RESUMO
The SENS-IS test protocol for the in vitro detection of sensitizers is based on a reconstructed human skin model (Episkin) as the test system and on the analysis of the expression of a large panel of genes. Its excellent performance was initially demonstrated with a limited set of test chemicals. Further studies (described here) were organized to confirm these preliminary results and to obtain a detailed statistical analysis of the predictive capacity of the assay. A ring-study was thus organized and performed within three laboratories, using a test set of 19 blind coded chemicals. Data analysis indicated that the assay is robust, easily transferable and offers high predictivity and excellent within- and between-laboratories reproducibility. To further evaluate the predictivity of the test protocol according to Cooper statistics a comprehensive test set of 150 chemicals was then analyzed. Again, data analysis confirmed the excellent capacity of the SENS-IS assay for predicting both hazard and potency characteristics, confirming that this assay should be considered as a serious alternative to the available in vivo sensitization tests.
Assuntos
Alérgenos/toxicidade , Alternativas aos Testes com Animais , Dermatite de Contato , Epiderme/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Laboratórios , Modelos Biológicos , Reprodutibilidade dos TestesRESUMO
Analysis of genes modulated during the sensitization process either on mice (LLNA) or human (blisters) combined with data mining has allowed the definition of a comprehensive panel of sensitization biomarkers. This set of genes includes already identified markers such as the ARE family and others not yet associated with the sensitization process (the so-called SENS-IS gene subset). The expression of this set of genes has been measured on reconstituted human epidermis models (Episkin) exposed to various sensitizers and non-sensitizers. Fine analysis of their expression pattern indicates that it is the number of modulated genes rather than the intensity of up-regulation that correlates best with the sensitization potential of a chemical. Moreover, sensitizers that are weak inductors of ARE genes tend to be relevant modulators of the SENS-IS subset. By combining the expression data obtained with both gene subsets, it is now possible to identify a wide variety of sensitizers on a test system (in vitro reconstructed human epidermis) that is very similar to the in vivo situation and compatible with a large variety of test substance characteristics.
Assuntos
Expressão Gênica/efeitos dos fármacos , Irritantes/toxicidade , Testes de Irritação da Pele/métodos , Pele/efeitos dos fármacos , Alternativas aos Testes com Animais , Animais , Biomarcadores , Cisteína/química , Epiderme/efeitos dos fármacos , Marcadores Genéticos , Humanos , Camundongos , ToxicogenéticaRESUMO
Epstein-Barr Virus (EBV) is present in the neoplastic cells of around 20-30% of patients with Hodgkin Lymphoma (HL). Although, an immunosuppressive environment is currently described in HL patients, little is known concerning the regulatory mechanism induced by EBV proteins expression in tumour cells. This study aimed to investigate an association between regulatory Type 1 cells (Tr1) and EBV tissue positivity in HL patients. Transcriptomic analysis of both EBV-positive and EBV-negative tumours showed that EBV infection increased gene expression of Tr1-related markers (ITGA2, ITGB2, LAG3) and associated-immunosuppressive cytokines (IL10). This up-regulation was associated with an over-expression of several chemokine markers known to attract T-helper type 2 (Th2) and regulatory T cells thus contributing to immune suppression. This Tr1 cells recruitment in EBV-positive HL was confirmed by immunohistochemical analysis of frozen nodes biopsies and by flow cytometric analysis of peripheral blood mononuclear cells of EBV-positive patients. Additionally, we showed that IL10 production was significantly enhanced in tumours and blood of EBV-positive HL patients. Our results propose a new model in which EBV can recruit Tr1 cells to the nodes' microenvironment, suggesting that the expression of EBV proteins in tumour cells could enable the escape of EBV-infected tumour cells from the virus-specific CTL response.
Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Doença de Hodgkin/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Idoso , Biomarcadores/metabolismo , Quimiocinas/metabolismo , Criança , Citocinas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Linfócitos T Reguladores/virologia , Células Th2/imunologia , Células Th2/virologia , Regulação para Cima , Adulto JovemRESUMO
Hepatitis C virus (HCV) is an important causative agent of liver disease, but factors that determine the resolution or progression of infection are poorly understood. In this study, we suggested that existence of immunosuppressive mechanisms, supported by regulatory T cells and especially the regulatory T cell 1 subset (Tr1), may explain the impaired immune response during infection and thus the fibrosis aggravation to hepatocellular carcinoma (HCC). Using quantitative real-time PCR, we investigated the intra-hepatic presence of Tr1 cells in biopsies from a genotype 1b infected patient followed for an 18-year period from cirrhosis to HCC. We described a significant increase of gene expression in particular for the cytokines IL-10, TGF-ß, and their receptors that were perfectly correlated with an increased expression of the Tr1 specific markers (combined expression of CD4, CD18, and CD49b). This was strongly marked since the patient evolved in the pathology and could explain the failure of the treatment. In conclusion, evidence of regulatory T cell installation in the liver of chronically infected patient with cirrhosis and HCC suggests for the first time a key role for these cells in the course of HCV infection.
RESUMO
Although interleukin (IL)-7 is mostly known as a key regulator of lymphocyte homeostasis, we recently demonstrated that it also contributes to body weight regulation through a hypothalamic control. Previous studies have shown that IL-7 is produced by the human obese white adipose tissue (WAT) yet its potential role on WAT development and function in obesity remains unknown. Here, we first show that transgenic mice overexpressing IL-7 have reduced adipose tissue mass associated with glucose and insulin resistance. Moreover, in the high-fat diet (HFD)-induced obesity model, a single administration of IL-7 to C57BL/6 mice is sufficient to prevent HFD-induced WAT mass increase and glucose intolerance. This metabolic protective effect is accompanied by a significant decreased inflammation in WAT. In lymphocyte-deficient HFD-fed SCID mice, IL-7 injection still protects from WAT mass gain. However, IL-7-triggered resistance against WAT inflammation and glucose intolerance is lost in SCID mice. These results suggest that IL-7 regulates adipose tissue mass through a lymphocyte-independent mechanism while its protective role on glucose homeostasis would be relayed by immune cells that participate to WAT inflammation. Our observations establish a key role for IL-7 in the complex mechanisms by which immune mediators modulate metabolic functions.
Assuntos
Tecido Adiposo Branco/patologia , Dieta Hiperlipídica , Comportamento Alimentar , Resistência à Insulina , Interleucina-7/metabolismo , Linfócitos/metabolismo , Animais , Comportamento Alimentar/efeitos dos fármacos , Feminino , Intolerância à Glucose/complicações , Intolerância à Glucose/patologia , Intolerância à Glucose/prevenção & controle , Humanos , Inflamação/complicações , Interleucina-7/administração & dosagem , Interleucina-7/farmacologia , Linfócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Obesidade/complicações , Obesidade/patologia , Obesidade/prevenção & controle , Tamanho do Órgão/efeitos dos fármacos , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacologia , Receptores de Interleucina-7/metabolismo , Células EstromaisRESUMO
Epstein-Barr virus (EBV) is associated with several malignant diseases that can be distinguished by their patterns of viral latent gene expression. We developed here an original peptidic approach to favor the induction of a specific CD4+ T-cell response against EBV latency II malignancies (Hodgkin's lymphoma, nasopharyngeal carcinoma, T/NK lymphoma). Previously, we selected 6 peptides derived from EBV nuclear antigen-1, latency membrane proteins (LMP)-1, and LMP-2 highly promiscuous for major histocompatibility complex class II molecules and showed their ability to induce interferon-γ-secreting CD4+ T cells. We confirmed here that all peptides used in cocktail are recognized by human CD4+ memory T cells from healthy donors, inducing a broad T-helper (Th)1 cytokine secretion interferon-γ, interleukin-2. Furthermore, we have generated EBV-specific CD4+ T-cell lines and proved their cytotoxic potential, not only on original models expressing latency II antigens (EBV-transformed T cell or monocyte), but also on lymphoblastoid cell lines expressing latency III antigens (lymphoblastoid cell lines). In addition, granzyme B enzyme-linked immunospot assays suggested that a part of this specific cytotoxic activity could be linked to the granule lytic pathway. Very importantly, we have showed that neither phenotypical changes nor functional activities of CD4+CD25+CD127(low)-regulatory T cells were observed in response to EBV+ peptides, avoiding any risk of aggravation of the preexisting immunosuppressive environment reported in EBV-associated malignancies. In conclusion, our promiscuous peptide cocktail could be used safely in immunotherapeutic approaches against EBV latency II malignancies, mainly to prevent relapse in high-risk patients further to classic treatments.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Citocinas/biossíntese , Citotoxicidade Imunológica , Granzimas/metabolismo , Humanos , Memória Imunológica , Peptídeos/química , Células Th1/imunologia , Células Th1/metabolismoRESUMO
Alternative, non-invasive techniques are necessary to monitor the progression of liver disease during chronic hepatitis C. Firstly, because serum is the most accessible material for studies using qPCR in microplates, gene transcription was compared in 219 selected genes involved in the pathogenesis of hepatitis C virus (HCV) infection between sera, PBMCs and liver samples collected simultaneously from five patients infected chronically. Secondly, using sera, gene profiles were compared between HCV-infected patients (n = 10) and healthy controls (n = 10). In addition, the influence of alcohol intake was examined in patients infected with HCV genotype-1. Firstly, amplifiable mRNAs were obtained in all samples. After amplification, significant correlations were observed between: liver versus serum; liver versus PBMCs; and serum versus PBMCs (r(2) = 0.37, r(2) = 0.54, r(2) = 0.49, respectively). A comparison of gene transcription by gene involved in T- and B-cell markers, adhesion molecules, apoptosis, liver matrix turnover and inflammation, revealed comparable, significant correlations between serum and liver, (r(2) = 0.30, r(2) = 0.60, r(2) = 0.51, r(2) = 0.51, r(2) = 0.26, and r(2) = 0.61 respectively). Secondly, a quantitative analysis of gene expression in sera between genotype-1b-infected patients and healthy controls revealed that 41 genes involved closely in T-cell activation and apoptosis were over-expressed significantly in patients infected with HCV. In these patients, alcohol consumption was associated with an increased expression of six genes involved in the inflammatory response, together with a decrease of genes associated with dendritic cell function. It is concluded that in patients infected with HCV, serum can be used to evaluate expression of liver genes. Further prospective studies are clearly needed to validate the initial results and to define the relevant genes.
Assuntos
Perfilação da Expressão Gênica , Hepacivirus/fisiologia , Hepatite C Crônica/patologia , Interações Hospedeiro-Patógeno , Adulto , Feminino , Humanos , Leucócitos/química , Fígado/química , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Soro/química , Estatística como AssuntoRESUMO
Hepatitis C virus (HCV) becomes chronic in about 85 % of infected individuals, whereas only 15 % of infected people clear spontaneously the virus. The progression of hepatitis C to chronic status is associated to a profound down-regulation of CD4 and CD8 multispecific immune response. This immune defect may participate to the immune tolerance of VHC and consequently to its persistence. Recent findings indicate that T regulatory cells as Tr1 play an inhibitory role on T helper responses notably in the context of auto-immune or inflammatory disorders. The existence of immunosuppressive mechanisms supported by Tr1 lymphocytes and their IL-10 production represent an attractive hypothesis. We have previously evaluated the existence of regulatory T cells (Tr1) via high production of IL-10, in liver biopsies of three well-defined cohorts of HCV-1b infected patients. To this purpose, we compared liver biopsies of chronically infected patients including patients without liver lesions, with cirrhosis and with hepatocellular carcinoma (HCC). Using quantitative real time PCR, the results obtained demonstrate, an increased expression of interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta)_, in liver biopsies with more severe fibrosis. This observation was correlated with an increased expression during the pathogenesis progression, of the three specific markers of the Tr1 cells sub-population, recently described and confirming the Tr1 phenotype. Evidence of regulatory T cells installation in the liver of chronically infected patient and increased frequency in cirrhosis and HCC suggest a main role of these cells in the aggravation of the liver pathology. This study should bring insight of T regulatory cell implications in VHC persistence and in the pathology progression.
Assuntos
Citocinas/análise , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Cirrose Hepática/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Antígenos CD/análise , Antígenos CD/genética , Biópsia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Citocinas/genética , Primers do DNA/genética , Progressão da Doença , Hepatite C/imunologia , Hepatite C/patologia , Hepatite C Crônica/patologia , Humanos , Tolerância Imunológica , Interleucina-10/análise , Interleucina-10/genética , Fígado/patologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/genéticaRESUMO
The ex vivo response to three HLA-DR-restricted Nef peptides (Nef 66-97, Nef 133-159, Nef 180-202) and one HLA-DQ-restricted Nef peptide (Nef 56-68) was evaluated in 28 HIV-seropositive patients and 6 Long-term Non-Progressors (LTNPs). Analyzing specific proliferative response and IFN-gamma secretion, patients were identified as high responders, medium responders and non-responders to peptides. As high responder patients, LTNP patients showed strong proliferative response to all the Nef-peptides as strong IFN-gamma secretion. Twenty-four months later, all high responder patients were always without antiretroviral treatment whereas 50% of medium responders and at least 66% of low responder patients followed bi-therapy. CDC classification confirmed also unfavourable evolution for these two last groups. All high responder patients conserved stable CD4 counts, proliferative response to Nef peptides as strong IFN-gamma secretion during this 24-month period. So, early good T CD4 response to peptides of the Nef protein could thus be regarded as a factor of good prognosis in HIV infection and a tool of importance in the decision to put or not a patient under treatment.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Produtos do Gene nef/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Linfócitos T CD4-Positivos/virologia , Progressão da Doença , Infecções por HIV/prevenção & controle , Infecções por HIV/terapia , Humanos , RNA Viral/sangue , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
To understand the inter-individual and virus-independent variability of CD4+ T cell responses to HCV components, we evaluated the effect on these responses of HLA II molecules in uninfected healthy donors. Using HLA II-specific binding assays, we identified, in the Core and NS3 proteins, 21 long fragments and 24 15-mer peptides that bound to four to eight of the most preponderant HLA II molecules. We then evaluated the priming capacity of eight long promiscuous peptides in 12 HLA-unrelated healthy donors. The NS3 1250-1264 peptide primed T cells in all the naive donors, while five others were stimulating in at least half of the individuals. We also report sequences that bind to multiple HLA II molecules but are weakly immunogenic. We therefore conclude that (i) broad HLA II specificity is only a prerequisite for a peptide to be stimulating in multiple individuals, and (ii) promiscuous peptides widely differ in their capacity to prime CD4+ T cells from uninfected healthy donors. We suggest that these priming differences result from inter-individual variations in the peptide-specific T cell repertoire. Interestingly, five of the most immunogenic peptides we identified correspond to frequently targeted T cell epitopes in infected patients.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Proteínas do Core Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/metabolismo , Genótipo , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Interferon gama/metabolismo , Células L , Ativação Linfocitária/imunologia , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , TransfecçãoRESUMO
The Epstein-Barr virus (EBV) is associated with several malignant diseases, which can be distinguished by their patterns of viral latent gene expression. The latency II program is limited to the expression of the nonimmunodominant antigens EBNA1, LMP1 and LMP2 and is seen in EBV-positive Hodgkin disease, nasopharyngeal carcinomas, and peripheral T/NK-cell lymphomas. CD4 T cells may play a crucial role in controlling these EBV latency II malignancies. In this study, we used the prediction software TEPITOPE to predict promiscuous major histocompatibility complex class II epitopes derived from the latency II antigens EBNA1, LMP1, and LMP2. The predicted peptides were then submitted to peptide-binding assays on HLA II purified molecules, which allowed the selection of 6 peptides (EBNA1: 3; LMP1: 1; and LMP2: 2) with a highly promiscuous capability of binding. This peptide cocktail was immunogenic in a model of HLA-DR1 transgenic mice, leading to a specific cellular and humoral TH1 response. The peptides were also recognized by human CD4 T cells from individuals expressing various HLA II genotypes. This promiscuous peptide cocktail could be immunogenic in the majority of the population and may be used as a peptide-based vaccine in EBV latency II malignancies.
Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Vacinas contra Herpesvirus/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Doença de Hodgkin/imunologia , Doença de Hodgkin/prevenção & controle , Adulto , Animais , Linfócitos T CD4-Positivos/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/química , Feminino , Antígeno HLA-DR1/genética , Vacinas contra Herpesvirus/química , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Peptídeos/química , Peptídeos/imunologia , Software , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/imunologiaRESUMO
Epstein-Barr virus (EBV) is associated with several human malignancies where it expresses limited subsets of latent proteins. Of the latent proteins, latent membrane protein 1 (LMP1) is a potent transforming protein that constitutively induces multiple cell signaling pathways and contributes to EBV-associated oncogenesis. Regulation of LMP1 expression has been extensively described during the type III latency of EBV. Nevertheless, in the majority of EBV-associated tumors, the virus is commonly found to display a type II latency program in which it is still unknown which viral or cellular protein is really involved in maintaining LMP1 expression. Here, we demonstrate that LMP1 activates its own promoter pLMP1 through the JNK signaling pathway emerging from the TES2 domain. Our results also reveal that this activation is tightly controlled by LMP1, since pLMP1 is inhibited by LMP1-activated NF-kappaB signaling pathway. By using our physiological models of EBV-infected cells displaying type II latency as well as lymphoblastoid cell lines expressing a type III latency, we also demonstrate that this balanced autoregulation of LMP1 is shared by both latency programs. Finally, we show that this autoactivation is the most important mechanism to maintain LMP1 expression during the type II latency program of EBV.
Assuntos
Herpesvirus Humano 4/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/fisiologia , Transdução de Sinais , Proteínas da Matriz Viral/metabolismo , Latência Viral , Ativação Enzimática , Humanos , Rim/metabolismo , Rim/virologia , Luciferases/metabolismo , Linfócitos/metabolismo , Linfócitos/virologia , Proteínas da Matriz Viral/genéticaRESUMO
The identification of MHC class II-restricted peptides has become a priority for the development of peptide-based prophylactic and therapeutic vaccines. The aim of this study was to assess the correlations between peptide-binding assays on purified HLA II molecules and immunization of human HLA II transgenic mice deficient in murine class II molecules (Abeta degrees ). We used as models two MHC class II-restricted peptides, one derived from the HIV Nef regulatory protein (Nef (56-68)) and the other from the Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28GST (190-211)). High correlations were found between the two approaches, which showed that the Nef (56-68) and Sm28GST (190-211) peptides may represent promiscuous ligands for HLA-DQ and for HLA-DR molecules, respectively. We suggest a rational method based on the combination of peptide-binding assays and HLA II transgenic mice experiments as consistent and complementary tools for selecting T helper epitopes.
Assuntos
Genes MHC da Classe II , Fragmentos de Peptídeos/imunologia , Vacinas/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Epitopos de Linfócito T , Produtos do Gene nef/imunologia , Glutationa Transferase/imunologia , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência MolecularRESUMO
New vaccines based on subunits, synthetic peptides, or DNA need innovative adjuvants or antigen delivery systems: Spherulites are multilamellar vesicles made of incompatible lipid bilayers without any aqueous core. In this study, we evaluated their ability to induce immune responses against human serum albumin (HSA). Mice were immunized by the intraperitoneal/intravenous route or subcutaneously with HSA without any adjuvant or in its encapsulated form. We showed that Spherulites strongly enhanced the seric antibody responses and led to a mixed isotypic distribution characterized by an IgG(2a) potentiation. This demonstrated that Spherulites can improve the presentation of weak antigens to the immune system.
Assuntos
Formação de Anticorpos , Antígenos/administração & dosagem , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Imunoglobulina G/imunologia , Lipossomos/administração & dosagem , Albumina Sérica/administração & dosagem , Animais , Antígenos/imunologia , Feminino , Humanos , Injeções Intraperitoneais , Injeções Intravenosas , Injeções Subcutâneas , Lipossomos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Albumina Sérica/imunologiaRESUMO
Interleukin (IL-)7 and thyroxin (T4) favor Schistosoma mansoni development. Their effect is similar, rather than identical; moreover, cotreatment acts synergistically on parasites. This questioned a common mediator to their action, which we hypothesized was host glucose metabolism. Infection with S. mansoni resulted in an early peak in glycemia immediately followed by a peak of insulinemia (D7-21). In IL-7 + T4 cotreated infected animals, the peak of insulin was abrogated. We further assessed the consequences of experimentally induced glucose- or insulin-level variations on parasite development. Insulin treatment from day 14 to day 21 post-infection (PI) led to increased worm burden and parasite size, thus mimicking the effect of T4 on schistosome development. Interestingly, insulin treatment did not modify glycemia yet abrogated the hyperinsulinemia, normally occurring during infection. Finally, these treatments were associated with an alteration of the expression of parasite genes involved in glucose uptake. These experiments characterize the elaborate links between parasite and host metabolism and their reciprocal influences.
Assuntos
Glucose/metabolismo , Interleucina-7/fisiologia , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose mansoni/parasitologia , Tiroxina/fisiologia , Animais , Biomphalaria , Cricetinae , Feminino , Regulação da Expressão Gênica , Glucose/farmacologia , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/genética , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Interações Hospedeiro-Parasita , Hiperglicemia/metabolismo , Insulina/sangue , Insulina/metabolismo , Insulina/farmacologia , Interleucina-7/farmacologia , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/metabolismo , Organismos Livres de Patógenos Específicos , Tiroxina/farmacologiaRESUMO
HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice were created and their immunological potential evaluated in response to hepatitis B DNA vaccine. Every single immunized mouse developed hepatitis B virus-specific antibodies, HLA-DR1-restricted helper, and HLA-A2.1-restricted cytolytic T cell responses directed at the same immunodominant epitopes as those identified in naturally infected or vaccinated humans. These mice were specifically protected against a hepatitis B-recombinant vaccinia virus infection with a 10,000-fold or more reduction of the virus load at day 4 post-challenge. These mice represent a unique in vivo experimental model for human immune function studies without any interference with mouse MHC response which dwarfed the prediction of human responses. Furthermore, they enable the complete monitoring of immune adaptative responses for preclinical screening of candidate vaccines.
Assuntos
Antígeno HLA-A2/imunologia , Antígeno HLA-DR1/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Citometria de Fluxo , Genes MHC Classe I/imunologia , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígeno HLA-A2/genética , Antígeno HLA-DR1/genética , Hepatite B/imunologia , Hepatite B/prevenção & controle , Vírus da Hepatite B/imunologia , Humanos , Imunofenotipagem , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Animais , Dados de Sequência Molecular , Vacinação , Vacinas de DNA/imunologiaRESUMO
Transgenic mice expressing human HLA class II molecules provide a useful model for identifying HLA-restricted CD4+ epitopes. However, the influence of endogenous murine H-2-restricted T cell responses on HLA-restricted responses is not known. In the present study, we show that HLA-DR1 transgenic mice deficient for H-2 class II expression (HLA-DR1+/+/IAbeta0/0) exhibit an equivalent expression level of the transgene HLA-DR1 and a similar diversity in the TCR repertoire, but a slightly different number of CD4+ peripheral T cells, when compared to HLA-DR1 transgenic mice in which H-2 class II molecules were retained (HLA-DR1+/+/IAbeta+/+). More importantly, a strong antigen-specific HLA-DR1-restricted response was observed in nearly all HLA-DR1+/+/IAbeta0/0 mice immunized with HBV envelope protein (HBs) or capsid protein (HBc), whereas weak HBs- or HBc-specific HLA-DR1-restricted responses were detected in half of the immunized HLA-DR1+/+/IAbeta+/+ mice. Conversely, strong HBs- or HBc-specific H-2-restricted T cell responses were detected in HLA-DR1+/+/IAbeta+/+ mice but not in HLA-DR1+/+/IAbeta0/0 mice. Our results indicate that the coexpression of endogenous H-2 class II molecules reduces the intensity of HLA-DR1-restricted antigen-specific responses in transgenic mice, by favoring murine over human MHC recognition and education. Thus, HLA-DR1+/+/IAbeta0/0 mice represent a better model for identifying and characterizing HLA-DR1-restricted epitopes relevant for human disease.
Assuntos
Antígenos H-2/fisiologia , Antígeno HLA-DR1/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Citocinas/biossíntese , Mapeamento de Epitopos , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Humanos , Camundongos , Camundongos TransgênicosRESUMO
We describe novel peptide-protein microarrays, which were fabricated using semicarbazide glass slides that permitted the immobilization of glyoxylyl peptides by site-specific ligation and the immobilization of proteins by physisorption. The arrays permitted the simultaneous serodetection of antibodies directed against hepatitis C virus (HCV core p21 15-45 peptide, NS4 1925-1947 peptide, core, NS3, NS4, and mixture of core, NS3, NS4, and NS5 antigens), hepatitis B virus (HBc, HBe, and HBs), human immunodeficiency virus (Gp41 and Gp120 for HIV-I and Gp36 for HIV-II), Epstein-Barr virus (VCAp18 153-176 peptide), and syphilis (rTpN47 and rTpN17) antigens using an immunofluorescence assay. Peptide-protein microarrays displayed high signal-to-noise ratios, sensitivities, and specificities for the detection of antibodies as revealed by the analysis of a collection of human sera referenced against these five pathogens.
Assuntos
Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/virologia , Análise Serial de Proteínas/métodos , Doenças Transmissíveis/diagnóstico , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Hepatite/diagnóstico , Hepatite/metabolismo , Hepatite/virologia , Vírus de Hepatite/genética , Vírus de Hepatite/isolamento & purificação , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Humanos , Peptídeos/química , Peptídeos/genética , Testes Sorológicos/métodos , Treponema pallidum/química , Treponema pallidum/genética , Treponema pallidum/isolamento & purificaçãoRESUMO
We have described in the accompanying article the preparation of peptide-protein semicarbazide microarrays and their use for the simultaneous serodetection of antibodies directed against different pathogens. Here, we present a comparative study between semicarbazide and amine glass slides in an immunofluorescent serodetection assay using HIV (Gp120, Gp41), HCV (mix-HCV, core, NS3, and NS4), and HBV (HBs) recombinant antigens. Amine and semicarbazide surfaces displayed the same sensitivity for antibodies detection just after printing. However, the reactivity of protein antigens changed rapidly upon aging on amine slides but not on semicarbazide slides. Peptide or protein semicarbazide microarrays were found to be remarkably stable for months. Additional data concerning the characterization of the semicarbazide surface (homogeneity of the slides, chemical stability, contact angle measurements, atomic force microscopy studies, reproducibility of serodetection results) are also presented and discussed.
Assuntos
Aminas/química , Peptídeos/química , Análise Serial de Proteínas/métodos , Semicarbazidas/química , Testes Sorológicos/métodos , Adsorção , Estabilidade de Medicamentos , Vidro/química , Humanos , Microscopia de Força Atômica , Análise Serial de Proteínas/instrumentação , Testes Sorológicos/instrumentaçãoRESUMO
The latent membrane protein-1 (LMP1) is an integral membrane molecule expressed by Epstein-Barr virus (EBV) during viral latency and displays properties of a constitutively activated member of the TNF receptor family. LMP1 is required for B-cell or monocyte immortalization induced by EBV and is sufficient to transform rodent fibroblasts. Transforming potential of LMP1 is mediated by its cytoplasmic C-terminal domain, which activates various cellular signaling pathways including NFkappaB and JNK. In this report, we constructed mutants of LMP1 with preserved membrane spanning domain but mutated in the C-terminal domain and a second truncated C-terminal LMP1 fused to the enhanced green fluorescent protein. This latter mutant, termed LMP1-CT, impairs signaling by ectopic LMP1 as well as endogenous EBV-expressed wild-type (wt) LMP1. In contrast to dominant-negative mutants of LMP1 with preserved membrane spanning domains, LMP1-CT was unable to bind wt LMP1 to form an inactive complex. Its dominant-negative effects were due to binding and sequestration of LMP1 adapters TRAF2 and TRADD as assessed by coimmunoprecipitation experiments and confocal analysis. The effect was selective since LMP1-CT did not inhibit IL-1beta-induced signaling, whereas it impaired TNF-triggered NFkappaB and JNK signals without affecting TNF-induced apoptosis. In addition and in contrast to LMP1 constructs with membrane localization, LMP-CT did not display cytostatic properties in noninfected cells. Importantly, LMP1-CT inhibited survival induced by LMP1 in an EBV-transformed T-cell line expressing the type II viral latency commonly found in the majority of EBV-associated human tumors. These data demonstrate that LMP1-CT is a new tool to explore the differences between LMP1 and TNF signaling and may facilitate the design of molecules with potential therapeutic roles.