RESUMO
BACKGROUND: Growing evidence has suggested that Type I Interferon (I-IFN) plays a potential role in the pathogenesis of Down Syndrome (DS). This work investigates the underlying function of MX1, an effector gene of I-IFN, in DS-associated transcriptional regulation and phenotypic modulation. METHODS: We performed assay for transposase-accessible chromatin with high-throughout sequencing (ATAC-seq) to explore the difference of chromatin accessibility between DS derived amniocytes (DSACs) and controls. We then combined the annotated differentially expressed genes (DEGs) and enriched transcriptional factors (TFs) targeting the promoter region from ATAC-seq results with the DEGs in RNA-seq, to identify key genes and pathways involved in alterations of biological processes and pathways in DS. RESULTS: Binding motif analysis showed a significant increase in chromatin accessibility of genes related to neural cell function, among others, in DSACs, which is primarily regulated by members of the activator protein-1 (AP-1) transcriptional factor family. Further studies indicated that MX Dynamin Like GTPase 1 (MX1), defined as one of the key effector genes of I-IFN, is a critical upstream regulator. Its overexpression induced expression of AP-1 TFs and mediated inflammatory response, thus leading to decreased cellular viability of DS cells. Moreover, treatment with specific AP-1 inhibitor T-5224 improved DS-associated phenotypes in DSACs. CONCLUSIONS: This study demonstrates that MX1-mediated AP-1 activation is partially responsible for cellular dysfunction of DS. T-5224 effectively ameliorated DS-associated phenotypes in DSACs, suggesting it as a potential treatment option for DS patients.
Assuntos
Síndrome de Down , Fator de Transcrição AP-1 , Humanos , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , RNA-Seq , Síndrome de Down/tratamento farmacológico , Síndrome de Down/genética , Cromatina , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismoRESUMO
One of the major causes of early pregnancy loss is heat stress. In ruminants, interferon tau (IFNT) is the embryo signal to the mother. Once the interferon signaling pathway is activated, it drives gene expression for interferon-stimulated genes (ISGs) and alters neutrophils responses. The aim of the present study was to evaluate interferon (IFN) pathway, ISGs and gene expression in polymorphonuclear leukocytes (PMN) and oxidative stress in dairy cows under heat stress. Pregnant cows had their estrous cycle synchronized and randomly assigned to a comfort or heat stress group. Blood samples were collected at artificial insemination (AI) and on Days 10, 14 and 18 following AI. Pregnant cows were pregnancy checked by ultrasound on Day 30 and confirmed on Day 60 post-AI. Results are presented as mean ± SEM. The corpus luteum (CL) diameter was not different between groups of pregnant cows; concentration of progesterone of pregnant cows on Day 18 following AI was greater in comfort group compared to heat stressed group. Comfort pregnant cows had higher expression of all analyzed genes from interferon pathway, except for IFNAR1, on both Days 14 and 18. Conversely, heat stressed cows did not show altered expression of IFNT pathway genes and ISGs between Days 10, 14, and 18 after AI. The oxidative stress, determined as malondialdehyde (MDA) levels, was greater in heat stress group on Days 10, 14 and 18, independent of pregnancy status. Heat stress negatively influences expression of ISGs, IFN pathway gene expression in neutrophils, and oxidative stress. Our data suggest that lower conception rates in cows under heat stress are multifactorial, with the association of interferon pathway activation and the unbalanced oxidative stress being main contributing factors.
Assuntos
Resposta ao Choque Térmico/genética , Interferons/metabolismo , Neutrófilos/metabolismo , Estresse Oxidativo , Animais , Bovinos , Corpo Lúteo/diagnóstico por imagem , Corpo Lúteo/fisiologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Inseminação Artificial/veterinária , Malondialdeído/sangue , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Neutrófilos/citologia , Gravidez , Progesterona/sangue , Temperatura , UltrassonografiaRESUMO
In salmon farming, viruses are responsible for outbreaks that produce significant economic losses for which there is a lack of control tools other than vaccines. Type I interferon has been successfully used for treating some chronic viral infections in humans. However, its application in salmonids depends on the proper design of a vehicle that allows its massive administration, ideally orally. In mammals, administration of recombinant probiotics capable of expressing cytokines has shown local and systemic therapeutic effects. In this work, we evaluate the use of Lactococcus lactis as a type I Interferon expression system in Atlantic salmon, and we analyze its ability to stimulate the antiviral immune response against IPNV, in vivo and in vitro. The interferon expressed in L. lactis, even though it was located mainly in the bacterial cytoplasm, was functional, stimulating Mx and PKR expression in CHSE-214 cells, and reducing the IPNV viral load in SHK-1 cells. In vivo, the oral administration of this L. lactis producer of Interferon I increases Mx and PKR expression, mainly in the spleen, and to a lesser extent, in the head kidney. The oral administration of this strain also reduces the IPNV viral load in Atlantic salmon specimens challenged with this pathogen. Our results show that oral administration of L. lactis producing Interferon I induces systemic effects in Atlantic salmon, allowing to stimulate the antiviral immune response. This probiotic could have effects against a wide variety of viruses that infect Atlantic salmon and also be effective in other salmonids due to the high identity among their type I interferons.
Assuntos
Infecções por Birnaviridae/prevenção & controle , Proteínas de Peixes/metabolismo , Imunidade Inata , Vírus da Necrose Pancreática Infecciosa/patogenicidade , Interferon Tipo I/metabolismo , Lactococcus lactis/metabolismo , Probióticos , Salmo salar/microbiologia , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/microbiologia , Infecções por Birnaviridae/virologia , Linhagem Celular , Proteínas de Peixes/genética , Pesqueiros , Interações Hospedeiro-Patógeno , Vírus da Necrose Pancreática Infecciosa/crescimento & desenvolvimento , Vírus da Necrose Pancreática Infecciosa/imunologia , Interferon Tipo I/genética , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Proteínas de Resistência a Myxovirus/metabolismo , Salmo salar/genética , Salmo salar/imunologia , Salmo salar/virologia , Carga Viral , eIF-2 Quinase/metabolismoRESUMO
BACKGROUND: Genetic background may be an important host determinant of respiratory syncytial virus (RSV) disease severity, but full characterization of susceptibility genes remains unclear. This study aimed to assess the presence of specific single-nucleotide polymorphisms (SNPs) in selected genes codifying for different components of the antiviral innate immune response, to determine their role for developing RSV life-threatening disease (LTD). METHODS: Prospective cohort study including previously healthy full-term infants hospitalized with a first RSV infection during 2017-2018. RSV detection, quantification and subgroup determination, and genotyping for SNPs in Toll-like receptor 4 (TLR4 rs4986790, rs4986791), Toll-like receptor 8 (TLR8 rs3761624), macrophage receptor with collagenous structure(MARCO rs1318645) and myxovirus resistance 1(MX1 rs469390) were performed by real-time polymerase chain reaction in nasopharyngeal aspirates obtained on admission. Patients with LTD were those admitted to the intensive care unit requiring ventilatory support. RESULTS: Seventy-five patients were studied, 15 (20%) developed LTD. Infants with concurrent SNPs in MX1 and TLR8, MARCO and TLR8 or MARCO, MX1 and TLR8 had an increased risk of developing LTD. Multivariable logistic regression analysis confirmed this significant association (odds ratio [OR] = 3.75, P = 0.046; OR = 3.92, P = 0.040; OR = 5.56, P = 0.010, respectively). No differences were seen in viral load of patients with LTD compared with those with better outcome (P = 0.737). In addition, no differences in viral load were seen in patients with the described high-risk SNPs compared with those without these polymorphisms. CONCLUSIONS: Life-threatening RSV infection in previously healthy infants was significantly associated with the presence of combined SNPs in MARCO, MX1 and TLR8.
Assuntos
Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Infecções por Vírus Respiratório Sincicial/genética , Criança Hospitalizada , Feminino , Humanos , Imunidade Inata , Lactente , Masculino , Proteínas de Resistência a Myxovirus/genética , Estudos Prospectivos , Receptores Imunológicos/genética , Infecções por Vírus Respiratório Sincicial/imunologia , Receptor 4 Toll-Like/genética , Receptor 8 Toll-Like/genética , Carga ViralRESUMO
This study characterised the expression of interferon (IFN)-τ-stimulated genes (ISGs) and Type I IFN receptors in circulating polymorphonuclear cells (PMNs) of beef heifers and compared it with expression in peripheral blood mononuclear cells (PBMCs) up to Day 20 of gestation. Nelore heifers (n=26) were subjected to fixed-time AI (FTAI) on Day 0. PMNs and PBMCs were isolated on Days 0, 10, 14, 16, 18 and 20 after FTAI. The abundance of target transcripts (ubiquitin-like protein (ISG15), 2'-5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance 1 (MX1), myxovirus resistance 2 (MX2), IFN receptor I (IFNAR1) and IFN receptor 2 (IFNAR2)) was determined using real-time quantitative polymerase chain reaction and compared between pregnant (n=8) and non-pregnant (n=9) females. In both PBMCs and PMNs, ISG15 and OAS1 expression was greater in pregnant than non-pregnant heifers on Days 18 and 20. There were no significant differences in the expression of ISGs between PBMCs and PMNs. A time effect on expression was found for IFNAR1 in PBMCs and IFNAR2 in PMNs, with decreased expression of both genes on Days 18 and 20. When the expression of these genes was compared between cell types only in pregnant heifers, IFNAR2 expression in PMNs had an earlier decrease when compared to its expression in PBMCs, starting from Day 18. In conclusion, PMNs do not respond earlier to the conceptus stimulus, and ISG15 and OAS1 expression in both PMNs and PBMCs can be used as a suitable marker for pregnancy diagnosis on Days 18 and 20. In addition, gestational status did not affect IFNAR1 and IFNAR2 expression, but IFNAR2 showed a distinct response between PMNs and PBMCs of pregnant heifers.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Neutrófilos/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Ubiquitinas/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , Animais , Bovinos , Feminino , Proteínas de Resistência a Myxovirus/genética , Gravidez , Progesterona/sangue , Receptor de Interferon alfa e beta/genética , Ubiquitinas/genéticaRESUMO
The inflammatory tumor microenvironment is a fertile niche accelerating prostate cancer (PCa). We have reported that heme-oxygenase (HO-1) had a strong anti-tumoral effect in PCa. We previously undertook an in-depth proteomics study to build the HO-1 interactome in PCa. In this work, we used a bioinformatics approach to address the biological significance of HO-1 interactors. Open-access PCa datasets were mined to address the clinical significance of the HO-1 interactome in human samples. HO-1 interactors were clustered into groups according to their expression profile in PCa patients. We focused on the myxovirus resistance gene (MX1) as: (1) it was significantly upregulated under HO-1 induction; (2) it was the most consistently downregulated gene in PCa vs. normal prostate; (3) its loss was associated with decreased relapse-free survival in PCa; and (4) there was a significant positive correlation between MX1 and HMOX1 in PCa patients. Further, MX1 was upregulated in response to endoplasmic reticulum stress (ERS), and this stress triggered apoptosis and autophagy in PCa cells. Strikingly, MX1 silencing reversed ERS. Altogether, we showcase MX1 as a novel HO-1 interactor and downstream target, associated with ERS in PCa and having a high impact in the clinical setting.
Assuntos
Biologia Computacional/métodos , Heme Oxigenase-1/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Neoplasias da Próstata/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Mineração de Dados , Bases de Dados Genéticas , Estresse do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica , Heme Oxigenase-1/genética , Humanos , Masculino , Proteínas de Resistência a Myxovirus/genética , Células PC-3 , Neoplasias da Próstata/genética , Análise de Sobrevida , Microambiente TumoralRESUMO
OBJECTIVE: Primary Sjögren's syndrome (pSjS) is a chronic autoimmune disease that causes dry eye and mouth. No laboratory parameters to monitor the activation of this disease have been identified. Therefore, any possible relationships between salivary and blood myxovirus resistance 1 (MX1) and pSjS must be prospectively studied. METHODS: Thirty female patients with pSjS, 30 women with rheumatoid arthritis (RA) without secondary Sjögren's syndrome (SjS) and 28 healthy control women were enrolled in this investigation. Analyses of MX1 by the enzyme-linked immunosorbent assay (ELISA) method, SS-A (Ro) and SS-B (La) tests by the strip immunoblot method, anti-nuclear antibody (ANA) tests by immunofluorescence and the measurement of serum rheumatoid factor (RF), C3, C4, immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) were performed. RESULTS: The serum level of MX1 in patients without Raynaud phenomenon was higher than in those with Raynaud phenomenon (p:0.029, p<0.05, statistically significant). There was a statistically significant positive association between hemoglobin levels and MX1 serum levels. No statistically significant association was found among the other parameters. Low MX1 levels were shown to be associated with both a low disease activity score based on the European League Against Rheumatism (EULAR) Sjögren's Syndrome Disease Activity Index (ESSDAI) and hydroxychloroquine use in all patients. CONCLUSION: MX1 levels have a considerable impact on the assessment of the disease activity in SjS. We believe that more-comprehensive studies should be performed on patients with pSjS who do not use hydroxychloroquine to prove this relationship and that MX1 levels should be used as a routine marker for the assessment of pSjS disease activity. Further studies are needed to create awareness of the role that MX1 has in the diagnosis of pSjS, which may help to uncover novel pathways for new therapeutic modalities.
Assuntos
Isotipos de Imunoglobulinas/sangue , Proteínas de Resistência a Myxovirus/imunologia , Saliva/química , Síndrome de Sjogren/metabolismo , Adulto , Anticorpos Antinucleares/sangue , Biomarcadores/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Pessoa de Meia-Idade , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/imunologiaRESUMO
OBJECTIVE: Primary Sjögren's syndrome (pSjS) is a chronic autoimmune disease that causes dry eye and mouth. No laboratory parameters to monitor the activation of this disease have been identified. Therefore, any possible relationships between salivary and blood myxovirus resistance 1 (MX1) and pSjS must be prospectively studied. METHODS: Thirty female patients with pSjS, 30 women with rheumatoid arthritis (RA) without secondary Sjögren's syndrome (SjS) and 28 healthy control women were enrolled in this investigation. Analyses of MX1 by the enzyme-linked immunosorbent assay (ELISA) method, SS-A (Ro) and SS-B (La) tests by the strip immunoblot method, anti-nuclear antibody (ANA) tests by immunofluorescence and the measurement of serum rheumatoid factor (RF), C3, C4, immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) were performed. RESULTS: The serum level of MX1 in patients without Raynaud phenomenon was higher than in those with Raynaud phenomenon (p:0.029, p<0.05, statistically significant). There was a statistically significant positive association between hemoglobin levels and MX1 serum levels. No statistically significant association was found among the other parameters. Low MX1 levels were shown to be associated with both a low disease activity score based on the European League Against Rheumatism (EULAR) Sjögren's Syndrome Disease Activity Index (ESSDAI) and hydroxychloroquine use in all patients. CONCLUSION: MX1 levels have a considerable impact on the assessment of the disease activity in SjS. We believe that more-comprehensive studies should be performed on patients with pSjS who do not use hydroxychloroquine to prove this relationship and that MX1 levels should be used as a routine marker for the assessment of pSjS disease activity. Further studies are needed to create awareness of the role that MX1 has in the diagnosis of pSjS, which may help to uncover novel pathways for new therapeutic modalities.
Assuntos
Humanos , Feminino , Adulto , Pessoa de Meia-Idade , Saliva/química , Isotipos de Imunoglobulinas/sangue , Síndrome de Sjogren/metabolismo , Proteínas de Resistência a Myxovirus/imunologia , Imunoglobulina G , Imunoglobulina M/sangue , Ensaio de Imunoadsorção Enzimática , Biomarcadores/análise , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/imunologia , Anticorpos Antinucleares/sangueRESUMO
Type-I interferon (IFN-I) production is an early response to viral infection and pathogenic viruses have evolved multiple strategies to evade this cellular defense. Some viruses can establish and maintain persistent infections by altering the IFN-I signaling pathway. Here, we studied IFN-I synthesis and response in an in vitro model of persistent infection by respiratory syncytial virus (RSV) in a murine macrophage-like cell line. In this model, interferon regulatory factor 3 was constitutively active and located at nuclei of persistently infected cells, inducing expression of IFN-beta mRNA and protein. However, persistently infected macrophages did not respond in an autocrine manner to the secreted-IFN-beta or to recombinant-IFN-beta, since phosphorylated-STAT1 was not detected by western blot and transcription of the interferon-stimulated genes (ISGs) Mx1 and ISG56 was not induced. Treatment of non-infected macrophages with supernatants from persistently infected cells induced STAT1 phosphorylation and ISGs expression, mediated by the IFN-I present in the supernatants, because blocking the IFN-I receptor inhibited STAT1 phosphorylation. Results suggest that the lack of autocrine response to IFN-I by the host cell may be one mechanism for maintenance of RSV persistence. Furthermore, STAT1 phosphorylation and ISGs expression induced in non-infected cells by supernatants from persistently infected macrophages suggest that RSV persistence may trigger a proinflammatory phenotype in non-infected cells as part of the pathogenesis of RSV infection.
Assuntos
Interferon beta/biossíntese , Macrófagos/virologia , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Vírus Sinciciais Respiratórios/isolamento & purificação , Animais , Western Blotting , Células Cultivadas , Perfilação da Expressão Gênica , Camundongos , Proteínas de Resistência a Myxovirus/análise , RNA Mensageiro/análise , Fator de Transcrição STAT1/análise , Fatores de Transcrição/análiseRESUMO
The bovine viral diarrhea virus (BVDV-1) is a pathogen responsible for high economic losses in the cattle industry worldwide. This virus has the capacity to modulate the immune system of several higher vertebrates, but there is little information available on the cell infection mechanism. To further investigate the effects of BVDV-1 on the activation of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the proinflammatory and antiviral cytokine expression profiles were analyzed. The results showed that BVDV-1 was able to induce the production of BCL3, IL-1ß, IL-8, IL-15, IL-18, Mx-1, IRF-1, and IRF-7 in a way similar to polyinosinic-polycytidylic acid. Interestingly, all BVDV-1 activities were blocked by pharmacological inhibitors of the NF-κB signaling pathway. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that BVDV-1 regulates gene expression in bovines through the activation of several key transcription factors. Collectively, these data identified BVDV-1 as a viral regulator of immune marker expression, even from early infection. Additionally, this is the first report to find BVDV-1 modulating the activation of cytokine production and transcriptions factors mainly through the NF-κB pathway in vertebrates.
Assuntos
Vírus da Diarreia Viral Bovina/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Interleucinas/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Nitrilas/farmacologia , Sulfonas/farmacologia , Animais , Proteína 3 do Linfoma de Células B , Biomarcadores/metabolismo , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina/crescimento & desenvolvimento , Vírus da Diarreia Viral Bovina/imunologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interleucinas/genética , Interleucinas/imunologia , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Lichen planus (LP) is a common inflammatory skin disease of unknown etiology. Reports of a common transactivation of quiescent human endogenous retroviruses (HERVs) support the connection of viruses to the disease. HERVs are ancient retroviral sequences in the human genome and their transcription is often deregulated in cancer and autoimmune diseases. We explored the transcriptional activity of HERV sequences as well as the antiviral restriction factor and interferon-inducible genes in the skin from LP patients and healthy control (HC) donors. The study included 13 skin biopsies from patients with LP and 12 controls. Real-time PCR assay identified significant decrease in the HERV-K gag and env mRNA expression levels in LP subjects, when compared to control group. The expressions of HERV-K18 and HERV-W env were also inhibited in the skin of LP patients. We observed a strong correlation between HERV-K gag with other HERV sequences, regardless the down-modulation of transcripts levels in LP group. In contrast, a significant up-regulation of the cytidine deaminase APOBEC 3G (apolipoprotein B mRNA-editing), and the GTPase MxA (Myxovirus resistance A) mRNA expression level was identified in the LP skin specimens. Other transcript expressions, such as the master regulator of type I interferon-dependent immune responses, STING (stimulator of interferon genes) and IRF-7 (interferon regulatory factor 7), IFN-ß and the inflammassome NALP3, had increased levels in LP, when compared to HC group. Our study suggests that interferon-inducible factors, in addition to their role in innate immunity against exogenous pathogens, contribute to the immune control of HERVs. Evaluation of the balance between HERV and interferon-inducible factor expression could possibly contribute to surveillance of inflammatory/malignant status of skin diseases.
Assuntos
Retrovirus Endógenos/metabolismo , Produtos do Gene env/metabolismo , Produtos do Gene gag/metabolismo , Interferon beta/metabolismo , Líquen Plano/imunologia , Proteínas de Membrana/metabolismo , Proteínas da Gravidez/metabolismo , Pele/metabolismo , Superantígenos/metabolismo , Desaminase APOBEC-3G , Adulto , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Retrovirus Endógenos/genética , Feminino , Regulação Viral da Expressão Gênica , Produtos do Gene env/genética , Produtos do Gene gag/genética , Humanos , Vigilância Imunológica , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/genética , Líquen Plano/genética , Líquen Plano/virologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas da Gravidez/genética , Pele/virologia , Superantígenos/genética , Ativação Transcricional , Regulação para Cima , Adulto JovemRESUMO
Certain host single nucleotide polymorphisms (SNPs) affect the likelihood of a sustained virological response (SVR) to treatment in subjects infected with hepatitis C virus (HCV). SNPs in the promoters of interleukin (IL)-10 (-1082 A/G, rs1800896), myxovirus resistance protein 1 (-123 C/A, rs17000900 and -88 G/T, rs2071430) and tumour necrosis factor (TNF) (-308 G/A, rs1800629 and -238 G/A, rs361525) genes and the outcome of PEGylated α-interferon plus ribavirin therapy were investigated. This analysis was performed in 114 Brazilian, HCV genotype 1-infected patients who had a SVR and in 85 non-responders and 64 relapsers. A significantly increased risk of having a null virological response was observed in patients carrying at least one A allele at positions -308 [odds ratios (OR) = 2.58, 95% confidence intervals (CI) = 1.44-4.63, p = 0.001] or -238 (OR = 7.33, 95% CI = 3.59-14.93, p < 0.001) in the TNF promoter. The risk of relapsing was also elevated (-308: OR = 2.87, 95% CI = 1.51-5.44, p = 0.001; -238: OR = 4.20, 95% CI = 1.93-9.10, p < 0.001). Multiple logistic regression of TNF diplotypes showed that patients with at least two copies of the A allele had an even higher risk of having a null virological response (OR = 16.43, 95% CI = 5.70-47.34, p < 0.001) or relapsing (OR = 6.71, 95% CI = 2.18-20.66, p = 0.001). No statistically significant association was found between the other SNPs under study and anti-HCV therapy response.
Assuntos
Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Antivirais/administração & dosagem , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/administração & dosagem , Regiões Promotoras Genéticas , Ribavirina/administração & dosagem , Fator de Necrose Tumoral alfa/genética , Quimioterapia Combinada , Genótipo , Hepatite C Crônica/genética , /genética , Proteínas de Resistência a Myxovirus/genética , Polimorfismo de Nucleotídeo Único , Falha de Tratamento , Carga ViralRESUMO
Certain host single nucleotide polymorphisms (SNPs) affect the likelihood of a sustained virological response (SVR) to treatment in subjects infected with hepatitis C virus (HCV). SNPs in the promoters of interleukin (IL)-10 (-1082 A/G, rs1800896), myxovirus resistance protein 1 (-123 C/A, rs17000900 and -88 G/T, rs2071430) and tumour necrosis factor (TNF) (-308 G/A, rs1800629 and -238 G/A, rs361525) genes and the outcome of PEGylated α-interferon plus ribavirin therapy were investigated. This analysis was performed in 114 Brazilian, HCV genotype 1-infected patients who had a SVR and in 85 non-responders and 64 relapsers. A significantly increased risk of having a null virological response was observed in patients carrying at least one A allele at positions -308 [odds ratios (OR) = 2.58, 95% confidence intervals (CI) = 1.44-4.63, p = 0.001] or -238 (OR = 7.33, 95% CI = 3.59-14.93, p < 0.001) in the TNF promoter. The risk of relapsing was also elevated (-308: OR = 2.87, 95% CI = 1.51-5.44, p = 0.001; -238: OR = 4.20, 95% CI = 1.93-9.10, p < 0.001). Multiple logistic regression of TNF diplotypes showed that patients with at least two copies of the A allele had an even higher risk of having a null virological response (OR = 16.43, 95% CI = 5.70-47.34, p < 0.001) or relapsing (OR = 6.71, 95% CI = 2.18-20.66, p = 0.001). No statistically significant association was found between the other SNPs under study and anti-HCV therapy response.
Assuntos
Antivirais/administração & dosagem , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/administração & dosagem , Regiões Promotoras Genéticas , Ribavirina/administração & dosagem , Fator de Necrose Tumoral alfa/genética , Quimioterapia Combinada , Feminino , Genótipo , Hepatite C Crônica/genética , Humanos , Interleucina-10/genética , Masculino , Pessoa de Meia-Idade , Proteínas de Resistência a Myxovirus/genética , Polimorfismo de Nucleotídeo Único , Falha de Tratamento , Carga ViralRESUMO
Programs for the prevention of mother-to-child transmission of HIV have reduced the transmission rate of perinatal HIV infection and have thereby increased the number of HIV-exposed uninfected (HEU) infants. Natural immunity to HIV-1 infection in both mothers and newborns needs to be further explored. In this study, we compared the expression of antiviral restricting factors in HIV-infected pregnant mothers treated with antiretroviral therapy (ART) in pregnancy (n=23) and in cord blood (CB) (n=16), placental tissues (n=10-13) and colostrum (n=5-6) samples and compared them to expression in samples from uninfected (UN) pregnant mothers (n=21). Mononuclear cells (MNCs) were prepared from maternal and CB samples following deliveries by cesarean section. Maternal (decidua) and fetal (chorionic villus) placental tissues were obtained, and colostrum was collected 24 h after delivery. The mRNA and protein expression levels of antiviral factors were then evaluated. We observed a significant increase in the mRNA expression levels of antiviral factors in MNCs from HIV-infected mothers and CB, including the apolipoprotein B mRNA-editing enzyme 3G (A3G), A3F, tripartite motif family-5α (TRIM-5α), TRIM-22, myxovirus resistance protein A (MxA), stimulator of interferon (IFN) genes (STING) and IFN-ß, compared with the levels detected in uninfected (UN) mother-CB pairs. Moreover, A3G transcript and protein levels and α-defensin transcript levels were decreased in the decidua of HIV-infected mothers. Decreased TRIM-5α protein levels in the villi and increased STING mRNA expression in both placental tissues were also observed in HIV-infected mothers compared with uninfected (UN) mothers. Additionally, colostrum cells from infected mothers showed increased tetherin and IFN-ß mRNA levels and CXCL9 protein levels. The data presented here indicate that antiviral restricting factor expression can be induced in utero in HIV-infected mothers. Future studies are warranted to determine whether this upregulation of antiviral factors during the perinatal period has a protective effect against HIV-1 infection.
Assuntos
Sangue Fetal/metabolismo , Regulação da Expressão Gênica/imunologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , Imunidade Inata/imunologia , Viremia/prevenção & controle , Desaminase APOBEC-3G , Fatores de Restrição Antivirais , Western Blotting , Brasil , Proteínas de Transporte/metabolismo , Vilosidades Coriônicas/metabolismo , Colostro/metabolismo , Citidina Desaminase/metabolismo , Primers do DNA/genética , Decídua/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Interferon beta/economia , Interferon beta/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas de Membrana/metabolismo , Antígenos de Histocompatibilidade Menor , Mães , Proteínas de Resistência a Myxovirus/metabolismo , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/metabolismo , Estatísticas não Paramétricas , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Viremia/metabolismoRESUMO
OBJECTIVES: Suppressor of cytokine signaling 3, myxovirus resistance protein and osteopontin gene polymorphisms may influence the therapeutic response in patients with chronic hepatitis C, and an association with IL28 might increase the power to predict sustained virologic response. Our aims were to evaluate the association between myxovirus resistance protein, osteopontin and suppressor of cytokine signaling 3 gene polymorphisms in combination with IL28B and to assess the therapy response in hepatitis C patients treated with pegylated-interferon plus ribavirin. METHOD: Myxovirus resistance protein, osteopontin, suppressor of cytokine signaling 3 and IL28B polymorphisms were analyzed by PCR-restriction fragment length polymorphism, direct sequencing and real-time PCR. Ancestry was determined using genetic markers. RESULTS: We analyzed 181 individuals, including 52 who were sustained virologic responders. The protective genotype frequencies among the sustained virologic response group were as follows: the G/G suppressor of cytokine signaling 3 (rs4969170) (62.2%); T/T osteopontin (rs2853744) (60%); T/T osteopontin (rs11730582) (64.3%); and the G/T myxovirus resistance protein (rs2071430) genotype (54%). The patients who had ≥3 of the protective genotypes from the myxovirus resistance protein, the suppressor of cytokine signaling 3 and osteopontin had a greater than 90% probability of achieving a sustained response (p<0.0001). The C/C IL28B genotype was present in 58.8% of the subjects in this group. The sustained virological response rates increased to 85.7% and 91.7% by analyzing C/C IL28B with the T/T osteopontin genotype at rs11730582 and the G/G suppressor of cytokine signaling 3 genotype, respectively. Genetic ancestry analysis revealed an admixed population. CONCLUSION: Hepatitis C genotype 1 patients who were responders to interferon-based therapy had a high frequency of multiple protective polymorphisms in the myxovirus resistance protein, osteopontin and suppressor of cytokine signaling 3 genes. The combined analysis of the suppressor of cytokine signaling 3 and IL28B genotypes more effectively predicted sustained virologic response than IL28B analysis alone.
Assuntos
Hepatite C Crônica/tratamento farmacológico , Interleucinas/genética , Proteínas de Resistência a Myxovirus/genética , Osteopontina/genética , Polimorfismo Genético/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Adulto , Antivirais/uso terapêutico , Feminino , Frequência do Gene , Marcadores Genéticos , Genótipo , Hepacivirus/efeitos dos fármacos , Humanos , Interferon alfa-2 , Interferon-alfa/uso terapêutico , Interferons , Masculino , Pessoa de Meia-Idade , Proteínas de Resistência a Myxovirus/efeitos dos fármacos , Osteopontina/efeitos dos fármacos , Polietilenoglicóis/uso terapêutico , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/uso terapêutico , Ribavirina/uso terapêutico , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/efeitos dos fármacos , Resultado do TratamentoRESUMO
OBJECTIVES: Suppressor of cytokine signaling 3, myxovirus resistance protein and osteopontin gene polymorphisms may influence the therapeutic response in patients with chronic hepatitis C, and an association with IL28 might increase the power to predict sustained virologic response. Our aims were to evaluate the association between myxovirus resistance protein, osteopontin and suppressor of cytokine signaling 3 gene polymorphisms in combination with IL28B and to assess the therapy response in hepatitis C patients treated with pegylated-interferon plus ribavirin. METHOD: Myxovirus resistance protein, osteopontin, suppressor of cytokine signaling 3 and IL28B polymorphisms were analyzed by PCR-restriction fragment length polymorphism, direct sequencing and real-time PCR. Ancestry was determined using genetic markers. RESULTS: We analyzed 181 individuals, including 52 who were sustained virologic responders. The protective genotype frequencies among the sustained virologic response group were as follows: the G/G suppressor of cytokine signaling 3 (rs4969170) (62.2%); T/T osteopontin (rs2853744) (60%); T/T osteopontin (rs11730582) (64.3%); and the G/T myxovirus resistance protein (rs2071430) genotype (54%). The patients who had ≥3 of the protective genotypes from the myxovirus resistance protein, the suppressor of cytokine signaling 3 and osteopontin had a greater than 90% probability of achieving a sustained response (p<0.0001). The C/C IL28B genotype was present in 58.8% of the subjects in this group. The sustained virological response rates increased to 85.7% and 91.7% by analyzing C/C IL28B with the T/T osteopontin genotype at rs11730582 and the G/G suppressor of cytokine signaling 3 genotype, respectively. Genetic ancestry analysis revealed an admixed population. CONCLUSION: Hepatitis C genotype 1 patients who were responders to interferon-based therapy had a high frequency of multiple protective polymorphisms in the myxovirus ...
Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Hepatite C Crônica/tratamento farmacológico , Interleucinas/genética , Proteínas de Resistência a Myxovirus/genética , Osteopontina/genética , Polimorfismo Genético/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Antivirais/uso terapêutico , Frequência do Gene , Marcadores Genéticos , Genótipo , Hepacivirus/efeitos dos fármacos , Interferon-alfa/uso terapêutico , Proteínas de Resistência a Myxovirus/efeitos dos fármacos , Osteopontina/efeitos dos fármacos , Valor Preditivo dos Testes , Polietilenoglicóis/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/uso terapêutico , Ribavirina/uso terapêutico , Proteínas Supressoras da Sinalização de Citocina/efeitos dos fármacos , Resultado do TratamentoRESUMO
BACKGROUND: Overexpression of type I interferon (IFN-I)-induced genes is a common feature of systemic lupus erythematosus (SLE) and its experimental models, but the participation of endogenous overproduction of IFN-I on it is not clear. To explore the possibility that abnormally increased IFN-I receptor (IFNAR) signaling could participate in IFN-I-induced gene overexpression of SLE, we examined the phosphorylation status of the IFNAR-associated signaling partners Jak1 and STAT2, and its relation with expression of its physiologic inhibitor SOCS1 and with plasma levels of IFNα and IFN-like activity. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood mononuclear cells (PBMC) from SLE patients with or without disease activity and healthy controls cultured in the presence or in the absence of IFNß were examined by immunoprecipitation and/or western blotting for expression of the two IFNAR chains, Jak1, Tyk2, and STAT2 and their phosphorylated forms. In SLE but not in healthy control PBMC, Jak1 and STAT2 were constitutively phosphorylated, even in the absence of disease activity (basal pJak1: controls vs. active SLE p<0.0001 and controls vs. inactive SLE p = 0.0006; basal pSTAT2: controls vs. active and inactive SLE p<0.0001). Although SOCS1 protein was slightly but significantly decreased in SLE in the absence or in the presence of IFNß (p = 0.0096 to p<0.0001), in SOCS1 mRNA levels were markedly decreased (p = 0.036 to p<0.0001). IFNß induced higher levels of the IFN-I-dependent MxA protein mRNA in SLE than in healthy controls, whereas the opposite was observed for SOCS1. Although there was no relation to increased serum IFNα, active SLE plasma could induce expression of IFN-dependent genes by normal PBMC. CONCLUSIONS/SIGNIFICANCE: These findings suggest that in some SLE patients IFN-I dependent gene expression could be the result of a low IFNAR signaling threshold.
Assuntos
Janus Quinase 1/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT2/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Células Cultivadas , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Interferon-alfa/sangue , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Pessoa de Meia-Idade , Proteínas de Resistência a Myxovirus , Fosforilação , Receptor de Interferon alfa e beta/metabolismo , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ativação Transcricional , Adulto JovemRESUMO
Interferons (IFNs) are potent biologically active proteins that are widely used as biopharmaceuticals, so their potency must be correctly identified. Usually, the biological activity is quantified by a bioassay based on its capacity to induce an antiviral state in target cells, but this type of assays is subject to virus manipulation-related issues and they show considerable intra- and inter-assay variability. In this work, we generated a reporter gene assay (RGA) supported on the WISH-Mx/eGFP reporter cell line to determine human type I IFN activity. WISH cells were stably transfected with the enhanced green fluorescent protein (eGFP) gene under the control of type I IFN-inducible Mx2 promoter. This system implies the use of a standardized cell line for human IFN-potency analysis such as WISH cells and the simultaneous use of the sensitive reporter gene eGFP, having also several advantages when compared to antiviral activity assays and other RGAs: it can determine the potency of hIFN-α and hIFN-ß using only one cell line showing the highest expression of eGFP after 28h and being only observed in cells treated with type I IFNs due to the specificity of the Mx promoter. It is a sensitive assay and it represents a safe alternative when compared with the conventional antiviral tests. The cell line showed the same sensitivity along 57 generations, allowing its use during two months of successive culture. The inter- and intra-assay coefficients of variation were lower than 20%, demonstrating its reproducibility. In addition, this reporter cell line can be used for the conventional antiviral assay, either for hIFN-α or hIFN-ß. In conclusion, we have developed an alternative reporter system for the analysis of type I IFNs, in which its performance make it a suitable candidate to replace or complement conventional bioassays that are currently employed to measure IFN potency.
Assuntos
Antivirais/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Citometria de Fluxo/métodos , Proteínas de Ligação ao GTP/genética , Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Proteínas de Resistência a Myxovirus , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Transfecção , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacosRESUMO
The Mx (myxovirus resistance) gene codes for a protein with antiviral activity. Non-synonymous G/A polymorphism at position 2032 of chicken Mx cDNA results in a change at amino acid 631 of the Mx protein. This mutation has been shown to affect the antiviral activity of the Mx molecule, although recent studies have not confirmed this effect in response to some influenza strains. Nevertheless, the G/A polymorphism could be important for the chicken's response to other viruses. A robust PCR-RFLP protocol for genotyping chicken Mx gene polymorphism associated with the S631N mutation was developed. The F primer anneals to the last intron of the Mx gene, and the R primer anneals to the last exon of the gene, with an expected PCR product of 299 bp. PCR products were digested with Hpy8I. This enzyme cuts the sequence 5'-GTN|NAC-3', 2 bp downstream of the Mx polymorphism for the G allele, whereas the fragment containing the A allele is not cleaved. One hundred and twenty-seven chickens (commercial broilers, White Leghorn and New Hampshire) were genotyped using this protocol, and genotyping data were validated by sequencing. Full identity of results between the two genotyping methods was observed for all 127 samples, proving the reliability and robustness of this PCR-RFLP protocol.