RESUMO
Cellular 5'-3' exoribonuclease 1 (XRN1) is best known for its role as a decay factor, which by degrading 5' monophosphate RNA after the decapping of DCP2 in P-bodies (PBs) in Drosophila, yeast, and mammals. XRN1 has been shown to degrade host antiviral mRNAs following the influenza A virus (IAV) PA-X-mediated exonucleolytic cleavage processes. However, the mechanistic details of how XRN1 facilitates influenza A virus replication remain unclear. In this study, we discovered that XRN1 and nonstructural protein 1 (NS1) of IAV are directly associated and colocalize in the PBs. Moreover, XRN1 downregulation impaired viral replication while the viral titers were significantly increased in cells overexpressing XRN1, which suggest that XRN1 is a positive regulator in IAV life cycle. We further demonstrated that the IAV growth curve could be suppressed by adenosine 3',5'-bisphosphate (pAp) treatment, an inhibitor of XRN1. In virus-infected XRN1 knockout cells, the phosphorylated interferon regulatory factor 3 (p-IRF3) protein, interferon beta (IFN-ß) mRNA, and interferon-stimulated genes (ISGs) were significantly increased, resulting in the enhancement of the host innate immune response and suppression of viral protein production. Our data suggest a novel mechanism by which the IAV hijacks the cellular XRN1 to suppress the host innate immune response and to facilitate viral replication. IMPORTANCE A novel mechanistic discovery reveals that the host decay factor XRN1 contributes to influenza A virus replication, which exploits XRN1 activity to inhibit RIG-I-mediated innate immune response. Here, we identified a novel interaction between viral NS1 and host XRN1. Knockdown and knockout of XRN1 expression in human cell lines significantly decreased virus replication while boosting RIG-I-mediated interferon immune response, suggesting that XRN1 facilitates influenza A virus replication. The pAp effect as XRN1 inhibitor was evaluated; we found that pAp was capable of suppressing viral growth. To our knowledge, this study shows for the first time that a negative-strand and nucleus-replicating RNA virus, as influenza A virus, can hijack cellular XRN1 to suppress the host RIG-I-dependent innate immune response. These findings provide new insights suggesting that host XRN1 plays a positive role in influenza A virus replication and that the inhibitor pAp may be used in novel antiviral drug development.
Assuntos
Exorribonucleases/genética , Exorribonucleases/imunologia , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Interferon beta/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Replicação Viral , Células A549 , Regulação para Baixo , Humanos , Imunidade Inata , Vírus da Influenza A/imunologia , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/imunologiaRESUMO
OBJECTIVE: To evaluate clinical associations of anti-PM/Scl antibodies in patients with SSc in a multicentre international cohort, with particular focus on unresolved issues, including scleroderma renal crisis (RC), malignancies, and functional outcome of interstitial lung disease (ILD). METHODS: (1) Analysis of SSc patients from the EUSTAR database: 144 anti-PM/Scl+ without SSc-specific autoantibodies were compared with 7202 anti-PM/Scl-, and then to 155 anti-Pm/Scl+ with SSc-specific antibodies. (2) Case-control study: additional data were collected for 165 anti-PM/Scl+ SSc patients (85 from the EUSTAR registry) and compared with 257 anti-PM/Scl- SSc controls, matched for sex, cutaneous subset, disease duration and age at SSc onset. RESULTS: Patients with isolated anti-PM/Scl+, as compared with anti-Pm/Scl-, had higher frequency of muscle involvement, ILD, calcinosis and cutaneous signs of DM, but similar frequency of SRC and malignancies (either synchronous with SSc onset or not). The presence of muscle involvement was associated with a more severe disease phenotype. Although very frequent, ILD had a better functional outcome in cases than in controls. In patients with both anti-PM/Scl and SSc-specific antibodies, a higher frequency of typical SSc features than in those with isolated anti-PM/Scl was observed. CONCLUSION: The analysis of the largest series of anti-PM/Scl+ SSc patients so far reported helps to delineate a specific clinical subset with muscle involvement, cutaneous DM, calcinosis and ILD characterized by a good functional outcome. SRC and malignancies do not seem to be part of this syndrome.
Assuntos
Exorribonucleases/imunologia , Complexo Multienzimático de Ribonucleases do Exossomo/imunologia , Sistema de Registros , Escleroderma Sistêmico/imunologia , Adulto , Autoanticorpos , Europa (Continente)/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/epidemiologiaRESUMO
A repertoire of T cells with diverse antigen receptors is selected in the thymus. However, detailed mechanisms underlying this thymic positive selection are not clear. Here we show that the CCR4-NOT complex limits expression of specific genes through deadenylation of mRNA poly(A) tails, enabling positive selection. Specifically, the CCR4-NOT complex is up-regulated in thymocytes before initiation of positive selection, where in turn, it inhibits up-regulation of pro-apoptotic Bbc3 and Dab2ip. Elimination of the CCR4-NOT complex permits up-regulation of Bbc3 during a later stage of positive selection, inducing thymocyte apoptosis. In addition, CCR4-NOT elimination up-regulates Dab2ip at an early stage of positive selection. Thus, CCR4-NOT might control thymocyte survival during two-distinct stages of positive selection by suppressing expression levels of pro-apoptotic molecules. Taken together, we propose a link between CCR4-NOT-mediated mRNA decay and T cell selection in the thymus.
Assuntos
Apoptose/genética , Exorribonucleases/genética , Proteínas Repressoras/genética , Timócitos/imunologia , Timo/imunologia , Animais , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Diferenciação Celular , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Exorribonucleases/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Poli A/genética , Poli A/imunologia , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Proteínas Repressoras/imunologia , Transdução de Sinais , Timócitos/citologia , Timo/citologia , Timo/crescimento & desenvolvimento , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/imunologiaRESUMO
Autoimmune connective tissue diseases, including dermatomyositis and systemic sclerosis, have a heterogeneous clinical presentation and prognosis; moreover, their clinical features are often incomplete and overlap with other rheumatic disorders, which can make diagnosis and prognostic stratification challenging. Specific autoantibodies have been associated with certain clinical findings as well as prognostic implications, and many new associations have been made over the last decade. Although patient populations manifest considerable heterogeneity, autoantibodies can be used to help predict clinical features, prognosis, and response to therapy. In this review, the clinical and prognostic implications associated with disease-specific autoantibodies in dermatomyositis and scleroderma are summarized with an emphasis on how the clinician can use this information for patient care.
Assuntos
Autoanticorpos/sangue , Dermatomiosite/sangue , Helicase IFIH1 Induzida por Interferon/imunologia , Escleroderma Sistêmico/sangue , Fatores de Transcrição/imunologia , Adenosina Trifosfatases/imunologia , Anticorpos Antinucleares/sangue , Anticorpos Antifosfolipídeos/sangue , DNA Topoisomerases/imunologia , Proteínas de Ligação a DNA/imunologia , RNA Polimerases Dirigidas por DNA/imunologia , Exorribonucleases/imunologia , Complexo Multienzimático de Ribonucleases do Exossomo/imunologia , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/imunologia , Fenótipo , Prognóstico , Ribonucleoproteínas Nucleolares Pequenas/imunologia , Enzimas Ativadoras de Ubiquitina/imunologiaRESUMO
OBJECTIVES: We aimed to identify the possible clinical and laboratory predictors of calcinosis in a cohort of patients with a diagnosis of polymyositis (PM) and dermatomyositis (DM). METHODS: We carried out a retrospective analysis of a cohort of myositis patients attending our clinic between January 2013 and May 2014. RESULTS: 74 patients (58 females, 16 males) with PM (30 cases), DM (30 cases), overlap syndrome (13 cases) and inclusion body myositis (1 case) were enrolled. Sixteen patients (21.6%) had calcinosis that occurred a mean of 43.7 months after diagnosis of PDM. At multivariate analysis, patients with calcinosis experienced longer follow-up duration (p=0.006), anti-PM/Scl (p=0.033) and anti-NXP2 (p=0.024) positivity compared to patients without calcinosis. Furthermore, anti-NXP-2 positive C+ showed a diffuse form of calcinosis from the beginning and lower frequency of respiratory tract involvement. No single drug or associations of drugs was found effective in the treatment of calcinosis. CONCLUSIONS: A longer follow-up period of time, DM diagnosis and positivity for PM/Scl and NXP-2 could all be considered risk factors which foresee the development of calcinosis. Moreover, the positivity for antibodies to NXP-2 depicts a distinct phenotype of calcinosis with an early onset and quick widespread dissemination.
Assuntos
Calcinose/etiologia , Dermatomiosite/complicações , Polimiosite/complicações , Adenosina Trifosfatases/imunologia , Adulto , Autoanticorpos/sangue , Biomarcadores/sangue , Calcinose/sangue , Calcinose/tratamento farmacológico , Calcinose/imunologia , Distribuição de Qui-Quadrado , Proteínas de Ligação a DNA/imunologia , Dermatomiosite/sangue , Dermatomiosite/tratamento farmacológico , Dermatomiosite/imunologia , Exorribonucleases/imunologia , Complexo Multienzimático de Ribonucleases do Exossomo/imunologia , Feminino , Humanos , Japão , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Polimiosite/sangue , Polimiosite/tratamento farmacológico , Polimiosite/imunologia , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVE: To determine the relationships between systemic sclerosis (SSc)-related autoantibodies, as well as their clinical associations, in a well-characterized Australian patient cohort. METHODS: Serum from 505 Australian SSc patients were analyzed with a commercial line immunoassay (EuroLine; Euroimmun) for autoantibodies to centromere proteins CENP-A and CENP-B, RNA polymerase III (RNAP III; epitopes 11 and 155), the 90-kd nucleolar protein NOR-90, fibrillarin, Th/To, PM/Scl-75, PM/Scl-100, Ku, topoisomerase I (topo I), tripartite motif-containing protein 21/Ro 52, and platelet-derived growth factor receptor. Patient subgroups were identified by hierarchical clustering of the first 2 dimensions of a principal components analysis of quantitative autoantibody scores. Results were compared with detailed clinical data. RESULTS: A total of 449 of the 505 patients were positive for at least 1 autoantibody by immunoblotting. Heatmap visualization of autoantibody scores, along with principal components analysis clustering, demonstrated strong, mutually exclusive relationships between CENP, RNAP III, and topo I. Five patient clusters were identified: CENP, RNAP III strong, RNAP III weak, topo I, and other. Clinical features associated with CENP, RNAP III, and topo I were consistent with previously published reports concerning limited cutaneous and diffuse cutaneous SSc. A novel finding was the statistical separation of RNAP III into 2 clusters. Patients in the RNAP III strong cluster had an increased risk of gastric antral vascular ectasia, but a lower risk of esophageal dysmotility. Patients in the other cluster were more likely to be male and to have a history of smoking and a history of malignancy, but were less likely to have telangiectasia, Raynaud's phenomenon, and joint contractures. CONCLUSION: Five major autoantibody clusters with specific clinical and serologic associations were identified in Australian SSc patients. Subclassification and disease stratification using autoantibodies may have clinical utility, particularly in early disease.
Assuntos
Autoanticorpos/imunologia , Escleroderma Sistêmico/imunologia , Idoso , Antígenos Nucleares/imunologia , Austrália , Autoantígenos/imunologia , Proteína Centromérica A , Proteína B de Centrômero/imunologia , Proteínas Cromossômicas não Histona/imunologia , Estudos de Coortes , Contratura/etiologia , Contratura/imunologia , DNA Topoisomerases Tipo I/imunologia , Proteínas de Ligação a DNA/imunologia , Transtornos da Motilidade Esofágica/etiologia , Transtornos da Motilidade Esofágica/imunologia , Exorribonucleases/imunologia , Complexo Multienzimático de Ribonucleases do Exossomo/imunologia , Feminino , Ectasia Vascular Gástrica Antral/etiologia , Ectasia Vascular Gástrica Antral/imunologia , Humanos , Immunoblotting , Autoantígeno Ku , Masculino , Pessoa de Meia-Idade , Neoplasias/epidemiologia , Proteínas Pol1 do Complexo de Iniciação de Transcrição/imunologia , Análise de Componente Principal , RNA Polimerase III/imunologia , Proteínas de Ligação a RNA/imunologia , Doença de Raynaud/etiologia , Doença de Raynaud/imunologia , Receptores do Fator de Crescimento Derivado de Plaquetas/imunologia , Ribonucleoproteínas/imunologia , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/epidemiologia , Fatores Sexuais , Fumar/epidemiologia , Telangiectasia/etiologia , Telangiectasia/imunologiaRESUMO
2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.
Assuntos
2',5'-Oligoadenilato Sintetase/imunologia , Antivirais/farmacologia , Endorribonucleases/imunologia , Exorribonucleases/química , Imunidade Inata , Bibliotecas de Moléculas Pequenas/farmacologia , 2',5'-Oligoadenilato Sintetase/genética , Nucleotídeos de Adenina/imunologia , Nucleotídeos de Adenina/metabolismo , Antivirais/síntese química , Cristalografia por Raios X , Vírus da Encefalomiocardite/genética , Vírus da Encefalomiocardite/metabolismo , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/genética , Exorribonucleases/imunologia , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HeLa , Humanos , Interferon-alfa/farmacologia , Modelos Moleculares , Oligorribonucleotídeos/imunologia , Oligorribonucleotídeos/metabolismo , Poli I-C/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/metabolismo , Rhinovirus/genética , Rhinovirus/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: Detection of systemic sclerosis-associated antibodies (SSc-Ab) in routine clinical practice is mostly restricted to anti-centromere and anti-Scl-70 antibodies. However, also other antinuclear antibodies have been shown to be valuable diagnostic and prognostic markers for the disease. The aim of this study was to evaluate the diagnostic performance of measuring the most prevalent SSc-Ab with fluoroenzymeimmunoassay (FEIA) as an alternative for the combined conventional techniques (CCT). METHODS: Sera from 144 consecutive systemic sclerosis (SSc) patients previously tested by CCT (indirect immunofluorescence on HEp-2000, western blotting, protein radio immunoprecipitation and a well-documented line immunoassay) and an additional group of 266 disease controls (80 rheumatoid arthritis, 58 systemic lupus erythematosus, 50 spondyloarthropathy, 48 osteoarthritis, 18 polymyalgia rheumatica and 12 ANCA-associated vasculitis) were retrospectively evaluated. Anti-centromere-B, anti-Scl-70, anti-RNA polymerase III and anti-PM/Scl antibodies were measured using FEIA. RESULTS: Using cut-off values corresponding with likelihood ratios larger than 10 FEIA obtained the following sensitivities: 45.1% for anti-centromere-B, 15.3% for anti-Scl-70, 5.6% for anti-RNA polymerase III and 2.1% for anti-PM/Scl. The overall agreement between combined conventional techniques and FEIA was good for all individual reactivities (kappa>0.800). The overall diagnostic sensitivity of 68.1% and diagnostic specificity of 98.1% were comparable to those obtained by CCT. CONCLUSIONS: FEIA testing for anti-centromere-B, anti-Scl-70, anti-RNA polymerase III and anti-PM/Scl-100 shows good performance and represents an accurate alternative for the time-consuming combined conventional techniques.
Assuntos
Anticorpos Antinucleares/sangue , Autoantígenos/imunologia , Fluorimunoensaio , Técnicas Imunoenzimáticas , Escleroderma Sistêmico/diagnóstico , Biomarcadores/sangue , Western Blotting , Proteína B de Centrômero/imunologia , DNA Topoisomerases Tipo I , Exorribonucleases/imunologia , Complexo Multienzimático de Ribonucleases do Exossomo/imunologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Proteínas Nucleares/imunologia , Valor Preditivo dos Testes , RNA Polimerase III/imunologia , Ensaio de Radioimunoprecipitação , Estudos Retrospectivos , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/imunologia , Sensibilidade e EspecificidadeRESUMO
Long before the RNA degrading exosome was first described in the yeast Saccharomyces cerevisiae, the use of autoantibodies found in the sera of certain autoimmune patients allowed the identification of a complex of polypeptides which later appeared to be the human exosome. Today, the most extensively documented association of the exosome with disease is still its targeting by the immune system of such patients. The highest frequency of autoantibodies to components of the exosome complex is found in polymyositis-scleroderma overlap patients and therefore the exosome is termed PM/Scl autoantigen in the autoimmune field. More recently, one of the core components of the exosome was identified as a protein associated with chronic myelogenous leukemia. In this chapter we will describe the identification of the PM/Scl autoantigen from a historical perspective, discuss our current knowledge on the occurrence of autoantibodies to exosome components in autoimmune diseases and end with the data that connect the exosome with cancer.
Assuntos
Exorribonucleases , Exossomos , Autoanticorpos/imunologia , Autoantígenos/imunologia , Exorribonucleases/imunologia , Complexo Multienzimático de Ribonucleases do Exossomo , Humanos , Escleroderma SistêmicoRESUMO
Long before the RNA degrading exosome was first described in the yeast Saccharomyces cerevisiae, the use of autoantibodies found in the sera of certain autoimmune patients allowed the identification of a complex of polypeptides which later appeared to be the human exosome. Today, the most extensively documented association of the exosome with disease is still its targeting by the immune system of such patients. The highest frequency of autoantibodies to components of the exosome complex is found in polymyositis-scleroderma overlap patients and therefore the exosome is termed PM/Scl autoantigen in the autoimmune field. More recently, one of the core components of the exosome was identified as a protein associated with chronic myelogenous leukemia. In this chapter we will describe the identification of the PM/Scl autoantigen from a historical perspective, discuss our current knowledge on the occurrence of autoantibodies to exosome components in autoimmune diseases and end with the data that connect the exosome with cancer.
Assuntos
Doenças Autoimunes , Exorribonucleases/imunologia , Exossomos/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Exorribonucleases/química , Complexo Multienzimático de Ribonucleases do Exossomo , Exossomos/química , Humanos , Neoplasias/imunologia , Proteínas Nucleares/imunologia , RNA/metabolismoRESUMO
BACKGROUND: To date, no series has analysed long-term outcome in patients with polymyositis/dermatomyositis (PM/DM) with anti-PM-Scl antibody. OBJECTIVES: The aims of the present study were: (i) to assess clinical features and long-term outcome, including organ complications, functional course and mortality rate, in patients with isolated PM/DM with anti-PM-Scl antibody; and (ii) to evaluate prevalence, characteristics and long-term outcome of interstitial lung disease (ILD) in patients with isolated PM/DM with anti-PM-Scl antibody. METHODS: The medical records of 20 consecutive patients with isolated PM/DM with anti-PM-Scl antibody were reviewed. RESULTS: Two patients (10%) achieved remission of PM/DM, whereas 14 (70%) improved and four (20%) had a worsened clinical status. Short-term recurrences (during tapering of therapy) occurred in nine patients and long-term recurrences (after discontinuation of therapy) in three patients. Moreover, patients with PM/DM with anti-PM-Scl antibody exhibited severe complications, as follows: oesophageal involvement (n = 4) requiring enteral feeding in three cases, ventilatory insufficiency (n = 3) requiring mechanical ventilation in two cases; three other patients had cancer. Interestingly, patients with PM/DM with anti-PM-Scl antibody often presented symptoms that are usually found in antisynthetase syndrome, i.e. hyperkeratotic rhagadiform hand symptoms (n = 2; 10%), Raynaud's phenomenon (n = 8; 40%), arthralgia/arthritis (n = 7; 35%) and ILD (n = 12; 60%). In our cohort, the associated ILD often required combined therapy of steroids and immunosuppressive agents. CONCLUSIONS: Our series suggests that the presence of anti-PM-Scl antibody is not a good prognostic factor in patients with PM/DM, as there appears to be an association with lung and oesophageal involvement; in addition, anti-PM-Scl antibody may coexist with malignancy in patients with PM/DM. Furthermore, anti-PM-Scl antibody-positive patients with PM/DM often exhibit 'mechanic's hands', Raynaud's phenomenon and joint involvement. Our latter findings raise the possibility that the immunogenetic background influences the autoantibody status of these patients; HLA-DR3 has, in fact, been found in association with antisynthetase syndrome antibodies and with anti-PM-Scl antibodies.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Dermatomiosite/imunologia , Exorribonucleases/imunologia , Imunossupressores/uso terapêutico , Proteínas Nucleares/imunologia , Adolescente , Adulto , Idoso , Anticorpos Anti-Idiotípicos/sangue , Biomarcadores/sangue , Dermatomiosite/complicações , Dermatomiosite/tratamento farmacológico , Quimioterapia Combinada , Exorribonucleases/sangue , Complexo Multienzimático de Ribonucleases do Exossomo , Feminino , Humanos , Doenças Pulmonares Intersticiais/etiologia , Doenças Pulmonares Intersticiais/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/sangue , Prognóstico , Esteroides/uso terapêutico , Fatores de Tempo , Adulto JovemRESUMO
A characteristic serological feature of patients suffering from the overlap polymyositis and scleroderma (PM/Scl) syndrome are antibodies to the human counterpart of the yeast exosome referred to as the PM/Scl complex. Historically, the detection of anti-PM/Scl antibodies was laborious and relied largely on indirect immunofluorescence and immunodiffusion techniques. In 1992 the major autoantigen PM/Scl-100 was identified and cloned. Subsequently, the major epitopes were mapped and one of these, termed PM1-Alpha, became the antigen for a novel ELISA exhibiting high sensitivity and specificity for the detection of anti-PM/Scl antibodies. Comparative studies with other methods using other PM/Scl autoantigens have shown that the PM1-Alpha ELISA has higher sensitivity and specificity than assays that employed recombinant PM/Scl-75c and PM/Scl-100. Anti-PM1-Alpha antibodies were identified in 55.0% of sera from PM/Scl overlap syndrome patients, but were also seen in 7.9% of SSc and in 7.5% of PM patients. The frequency in other systemic autoimmune diseases and in infectious diseases was significant lower. In summary, the data derived from individual studies suggest that PM1-Alpha may become the "gold standard" for the detection of anti-PM/Scl antibodies.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Exorribonucleases/imunologia , Epitopos Imunodominantes/imunologia , Proteínas Nucleares/imunologia , Polimiosite/diagnóstico , Escleroderma Sistêmico/diagnóstico , Autoanticorpos/imunologia , Autoantígenos/imunologia , Ensaio de Imunoadsorção Enzimática/tendências , Exorribonucleases/química , Complexo Multienzimático de Ribonucleases do Exossomo , Humanos , Epitopos Imunodominantes/química , Metanálise como Assunto , Proteínas Nucleares/química , Especificidade de Órgãos , Peptídeos/química , Peptídeos/imunologia , Polimiosite/complicações , Polimiosite/imunologia , Curva ROC , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/imunologia , Sensibilidade e EspecificidadeRESUMO
Chronic myelogenous leukemia (CML) 28 is a broadly immunogenic antigen that can have an antileukemia effect. We adopted the SYFPEITHI database to predict human leukocyte antigen-A2 restricted CML28 peptide. We designed artificial antigen-presenting cells (aAPCs) by coating micro beads with human leukocyte antigen-A2-immunoglobulin dimer and CD28-specific antibody. We used the selected peptides-pulsed aAPCs to induce cytotoxic T lymphocytes (CTLs), to choose the CML28 peptide, which was best for inducing CTLs. The result showed that the peptides-pulsed aAPCs could induce CTLs and that the peptide VLTFALDSV was the best choice for significantly inducing specific CTLs.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Exorribonucleases/imunologia , Antígeno HLA-A2/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Epitopos/imunologia , Complexo Multienzimático de Ribonucleases do Exossomo , Antígeno HLA-A2/química , Humanos , Proteínas de Ligação a RNARESUMO
Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyte antigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleated cells. The identification of novel, tumor-specific epitopes is a critical step in the development of immunotherapeutics for breast cancer. To directly identify peptide epitopes unique to cancerous cells, secreted human class I HLA molecules (sHLA) were constructed by deletion of the transmembrane and cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed in breast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, nontumorigenic cell line MCF10A. Stable transfectants were seeded into bioreactors for production of > 25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy. Comparative analysis of HLA-A*0201 peptides revealed 5 previously uncharacterized epitopes uniquely presented on breast cancer cells. These peptides were derived from intracellular proteins with either well-defined or putative roles in breast cancer development and progression: Cyclin Dependent Kinase 2 (Cdk2), Ornithine Decarboxylase (ODC1), Kinetochore Associated 2 (KNTC2 or HEC1), Macrophage Migration Inhibitory Factor (MIF), and Exosome Component 6 (EXOSC6). Cellular recognition of the MIF, KNTC2, EXOSC6, and Cdk2 peptides by circulating CD8+ cells was demonstrated by tetramer staining and IFN-gamma ELISPOT. The identification and characterization of peptides unique to the class I of breast cancer cells provide putative targets for the development of immune diagnostic tools and therapeutics.
Assuntos
Neoplasias da Mama/imunologia , Epitopos de Linfócito T/análise , Antígenos HLA-A/imunologia , Sequência de Aminoácidos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Quinase 2 Dependente de Ciclina/imunologia , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas do Citoesqueleto , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/isolamento & purificação , Exorribonucleases/imunologia , Exorribonucleases/metabolismo , Feminino , Antígenos HLA-A/química , Antígeno HLA-A2 , Humanos , Interferon gama/metabolismo , Oxirredutases Intramoleculares/imunologia , Oxirredutases Intramoleculares/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Fatores Inibidores da Migração de Macrófagos/imunologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Espectrometria de Massas , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Ornitina Descarboxilase/imunologia , Ornitina Descarboxilase/metabolismoRESUMO
Induction of anti-tumor immune responses by dendritic cells (DCs) transduced with a recombinant adeno-associated virus type 2 (rAAV2) encoding tumor antigens is considered a promising approach for cancer vaccine development. CML28, a novel antigen with the properties of cancer/ testis (CT) antigens, is an attractive target for antigen-specific immunotherapy. Here we investigated the feasibility of inducing CML28-specific cytotoxic T lymphocyte (CTL) responses using DCs transduced with the rAAV2 vectors containing the CML28 gene (rAAV/CML28). Using an adenovirus-free packaging system, rAAV/CML28 was generated. The transduction efficiency of rAAV/CML28 in DCs increased in a multiplicity of infection (MOI)-dependent manner. The rAAV/CML28 transduction did not impair DC maturation, but even enhanced the CD80 expression. The rAAV/CML28-transduced DCs induced CML28-specific CTLs which exhibited a MHC class I-mediated antigen-specific lytic activity against CML28-bearing tumor cell lines (HepG2 and MCF-7) as well as the primary leukemia blasts. These findings suggest that rAAV/CML28-transduced DCs vaccine may serve as a feasible approach for the treatment of CML28-associated cancers.
Assuntos
Adenoviridae/genética , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Células Dendríticas/imunologia , Exorribonucleases/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Antígeno B7-1/imunologia , Linhagem Celular Tumoral , Células Dendríticas/metabolismo , Exorribonucleases/biossíntese , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo , Estudos de Viabilidade , Genes MHC Classe I , Humanos , Ativação Linfocitária , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNARESUMO
CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Células Dendríticas/imunologia , Exorribonucleases/genética , Exorribonucleases/imunologia , Proteínas Supressoras da Sinalização de Citocina/genética , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/administração & dosagem , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Complexo Multienzimático de Ribonucleases do Exossomo , Inativação Gênica , Vetores Genéticos/genética , Humanos , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Proteína 1 Supressora da Sinalização de Citocina , Linfócitos T Citotóxicos/efeitos dos fármacos , Transfecção/métodosRESUMO
Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. CML28, a recently discovered cancer-testis (CT) antigen from chronic myelogenous leukemia, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A*0201 is one of the most common histocompatibility molecule in Chinese, we aim at identifying CML28 peptides presented by HLA-A*0201. A panel of CML28-derived antigenic peptides was predicted using a computer-based program. Four peptides with highest predicted score were synthesized and tested for their binding affinities to HLA-A*0201 molecule. Then these peptides were assessed for their immunogenicity to elicit specific immune responses mediated by CTLs both in vitro, from PBMCs sourced from four healthy HLA-A*0201(+) donors, and in vivo, in HLA-A*0201 transgenic mice. One of the tested peptides, CML28((173-181)), induced peptide-specific CTLs in vitro as well as in vivo, which could specifically secrete IFN-gamma and lyse major histocompatibility complex (MHC)-matched tumor cell lines endogenously expressing CML28 antigen and CML28((173-181) )pulsed Jurkat-A2/Kb cells, respectively. These results demonstrate that CML28((173-181) )is a naturally processed and presented CTL epitope with HLA-A*0201 motif and has a promising immunogenicity both in vitro and in vivo. As CML28 is expressed in a large variety of histological tumors besides chronic myelogenous leukemia, we propose that the newly identified epitope, CML28((173-181)), would be of potential use in peptide-based, cancer-specific immunotherapy against a broad spectrum of tumors.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Epitopos de Linfócito T/imunologia , Exorribonucleases/imunologia , Antígenos HLA-A/metabolismo , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/farmacologia , Antígenos de Superfície/genética , Antígenos de Superfície/farmacologia , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Exorribonucleases/genética , Exorribonucleases/farmacologia , Complexo Multienzimático de Ribonucleases do Exossomo , Antígeno HLA-A2 , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Neoplasias/imunologia , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/farmacologia , Proteínas de Ligação a RNA , Linfócitos T Citotóxicos/efeitos dos fármacosRESUMO
This study was aimed to construct nucleic acid vaccine containing the coding region of the CML28 gene and to express it in human dendritic cells. The full length of CML28 cDNA was amplified from K562 by RT-PCR and subcloned into pGEM-T vector. The CML28 fragment was digested and subsequently inserted into the EcoRI-Xba I sites of pcDNA3.1HisA to construct the recombinant expression vector pcDNA3.1HisA-CML28, which was identified by restrition analysis and sequencing. Human dendritic cells (DC) were separated from peripheral blood mononuclear cells (PBMC) by culture with rhGM-CSF, rhIL-4 and assessed by flow cytometry. The constructed plasmid pcDNA3.1 HisA-CML28 was transfected into DC by electroporation. Western blot was used to detect the expression of fusion protein His-CML28. The results showed that recombinant plasmid pcDNA3.1HisA-CML28 contained the correct full CML28 cDNA identified by restriction analysis and sequencing, and can express the fusion protein His-CML28 in DCs. It is concluded that nucleic acid vaccine containing CML28 gene was constructed and expressed in DC successfully.