérgenos/administração & dosagem , Asma/imunologia , Interleucina-6/imunologia , Pulmão/imunologia , Aerossóis , Animais , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Quimiocinas/análise , Doença Crônica , Colágeno/análise , Citocinas/análise , Feminino , Fibrose , Substâncias de Crescimento/análise , Interleucina-6/genética , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Animais , Ovalbumina
10.
Clin Exp Immunol ; 130(3): 548-56, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12452848

RESUMO

In patients with systemic sclerosis (SSc), there are conflicting findings regarding which is predominant between type 1 and type 2 immune responses. To determine the balance between type 1 and type 2 T lymphocytes in peripheral blood from SSc patients, we investigated the expression of intracellular cytokines, such as interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-13, and chemokine receptors such as CXCR3 and CCR4 by flow cytometry. The frequency of IFN-gamma-producing cells among CD8+ cells was significantly increased in patients with diffuse cutaneous SSc (n = 11, P < 0.0001) and limited cutaneous SSc (lSSc; n= 16, P < 0.0001) compared with normal controls (n = 17) while there was no significant difference in the frequency of IL-4- or IL-13-producing cells. In contrast, the frequency of IFN-gamma- or IL-4-producing cells among CD4+ cells was similar between the three groups. Similar results were obtained when absolute numbers were assessed. The frequency of IFN-gamma-producing cells among CD8+ cells inversely correlated with percentage DLco in SSc patients (r = - 0.650, P < 0.005). CXCR3+ CD8+ cells selectively produced IFN-gamma, and the frequency of CXCR3+ CD45RO+ cells among CD8+ cells was higher in lSSc patients (n = 14, P < 0.01) than in normal controls (n = 22). In contrast, there was no significant difference in the frequencies of CXCR3- or CCR4-expressing CD45RO+ cells among CD4+ cells. These results demonstrate the predominance of type 1 cytokine-producing cells (Tc1 cells) in peripheral blood CD8+ T cells from SSc patients, but no definite Th1/Th2 imbalance in CD4+ T cells. Tc1 cells may be associated with pulmonary vascular damage in SSc.


Assuntos
Citocinas/análise , Receptores de Quimiocinas/análise , Escleroderma Sistêmico/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Biomarcadores/análise , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Humanos , Interferon gama/sangue , Interleucina-13/análise , Interleucina-2/análise , Interleucina-4/análise , Líquido Intracelular/imunologia , Masculino , Pessoa de Meia-Idade , Receptores CCR4 , Receptores CXCR3 , Estatísticas não Paramétricas , Células Th1/imunologia , Células Th2/imunologia
11.
Am J Kidney Dis ; 38(6): 1169-77, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11728947

RESUMO

p38 Mitogen-activated protein kinase (MAPK) is involved in the production and signal transduction of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and chemokines in vitro. However, the crucial role of p38 MAPK in the inflammatory processes of crescentic glomerulonephritis in vivo remains to be investigated. We showed a dramatic decrease in IL-1beta-induced phosphorylation of p38 MAPK, not extracellular signal-regulated kinases 1/2 or jun NH2-terminal kinase, in rat cultured mesangial cells by FR167653. We explored the effects of FR167653 as a specific inhibitor of p38 MAPK on renal injury and subsequent renal expression of chemokines in a progressive experimental crescentic glomerulonephritis model in Wistar-Kyoto rats. Rats developed crescentic glomerulonephritis leading to glomerulosclerosis and interstitial fibrosis by 56 days after the administration of nephrotoxic sera. The number of phosphorylated p38 MAPK-positive cells, detected mainly in crescents, correlated well with the percentage of crescents and number of ED-1-positive cells. Phosphorylated p38 MAPK-positive cells were downregulated in glomeruli in rats with the daily subcutaneous administration of FR167653 for 6 days. Concomitantly, renal expression of macrophage inflammatory protein-1alpha and monocyte chemoattractant protein-1/monocyte chemotactic and activating factor was markedly reduced by day 6. The severity of glomerulosclerosis and interstitial fibrosis significantly decreased by day 56, and renal function was preserved. These results suggest that p38 MAPK phosphorylation is pivotal for crescentic glomerulonephritis, followed by the subsequent expression of renal chemokines. This study provides evidence that regulation of p38 MAPK is a novel appealing therapeutic target for crescentic glomerulonephritis.


Assuntos
Proteínas de Transporte/metabolismo , Quimiocina CCL2/metabolismo , Glomerulonefrite/metabolismo , Proteínas Inflamatórias de Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Quimiocina CCL4 , Glomerulonefrite/patologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Fosforilação , Proteinúria/fisiopatologia , Pirazóis/farmacologia , Piridinas/farmacologia , Ratos , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por Mitógeno
12.
Clin Exp Immunol ; 126(2): 266-73, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11703370

RESUMO

To elucidate the in vivo mechanisms involved in the impairment in pulmonary defence as the result of treatment with glucocorticoids, we established fatal pneumonia with bacteraemia in dexamethasone (DEX)-treated mice by means of an intratracheal challenge of Pseudomonas aeruginosa. An increased neutrophil influx was observed in bronchoalveolar lavage (BAL) fluids from both untreated and DEX-treated mice. The complete suppression of an inducible isoform of nitric oxide synthase (iNOS) mRNA expression and tumour necrosis factor alpha (TNF-alpha) production during the early phase of pneumonia, but not CXC chemokine production, were found in the case of the DEX-treated mice. An immunohistochemical study with a specific antibody also revealed negative staining for nitrotyrosine in the lung tissue of DEX-treated mice, while the formation of nitrotyrosine, which indirectly indicates the generation of peroxynitrite with a potent bactericidal activity, was detected clearly in the bronchial epithelium as well as alveolar phagocytic cells of lung tissue from untreated mice. Furthermore, an intraperitoneal administration of S-methyl-isothiourea (SMT), a potent inhibitor of NOS, significantly decreased the survival and increased bacterial density in the case of untreated mice. In contrast, no significant effects on the survival and bacterial density in the lung and blood were found as the result of treatment with SMT in DEX-treated mice. Collectively, a complete repression of iNOS gene expression and a lack of the generation of peroxynitrite as well as an inhibition of TNF-alpha production in the lung appeared to be responsible for the progression of the fatal pneumonia due to P. aeruginosa in DEX-treated mice.


Assuntos
Dexametasona/toxicidade , Glucocorticoides/toxicidade , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Óxido Nítrico Sintase/genética , Ácido Peroxinitroso/biossíntese , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/patogenicidade , Animais , Quimiocinas CXC/biossíntese , Contagem de Colônia Microbiana , Feminino , Expressão Gênica/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos CBA , Óxido Nítrico Sintase Tipo II , Pneumonia Bacteriana/etiologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/biossíntese
13.
Cancer Gene Ther ; 8(10): 695-704, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11687892

RESUMO

The therapeutic efficacy of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system in many types of tumors is unsatisfactory due to the insufficient spread of gene transfer and insufficient cell killing. In the current study, we investigated whether adenovirally delivered monocyte chemoattractant protein (MCP)-1 potentiates the antitumor effects of the HSV-tk/GCV system in hepatocellular carcinoma (HCC) cells. Subcutaneous tumor foci of the human HCC cell line, HuH7, established in athymic mice were directly transduced with a recombinant adenovirus (rAd) harboring an HSV-tk gene driven by a human alpha-fetoprotein promoter, followed by GCV administration. Subsequently, another rAd expressing MCP-1 under the universal CAG promoter was injected. The growth of tumors was markedly suppressed by codelivering HSV-tk and MCP-1 genes compared to that by either HSV-tk/GCV or MCP-1 delivery. In the tumor tissues, monocyte/macrophage infiltration was detected immunohistochemically. The antitumor effects of the rAd expressing MCP-1 were markedly reduced by the administration of carrageenan, a compound known to inactivate macrophage. These results indicate that adenovirally delivered MCP-1 enhanced the antitumor effects of the HSV-tk/GCV system synergistically by recruitment/activation of macrophages in tumor tissues, suggesting an effective immunotherapy for HCC and other lineages of tumors when used adjuvantly with a suicide gene.


Assuntos
Antivirais/uso terapêutico , Carcinoma Hepatocelular/terapia , Quimiocina CCL2/genética , Escherichia coli/genética , Ganciclovir/uso terapêutico , Neoplasias Hepáticas/terapia , Simplexvirus/enzimologia , Timidina Quinase/genética , Animais , Apoptose , Carcinoma Hepatocelular/metabolismo , Quimiocina CCL2/metabolismo , Ensaio de Imunoadsorção Enzimática , Escherichia coli/enzimologia , Citometria de Fluxo , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Humanos , Técnicas Imunoenzimáticas , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peroxidase/metabolismo , Células Tumorais Cultivadas
14.
J Leukoc Biol ; 70(3): 455-60, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11527996

RESUMO

Simple methods that separate progenitor cells of different hemopoietic lineages would facilitate studies on lineage commitment and differentiation. We used an antibody specific for the chemokine receptor CCR1 to examine mononuclear cells isolated from cord blood samples. When CD34(+) cells were separated into CD34(+)CCR1(+) and CD34(+)CCR1(-) cells and plated in colony-forming assays, the granulocyte/macrophage progenitors were found almost exclusively in the CD34(+)CCR1(+) cells. In contrast, the CD34(+)CCR1(-) cells contained the majority of the erythroid progenitors. There was a highly significant difference (P<0.002) in the total percentage distribution of both granulocyte-macrophage colony-forming cells and erythroid burst-forming units between the two populations. This is the first report of separation of erythroid progenitors from granulocyte/macrophage progenitors using a chemokine receptor antibody in cord blood samples. These results suggest that at the clonogenic progenitor cell stage the expression of CCR1 might be lineage-specific. This method should prove useful for studies on erythroid progenitor and granulocyte/macrophage differentiation.


Assuntos
Técnicas de Cultura de Células/métodos , Células Precursoras Eritroides/citologia , Células Progenitoras Mieloides/citologia , Receptores de Quimiocinas/biossíntese , Anticorpos/imunologia , Antígenos CD34/análise , Biomarcadores/análise , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Quimiocina CCL4 , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura Livres de Soro , Células Precursoras Eritroides/química , Sangue Fetal/citologia , Citometria de Fluxo , Granulócitos/citologia , Granulócitos/imunologia , Humanos , Proteínas Inflamatórias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Células Progenitoras Mieloides/química , Células Progenitoras Mieloides/imunologia , RNA Mensageiro/biossíntese , Receptores CCR1 , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia
15.
Am J Physiol Gastrointest Liver Physiol ; 281(3): G735-42, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11518686

RESUMO

Although hypergastrinemia is frequently observed in individuals with a chronic Helicobacter pylori infection, its pathophysiological significance in gastric mucosal inflammation is unclear. The present study was designed to determine if gastrin induces the expression of CXC chemokines in gastric epithelial cells. Human and rat gastric epithelial cells, transfected with gastrin receptor, were stimulated with gastrin. The expression of mRNAs for human interleukin-8 (IL-8) and rat cytokine-induced neutrophil chemoattractant-1 and release of human IL-8 protein were then determined by Northern blot analysis and ELISA, respectively. Gastrin not only induced the expression of mRNAs for these chemokines but also stimulated IL-8 protein release. A luciferase assay using IL-8 promoter genes showed that nuclear factor (NF)-kappaB is absolutely required and activator protein-1 (AP-1) is partly required for the maximum induction of IL-8 by gastrin. An electrophoretic mobility shift assay revealed that gastrin is capable of activating both NF-kappaB and AP-1. In addition, the inhibition of NF-kappaB abrogated gastrin-induced chemokine expression. These results suggest that gastrin is capable of upregulating CXC chemokines in gastric epithelial cells and therefore may contribute to the progression of the inflammatory process in the stomach.


Assuntos
Quimiocinas CXC/biossíntese , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Gastrinas/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , NF-kappa B/metabolismo , Animais , Linhagem Celular , Quimiocina CXCL1 , Quimiocinas CXC/genética , Fatores Quimiotáticos/biossíntese , Fatores Quimiotáticos/genética , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Mucosa Gástrica/citologia , Mucosa Gástrica/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/genética , Humanos , Interleucina-1/farmacologia , Interleucina-8/biossíntese , Interleucina-8/genética , NF-kappa B/antagonistas & inibidores , Regiões Promotoras Genéticas/genética , Pirrolidinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores da Colecistocinina/genética , Receptores da Colecistocinina/metabolismo , Tiocarbamatos/farmacologia , Fator de Transcrição AP-1/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/farmacologia
16.
Leukemia ; 15(7): 1092-101, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11455979

RESUMO

Human haemopoietic stem and progenitor cells may be distinguished by the pattern of cell surface markers they display. The cells defined as 'stem' cells are heterogeneous and lack specific markers for their detection. However, they may be identified in in vitro assays such as the long-term culture initiating cell (LTC-IC) and in transplant assays involving immunosuppressed NOD/SCID mice. It is still not clear to what extent, if any, these cell populations overlap. The chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) prolongs survival of LTC-IC in suspension cultures and we now show that in longterm bone marrow cultures (LTBMC) maintenance of haemopoiesis was significantly better from the CD34+ cells which possess MIP-1alpha receptors (P < 0.006). We examined one MIP-1alpha receptor, CCR1, which is present on CD34+ cells from haemopoietic tissues. In LTBMC the production of GM-CFC from CD34+CCR1- cells was significantly higher (P < 0.02) than that from CD34+CCR1+ cultures and the incidence of LTC-IC was 3- to 6-fold higher in the CD34+CCR1- cell fraction. In contrast, the cells responsible for high levels of engraftment in NOD/SCID mice were contained in the CD34+CCR1+ cell fraction. The CD34+CCR1+ cells engrafted to high levels in NOD/SCID and generated large numbers of progenitor cells. Therefore, we conclude that LTC-IC and SRC may be distinguished on the basis of expression of the chemokine receptor CCR1.


Assuntos
Antígenos CD34/análise , Células-Tronco Hematopoéticas/química , Receptores de Quimiocinas/análise , Animais , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , Sangue Fetal/citologia , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Proteínas Inflamatórias de Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores CCR1
17.
Clin Cancer Res ; 7(6): 1812-20, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11410524

RESUMO

Interleukin 1alpha (IL-1alpha) is an important regulatory cytokine, the release of which after an injury can induce activation of transcription factors nuclear factor (NF)kappaB and activator protein (AP-1), which promote expression of genes involved in cell survival, proliferation, and angiogenesis. IL-1alpha is expressed autonomously by head and neck squamous cell carcinomas (HNSCCs) and a variety of other cancers, raising the possibility that IL-1alpha may serve as an autocrine factor that stimulates the activation of prosurvival transcription factors and target genes in cancer. In this study, we examined the role of IL-1alpha in the activation of NFkappaB and AP-1, the expression of proangiogenic cytokine IL-8, and in the survival and proliferation of HNSCC cell lines. HNSCCs were found to secrete and respond to functional IL-1alpha, in that culture supernatant from a high IL-1alpha-secreting line, UM-SCC-11B, could induce secretion of cytokine IL-8 by a low IL-1alpha-secreting line, UM-SCC-9; and the induction of IL-8 secretion could be blocked by the anti-IL-1alpha-neutralizing antibody or the IL-1 receptor antagonist (IL-1RA). Furthermore, IL-1alpha could induce the expression of IL-8 through an autocrine mechanism, in that transfection of UM-SCC-9 cells with a plasmid encoding IL-1alpha resulted in the increased coexpression of IL-1alpha and IL-8; whereas transfection with a plasmid encoding IL-1RA lacking the secretory leader sequence led to the decreased coexpression of IL-1alpha and IL-8. IL-1alpha was found to induce coexpression of IL-8 through the activation of NFkappaB and AP-1, in that mutation of the NFkappaB site within the IL-8 promoter abolished autocrine- and recombinant IL-1alpha-induced IL-8 reporter gene activity, whereas mutation in AP-1 partially decreased IL-8 reporter gene activity in UM-SCC-9 cells. Intracellular expression of IL-1RA decreased NFkappaB reporter gene activity, indicating that endogenously expressed IL-1alpha contributes to constitutive NFkappaB activation in this HNSCC line. Expression of IL-1alpha affected survival of UM-SCC-9, inasmuch as transfection of cells with plasmid encoding IL-1alpha or IL-1RA led to the increased or decreased survival of cells cotransfected with a beta-galactosidase reporter gene, respectively. IL-1alpha was also found to promote the increased growth of UM-SCC-9 cells in vitro. We demonstrate that exogenous and endogenous IL-1alpha contributes to the transcriptional activation of NFkappaB and AP-1, to the expression of IL-8, and to cell survival and the growth of HNSCC in vitro.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Interleucina-1/metabolismo , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição AP-1/metabolismo , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular , Corantes/farmacologia , Ensaio de Imunoadsorção Enzimática , Genes Reporter , Vetores Genéticos , Humanos , Interleucina-8/biossíntese , Mutação , Plasmídeos/metabolismo , Proteínas Recombinantes/metabolismo , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Fatores de Tempo , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas
18.
J Immunol ; 167(1): 366-74, 2001 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-11418672

RESUMO

The destructive pulmonary inflammation associated with Pseudomonas aeruginosa colonization is caused, in part, by the production of the chemokine IL-8, which recruits neutrophils into the lung. The Pseudomonas autoinducer, N-3-oxododecanoyl homoserine lactone (3-O-C12-HSL), is a small lipid-soluble molecule that is essential in the regulation of many P. aeruginosa virulence factors, but little is known about how it affects eukaryotic cells. In this report we demonstrate that 3-O-C12-HSL is a potent stimulator of both IL-8 mRNA and protein from human fibroblasts and epithelial cells in vitro. The IL-8 produced from these 3-O-C12-HSL-stimulated cells was found to be functionally active by inducing the chemotaxis of neutrophils. To determine a mechanism for this IL-8 induction, deletion constructs of the IL-8 promoter were examined. It was found that the DNA region between nucleotides -1481 and -546 and the transcription factor NF-kappaB were essential for the maximal induction of IL-8 by 3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a shift with both AP-2 and NF-kappaB consensus DNA. The activation of NF-kappaB and subsequent production of IL-8 were found to be regulated by a mitogen-activated protein kinase pathway. These findings support the concept that the severe lung damage that accompanies P. aeruginosa infections is caused by an exuberant neutrophil response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the mechanisms of 3-O-C12-HSL activation of lung structural cells may provide a means to help control lung damage during infections with P. aeruginosa.


Assuntos
4-Butirolactona/fisiologia , Proteínas de Ligação a DNA/fisiologia , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Homosserina/fisiologia , Interleucina-8/biossíntese , Pulmão/metabolismo , NF-kappa B/fisiologia , Pseudomonas aeruginosa/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Regiões 5' não Traduzidas/fisiologia , Linhagem Celular , Sistema Livre de Células/fisiologia , Células Cultivadas , Quimiotaxia de Leucócito/imunologia , Proteínas de Ligação a DNA/biossíntese , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Homosserina/análogos & derivados , Homosserina/farmacologia , Humanos , Interleucina-8/genética , Interleucina-8/fisiologia , Pulmão/citologia , Pulmão/imunologia , NF-kappa B/biossíntese , Neutrófilos/imunologia , Regiões Promotoras Genéticas/imunologia , Pseudomonas aeruginosa/patogenicidade , Fator de Transcrição AP-1/biossíntese , Fator de Transcrição AP-2 , Fatores de Transcrição/biossíntese , Transcrição Gênica/imunologia
19.
J Immunol ; 166(12): 7104-11, 2001 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-11390455

RESUMO

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-Delta(12,14)PGJ2 (15d-PGJ2) have been proposed as a new class of antiinflammatory compounds with possible clinical applications. As there is some controversy over the inhibitory effects of 15d-PGJ2 on chemokine gene expression, we investigated whether 15d-PGJ2 itself affected chemokine gene expression in human monocytes/macrophages and two monocytic cell lines. Here we demonstrate that the 15d-PGJ2 can induce IL-8 gene expression. In contrast, monocyte chemoattractant protein-1 gene expression was suppressed by 15d-PGJ2, while the expression of RANTES was unaltered. Furthermore, concomitant treatment of monocytes/macrophages with 15d-PGJ2 (2.5 x 10(-6) M) potentiated LPS-induced gene expression of IL-8 mRNA, but suppressed PMA-induction of IL-8 mRNA. In addition, treatment of U937 and THP-1 cells with 15d-PGJ2 also resulted in induction of IL-8 gene expression. Further studies demonstrated that 15d-PGJ2 regulated IL-8 gene expression via a ligand-specific and PPARgamma-dependent pathway. Our observations revealed a previous unappreciated function and mechanism of 15d-PGJ2-mediated regulation of cytokine gene expression in monocytes/macrophages.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Quimiocinas/biossíntese , Quimiocinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Prostaglandina D2/farmacologia , Receptores de Esteroides , Fatores de Transcrição COUP , Sistema Livre de Células/fisiologia , Células Cultivadas , Quimiocina CCL2/biossíntese , Quimiocina CCL5/biossíntese , Quimiotaxia de Leucócito/imunologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Humanos , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , Interleucina-8/genética , Ligantes , Lipopolissacarídeos/farmacologia , Monócitos/imunologia , Monócitos/metabolismo , Neutrófilos/imunologia , Peroxissomos/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/imunologia , Prostaglandina D2/análogos & derivados , Prostaglandina D2/metabolismo , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Transfecção
20.
J Virol ; 75(13): 6095-106, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11390611

RESUMO

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-DeltaN110), 222 (NS5A-DeltaN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-DeltaN110 and NS5A-DeltaN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-kappaB and AP-1 were important in NS5A-DeltaN222 transactivation in the presence of tumor necrosis factor alpha and that NF-IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.


Assuntos
Hepacivirus/efeitos dos fármacos , Interferons/antagonistas & inibidores , Interleucina-8/biossíntese , Proteínas não Estruturais Virais/fisiologia , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Resistência Microbiana a Medicamentos , Células HeLa , Humanos , Interleucina-8/genética , Dados de Sequência Molecular , Fosforilação , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Fator de Transcrição STAT1 , Transdução de Sinais , Transativadores/metabolismo , Ativação Transcricional , Fator de Necrose Tumoral alfa/farmacologia
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