RESUMO
Zika virus (ZIKV) is a major public health concern in the Americas. We report that ZIKV infection and RNA extracted from ZIKV infected cells potently activated the induction of type I interferons (IFNs). This effect was fully dependent on the mitochondrial antiviral signaling protein (MAVS), implicating RIG-I-like receptors (RLRs) as upstream sensors of viral RNA. Indeed, RIG-I and the related RNA sensor MDA5 contributed to type I IFN induction in response to RNA from infected cells. We found that ZIKV NS5 from a recent Brazilian isolate blocked type I IFN induction downstream of RLRs and also inhibited type I IFN receptor (IFNAR) signaling. We defined the ZIKV NS5 nuclear localization signal and report that NS5 nuclear localization was not required for inhibition of signaling downstream of IFNAR. Mechanistically, NS5 blocked IFNAR signaling by both leading to reduced levels of STAT2 and by blocking phosphorylation of STAT1, two transcription factors activated by type I IFNs. Taken together, our observations suggest that ZIKV infection induces a type I IFN response via RLRs and that ZIKV interferes with this response by blocking signaling downstream of RLRs and IFNAR.
Assuntos
Proteína DEAD-box 58/imunologia , Interferon Tipo I/metabolismo , RNA/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular , Brasil , Proteína DEAD-box 58/genética , Regulação para Baixo , Células HEK293 , Humanos , Interferon Tipo I/genética , Fosforilação , Receptores Imunológicos , Transdução de Sinais , Replicação Viral , Zika virus , Infecção por Zika virusRESUMO
The objective of this study was to identify and construct a human natural phage single-chain antibody (scFv) library of human anaplastic thyroid carcinoma (ATC) using phage display technology. Total RNA was extracted from lymphatic tissue near an ATC and used to amplify variable heavy chain (VH) and variable light chain (VL) fragments with added linker sequences using reverse transcription-polymerase chain reaction (RT-PCR). After purification, the VH and VL amplicons were used to produce scFv fragments with added SfiI and NotI restriction enzyme recognition sites using splicing-overlap-extension PCR. Following digestion, the scFv gene was cloned in the pCANTAB-5E plasmid, and the recombinant phagemids were transformed into the susceptible Escherichia coli TG1 strain. After infection by the helper phage M13K07, a human ATC phage antibody library was successfully constructed. Clear 28 S and 18 S bands could be seen in the total RNA from the library, and the sizes of the VH, VL, and scFv genes contained therein were approximately 370, 350, and 750 bp, respectively. In addition, the conversion efficiency as measured by the pUC19 standard plasmid was 10(8) CFU/µg, and the positive insert ratio was 86.4% (19/22). These results demonstrated the successful construction of a human ATC scFv antibody gene library, and might provide the experimental basis for the further screening and identification of a phage single-chain antibody with ATC cell-specificity.
Assuntos
Cadeias Pesadas de Imunoglobulinas/imunologia , Anticorpos de Cadeia Única/genética , Carcinoma Anaplásico da Tireoide/imunologia , Bacteriófagos/genética , Técnicas de Visualização da Superfície Celular , Escherichia coli , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Tecido Linfoide/imunologia , RNA/genética , RNA/imunologia , Anticorpos de Cadeia Única/imunologia , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/patologiaRESUMO
The recognition of pathogens is assigned to an evolutionarily conserved family of receptors, the Toll-like receptors (TLRs). The investigation of RNA-based immunology has been reinvigorated with the observation that TLR3s interact with RNA (dsRNA of viral origin, poly (I:C) and endogenous RNA). Many RNAs, therefore, join the list of endogenous ligands for TLRs. The further finding that nucleoside modification alters RNA-mediated TLR signaling presents a mechanism for the long-observed differences in immunogenicity. The involvement of RNA modification in the pathogenesis of diseases, and its implications in the therapeutics, are still being studied, and will have important implications in the future.
Assuntos
Membrana Celular/metabolismo , Endossomos/metabolismo , Doenças do Sistema Imunitário/genética , RNA Viral/metabolismo , RNA/metabolismo , Receptor 3 Toll-Like/imunologia , Animais , Humanos , Imunidade Inata/genética , Inflamação , Mutação/genética , RNA/imunologia , RNA Viral/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
La determinación de carga viral plasmática es utilizada para el seguimiento y monitoreo de pacientes infectados por el HIV-1. El objetivo del trabajo es evaluar la performance de PCR en tiempo real COBAS TaqMan 48 HIV-1 RNA en comparación con COBAS AMPLICOR HIV-1 MONITOR versión 1.5 para la cuantificación de RNA viral.(AU)
The quantification of HIV-1 viral load in plasma is a useful tool for the manafement of infected patients. Objective: to evaluate the performance of the COBAS TaquMan 48 HIV-1 real-time PCR in comparasion with the COBAS AMPLICOR HIV-1 MONITOR version 1.5 assay for quantification of viral load.(AU)
Assuntos
Humanos , Carga Viral , Monitorização Imunológica , HIV/patogenicidade , RNA/imunologia , Estudos de Validação como Assunto , Plasma , Coleta de Amostras Sanguíneas/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da PolimeraseRESUMO
This study was conducted to evaluate the immunogenicity of a DNA or RNA vaccines encoding Brucella abortus Cu-Zn superoxide dismutase (SOD) in cattle. Intramuscular injection of plasmid DNA carrying Brucella SOD gene (pcDNA-SOD) into animals elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD IgG antibody with predominance of immunoglobulin G1 (IgG1) isotype over IgG2. In addition, the DNA vaccine elicited a specific T-cell-proliferative response. Furthermore, intraperitoneal injection of cattle with recombinant Semliki Forest virus particles carrying recombinant RNA encoding SOD (SFV-SOD) did not lead to the induction of SOD IgG 1 or 2 antibody, but induced specific T-cell activation. Both vaccines were able to induce a non-significant secretion of gamma interferon and did not induce the secretion of IL-4 or tumor necrosis factor (TNF)-alpha. These results suggest that SOD gene in a genetic vaccine formulation (DNA or RNA) might be of potential us as a vaccine to induce cell-mediated immunity in cattle. To our knowledge, this is the first study to evaluate a genetic vaccine against Brucella in cattle.
Assuntos
Vacina contra Brucelose/imunologia , Brucella abortus/imunologia , Brucelose Bovina/prevenção & controle , RNA/imunologia , Superóxido Dismutase/imunologia , Vacinas de DNA/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Antibacterianos/biossíntese , Formação de Anticorpos , Vacina contra Brucelose/administração & dosagem , Brucella abortus/enzimologia , Brucella abortus/genética , Bovinos , Feminino , Imunidade Celular , Distribuição Aleatória , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Vacinas de DNA/administração & dosagemRESUMO
We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections.
Assuntos
Vacina contra Brucelose/imunologia , Brucella abortus/imunologia , Brucelose/prevenção & controle , RNA/imunologia , Vírus da Floresta de Semliki/genética , Superóxido Dismutase/imunologia , Vacinas Sintéticas/imunologia , Vírion/genética , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Superóxido Dismutase/genética , Linfócitos T Citotóxicos/imunologia , VacinaçãoRESUMO
Exogenous RNA molecules can be incorporated into eukaryotic cells and can exert a variety of biological effects. We have previously showed that exogenous RNAs obtained from lymphoid organs of animals immunized with synthetic peptides of HIV-1 are able to induce cell-mediated immune responses. In this study, animals were immunized with a synthetic peptide (pol: 476-484) of HIV-1, referred to as p9, which is a cytotoxic T lymphocyte (CTL) epitope. The RNA extracted from the lymphoid organs of animals immunized with p9 was termed p9-RNA. We have demonstrated that p9-RNA is active in inducing human CTL. The p9-RNA was also able to activate the RNA-dependent protein kinase (PKR) of human lymphocytes. The polyA(+) p9-RNA was the fraction responsible for the activation of this protein kinase. We also found that p9-RNA activates the transcription factor nuclear kappa B (NF-kappaB) by inducing the degradation of its inhibitor I-kappaB. Thus, these findings suggest that p9-RNA may act as a regulatory RNA and that the induction of CTL activity by p9-RNA could be mediated by PKR through NF-kappaB activation. It is known that CTL activity plays an important role in host defense against HIV-1 infection. Elucidating the molecular mechanism of p9-RNA could contribute to determining the basis for the use of p9-RNA as an immunomodulator in HIV-infected patients.
Assuntos
Antígenos HIV/imunologia , RNA/imunologia , Linfócitos T Citotóxicos/enzimologia , Linfócitos T Citotóxicos/imunologia , eIF-2 Quinase/metabolismo , Animais , Western Blotting , Ativação Enzimática , Epitopos de Linfócito T/imunologia , Cobaias , Humanos , Camundongos , Camundongos Endogâmicos , NF-kappa B/metabolismo , Peptídeos/síntese química , Peptídeos/imunologia , Fosforilação , RNA/isolamento & purificaçãoRESUMO
It is known that exogenous RNA molecules can be taken up by eukaryotic cells and can exert a variety of biological effects both in vitro and in vivo. The modulation of human lymphocytes by exogenous RNAs has medical implications. The exogenous RNA used in this study was obtained from lymphoid organs of animals immunized with the synthetic peptide p12 of HIV-1 and was referred to as p12-RNA. Human lymphocytes were transfected with the p12-RNA and the transfer of immunoreactivity of p12 was assessed by the lymphocyte proliferation and the leukocyte adherence inhibition assays. Our results indicate that the transfer of cellular immune response to the p12 occurred in 9 donors (60%) who were named responsive individuals whereas 6 donors (40%) were non-responsives. We also found that the calcium phosphate-mediated RNA uptake method is effective in converting non-responsive into responsive donors. The calcium phosphate-mediated RNA uptake may also be used to increase the efficiency of RNA transfection in other models with medical implications and to contribute to a better understanding of the molecular events involved in the uptake of RNA. Our findings give support for the use of exogenous RNAs obtained from lymphoid organs of immunized animals with synthetic peptides of HIV-1 in the immune reconstitution of individuals infected with HIV-1.
Assuntos
Fosfatos de Cálcio/farmacologia , Proteína gp160 do Envelope de HIV/imunologia , HIV-1/imunologia , Linfócitos/imunologia , Oligopeptídeos/imunologia , RNA/imunologia , Animais , Células Cultivadas , Cobaias , Proteína gp160 do Envelope de HIV/genética , HIV-1/genética , Humanos , Imunidade Celular , Imunização Secundária , Teste de Inibição de Aderência Leucocítica , Ativação Linfocitária , Linfócitos/efeitos dos fármacos , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/genética , Transfecção , Resultado do TratamentoRESUMO
Immunoprecipitation analysis of total HeLa cells RNA extract byprotein A-Sheparose purified autoantibodies and pCp 32P-3' end labeling RNAs revealed that U1, U2, U4 and U5 snRNAs are related with anti-Sm or U1nRNP autoantibodies, while the hY1, hY3, hY4 and hY5 scRNAs were related to anti-SSA/Ro autoantibodies present in sera of patient with Systemic Lupus Erythematosus. The authors detected molecular snRNAs and scRNAs specificities by autoantibodies in 71 sera, the molecular RNA specificity for anti-Sm (U1, U2, U4 and U5 snRNAs) was present in 39 percent; anti-SSA/Ro sera reacted against scRNAs (hY1, hY3, hY4 and hY5) in 36 percent, then anti-U1nRNP sera recognized U1 snRNA in 13 percent of sera and anti-rRNP related with rRNA were recognized in 8 percent. Twenty-nine SLE sera were RNA negative. A molecular characterization of the autoantibodies in sera from SLE patients may be a useful tool for clinical and laboratory diagnosis of SLE, and the use of autoantibodies es molecular probes allows to continue exploring some basic mechanism of gene expression