RESUMO
Advanced therapy medicinal products (ATMPs) are gene therapy, cell therapy, and tissue engineered products. To gain access to the market within the European Union, ATMPs must be authorized by the European Commission (EC). Especially for small and medium-sized enterprises (SMEs), the European centralized procedure of marketing authorization that is conducted by the European Medicines Agency (EMA) constitutes a major challenge, because SMEs often have little experience with regulatory procedures and many have limited financial possibilities. To tackle these challenges, a certification procedure exclusively for SMEs and their ATMP development was introduced by the EC. Independently from a marketing authorization application, development and/or production processes can be certified. An issued certificate demonstrates that the respective process meets the current regulatory and scientific requirements of the EMA, representing a valuable milestone for putative investors and licensees. This article highlights the background, the detailed procedure, the minimum requirements, as well as the costs of certification, while giving further noteworthy guidance for interested parties.
Assuntos
Certificação/normas , Qualidade de Produtos para o Consumidor/normas , Terapia Genética/normas , Programas Nacionais de Saúde , Rotulagem de Produtos/normas , Controle de Qualidade , Transplante de Células-Tronco/normas , Terapias em Estudo/normas , Engenharia Tecidual/normas , Comitês Consultivos , União Europeia , Humanos , Marketing de Serviços de Saúde/normasRESUMO
The new era of regenerative medicine has led to rapid development of new innovative therapies especially for diseases and tissue/organ defects for which traditional therapies and medicinal products have not provided satisfactory outcome. Although the clinical use and developments of cell-based medicinal products (CBMPs) could be witnessed already for a decade, robust scientific and regulatory provisions for these products have only recently been enacted. The new Regulation for Advanced Therapies (EC) 1394/2007 together with the revised Annex I, Part IV of Directive 2001/83/EC provides the new legal framework for CBMPs. The wide variety of cell-based products and the foreseen limitations (small sample sizes, short shelf life) vs. particular risks (microbiological purity, variability, immunogenicity, tumourigenicity) associated with CBMPs have called for a flexible, case-by-case regulatory approach for these products. Consequently, a risk-based approach has been developed to allow definition of the amount of scientific data needed for a Marketing Authorisation Application (MAA) of each CBMP. The article provides further insight into the initial risk evaluation, as well as to the quality, non-clinical, and clinical requirements of CBMPs. Special somatic cell therapies designed for active immunotherapy are also addressed.
Assuntos
Transplante de Células/legislação & jurisprudência , Ensaios Clínicos como Assunto/legislação & jurisprudência , Terapia Genética/legislação & jurisprudência , Marketing de Serviços de Saúde/legislação & jurisprudência , Engenharia Tecidual/legislação & jurisprudência , Europa (Continente) , Técnicas de Transferência de Genes , Guias como Assunto , Humanos , Garantia da Qualidade dos Cuidados de Saúde/legislação & jurisprudênciaRESUMO
Triggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E. Flory, A. Hoffmeyer, U. Smola, U. R. Rapp, and J. T. Bruder, J. Virol. 70:2260-2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Interleucina-2/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Clonagem Molecular , Ciclosporina/farmacologia , DNA/genética , DNA/metabolismo , Fator de Transcrição de Proteínas de Ligação GA , Humanos , Ionomicina/farmacologia , Ionóforos/farmacologia , Células Jurkat , Mutação Puntual , Proteínas Proto-Oncogênicas c-raf , Transdução de Sinais/genética , Linfócitos T , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Vaccinia-virus (VV) recombinants encoding either the nucleocapsid (N) or the spike (S) protein of MHV-JHM were constructed to study the role of the immune response against defined coronavirus antigens. For the S-protein, a fusogenic (Sfus+) or non fusogenic variant (Sfus-) of the gene was inserted into the VV genome. A strong protection against acute encephalomyelitis (AE) was mediated in Lewis rats which were immunized by VV-Sfus+ and challenged with an otherwise lethal dose of MHV-JHM before the induction of S-specific IgG antibodies. By contrast, a VV recombinant encoding a variant non fusogenic S-protein or the N-protein was not capable conferring protection. In addition, we demonstrated that MHV-JHM S-specific IgG antibodies elicited before MHV-JHM challenge modulated the disease process, changing it from an acute disease to subacute demyelinating encephalomyelitis (SDE).
Assuntos
Capsídeo/imunologia , Doenças Desmielinizantes/etiologia , Encefalomielite/prevenção & controle , Glicoproteínas de Membrana/imunologia , Vírus da Hepatite Murina/imunologia , Vacinação , Vacinas Sintéticas , Vaccinia virus/imunologia , Proteínas do Core Viral/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais , Doença Aguda , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Fusão Celular , Doença Crônica , Doenças Desmielinizantes/imunologia , Encefalomielite/imunologia , Imunidade Celular , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Glicoproteínas de Membrana/genética , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/patogenicidade , Vírus da Hepatite Murina/fisiologia , Ratos , Ratos Endogâmicos Lew , Recombinação Genética , Glicoproteína da Espícula de Coronavírus , Linfócitos T Auxiliares-Indutores/imunologia , Vacinação/efeitos adversos , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/toxicidade , Vaccinia virus/genética , Proteínas do Envelope Viral/genética , Vacinas Virais/imunologia , Vacinas Virais/toxicidade , Virulência , Latência ViralRESUMO
In this communication we present clear evidence, that the N-protein of MHV-JHM contains immunodominant CD4+ T-cell sites. These sites were recognized by the immune system of virus infected Lewis rats. In previous investigations we have shown, that CD4+ T-cell lines with specificity for defined viral proteins can be selected from diseased Lewis rats and mediate protection, if transferred to otherwise lethally infected animals. To define regions of the N-protein, which are immunodominant for the T-cell response, we employed bacterially expressed N-protein and truncated subfragments of N as an antigen. We demonstrate, that T-cells from MHV-JHM infected, diseased Lewis rats recognized with high prevalence the carboxyterminal subfragment C4-N (95 aa) and to some extent the adjacent C3-N protein. The same results were obtained with T-cells derived from rats immunized with bacterially expressed N-protein or from animals vaccinated by a stable N-protein expressing vaccinia recombinant. Finally, transfer of CD4+ line T-cells to MHV-JHM infected rats specific for C4-N mediated protection against acute disease.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Capsídeo/imunologia , Infecções por Coronavirus/imunologia , Encefalomielite/microbiologia , Epitopos Imunodominantes/imunologia , Vírus da Hepatite Murina/imunologia , Proteínas do Core Viral/imunologia , Animais , Infecções por Coronavirus/prevenção & controle , Encefalomielite/imunologia , Encefalomielite/prevenção & controle , Imunoterapia Adotiva , Vírus da Hepatite Murina/patogenicidade , Ratos , Ratos Endogâmicos Lew , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Vacinas Virais/imunologia , VirulênciaRESUMO
CD4+ T cell lines specific for murine hepatitis virus (MHV) - JHM or myelin basic protein (MBP) proliferated when cultured together with MHC class I and II positive syngeneic rat astrocytes and either inactivated virus or MBP as antigen. The magnitude of the T cell proliferative response was comparable to that seen when thymocytes were used as a source of antigen presenting cells (APC). In contrast, MHC class I and II positive astrocytes were unable to significantly stimulate the proliferation of highly purified populations of naive CD4+ and CD8+ T cells in an allogeneic mixed lymphocyte reaction (MLR). Both T cell populations proliferated when mixed with allogeneic lymph node cells. Infection of the astrocytes with a variant of MHV-JHM (PI-AS22D) did not alter this cells incapacity to stimulate the naive CD4+ and CD8+ T cells to proliferate.
Assuntos
Células Apresentadoras de Antígenos , Astrócitos/microbiologia , Vírus da Hepatite Murina/imunologia , Linfócitos T/imunologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Antígenos CD4/análise , Hepatite Viral Animal/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Interferon gama/farmacologia , Linfonodos/imunologia , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Proteínas Recombinantes , Subpopulações de Linfócitos T/imunologiaRESUMO
Carbamylated haemoglobin arises from the non-enzymatic modification of haemoglobin by cyanate derived from spontaneous dissociation of urea. We studied the in vitro and in vivo interference of carbamylated haemoglobin in the assay of HbA1c by CLHP (ion exchange), affinity chromatography (IMX, Abbott) and immunoturbidimetry (Tina-Quant, Boehringer). For patients with chronic renal failure on continuous ambulatory peritoneal dialysis, CLHP assay of HbA1c gave an error of about +0.35% per 10 mmol/L of urea serum concentration. The IMX and especially Tina-Quant assays for measuring HbA1c were not sensitive to cyanate interference and constitute interesting alternatives for monitoring glycaemic balance in patients with chronic renal failure.
Assuntos
Hemoglobinas Glicadas/análise , Falência Renal Crônica/sangue , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Complicações do Diabetes , Humanos , Imunoensaio/métodos , Técnicas In Vitro , Falência Renal Crônica/complicações , Nefelometria e Turbidimetria/métodos , Ureia/sangueAssuntos
Encéfalo/microbiologia , Glicoproteínas de Membrana/genética , Vírus da Hepatite Murina/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Glicoproteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Vírus da Hepatite Murina/patogenicidade , Reação em Cadeia da Polimerase , Ratos , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/química , Virulência/genética , Latência ViralAssuntos
Artrite Reumatoide/fisiopatologia , Condrocalcinose/fisiopatologia , Articulações/fisiopatologia , Macrófagos/fisiologia , Osteoartrite/fisiopatologia , Espondilite Anquilosante/fisiopatologia , Sinovite/fisiopatologia , Tripsina/metabolismo , alfa-Macroglobulinas/metabolismo , Animais , Humanos , Camundongos , Membrana Sinovial/fisiopatologiaAssuntos
Encéfalo/imunologia , Doenças Desmielinizantes/microbiologia , Encefalomielite/microbiologia , Hepatite Viral Animal/imunologia , Vírus da Hepatite Murina/patogenicidade , Animais , Anticorpos Antivirais/análise , Encéfalo/microbiologia , Células Cultivadas , Doenças Desmielinizantes/imunologia , Encefalomielite/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Vírus da Hepatite Murina/imunologia , Vírus da Hepatite Murina/isolamento & purificação , Neuroglia/imunologia , Ratos , Ratos Endogâmicos Lew , Proteínas Virais/análiseRESUMO
Immunoperoxidase studies were carried out on the pannus-cartilage junction (PCJ) of patients with rheumatoid arthritis (RA) and psoriatic arthritis to investigate the distribution of the enzyme inhibitors alpha 2 macroglobulin (alpha 2 M) and alpha 1 antitrypsin (alpha 1 AT). Comparisons were made with the equivalent synovial cartilage junctional area in osteoarthritis (OA). In all 12 patients with RA, prominent deposits of alpha 2 M and alpha 1 AT were found within the PCJ whereas in OA patients and the case of psoriatic arthritis fewer or no deposits were seen. Inhibitors were localised in pannus synovial lining cells, perivascular inflammatory cells, macrophage and fibroblast-like cells invading the cartilage as well as along the junctional cartilage matrix and in the adjacent pannus-free cartilage. Deposits of immunoglobulins were found in similar areas to enzyme inhibitors. The presence of enzyme inhibitors at the PCJ suggests that this area is not unprotected against enzymatic attack. Thus the concept that pannus is a prime area for cartilage damage because aggressive, invasive cells release destructive enzymes in an environment free from inhibitors should be reviewed.
Assuntos
Artrite Reumatoide/metabolismo , Cartilagem Articular/metabolismo , Exsudatos e Transudatos/metabolismo , Osteoartrite/metabolismo , alfa 1-Antitripsina/metabolismo , alfa-Macroglobulinas/metabolismo , Artrite/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoglobulinas/metabolismo , PsoríaseRESUMO
Coronavirus MHV-JHM infections of rats provide useful models to study the pathogenesis of virus-induced central nervous system disease. To analyze the role of the immune response against defined MHV-JHM antigens, we tested the protective efficacy of vaccinia virus (VV) recombinants expressing either the nucleocapsid (N) or the spike (S) protein. A strong protection was mediated in animals by immunization with recombinant VV encoding a wild-type S protein (VV-Swildtype), whereas VV recombinant expressing a mutant S354CR protein (VV-S354CR) had no protective effect. Recombinant VV encoding N protein (VV-N) induces a humoral and a CD4+ T cell response, but did not prevent acute disease regardless of the immunization protocol. In these experiments, challenge with an otherwise lethal dose of MHV-JHM was performed prior to the induction of virus-neutralizing antibodies and studies with the anti-CD8+ monoclonal antibody. MRC OX8 showed that elimination of the CD8+ subset of T cells abrogates the protective effect. This result indicates that CD8+ T cells primed by recombinant VV expressing wild-type S protein are a primary mechanism of immunological defense against MHV-JHM infection in rats.
Assuntos
Infecções por Coronaviridae/imunologia , Encefalomielite/imunologia , Vírus da Hepatite Murina/imunologia , Linfócitos T/imunologia , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/biossíntese , Antígenos CD8/análise , Imunização , Ratos , Ratos Endogâmicos Lew , Vaccinia virus/imunologiaRESUMO
Synovial tissue from patients with rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, and having menisectomies was examined by immunofluorescence for deposits of alpha-2-macroglobulin (alpha 2M). In inflammed tissues, alpha 2M was found in the synovial lining cells and in perivascular cells. The amount of alpha 2M correlated with the degree of inflammation. Similarly, free lining cells obtained by trypsination of the intact synovial membrane contained identical inclusions. alpha 2M was not detected in the menisectomy cases and in the less inflammatory osteoarthritic specimens. In-vitro studies demonstrated uptake of alpha 2M-trypsin complexes but not of native alpha 2M by most of the cultured synovial cells whether they came from rheumatoid patients or controls. The internalised complexes disappeared within 12 hours of culture. The results suggest that alpha 2M-proteinase complexes formed in the joint are taken up by phagocytic and perivascular cells in a similar way to immune complexes.
Assuntos
Doenças Reumáticas/metabolismo , Membrana Sinovial/metabolismo , alfa-Macroglobulinas/metabolismo , Artrite Reumatoide/metabolismo , Células Cultivadas , Imunofluorescência , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Osteoartrite/metabolismo , Tripsina/metabolismoRESUMO
A new cell line of transformed astrocytes was obtained from primary new born rat brain cultures. rat brain cultures. These transformed cell line possess a normal karyotype, a doubling time of 17-24 hrs. and astrocyte specific protein marquers: glial fibrillar acidic protein (GFA) and glutamine synthetase. The ganglioside pattern is more differentiated than that of other known astrocyte lines.
Assuntos
Astrócitos/citologia , Animais , Astrócitos/análise , Encéfalo/citologia , Divisão Celular , Linhagem Celular , Separação Celular/métodos , Cariotipagem , Microscopia de Contraste de Fase , RatosRESUMO
The murine coronavirus neurotropic strain JHM (MHV-JHM) nucleocapsid (N) protein induces a strong T-helper cell response in Lewis rats. It has been shown previously that N-specific CD4+ T cells can confer protection against acute disease upon transfer to otherwise lethally infected rats. To define the major antigenic regions that elicit this T cell response, truncated fragments of N protein were expressed from a bacterial expression vector and employed as T cell antigens. Lymphocytes from either MHV-JHM-infected or immunized rats were stimulated in culture with virus antigen, grown and tested for their specificity to the N protein fragments. The carboxy-terminally located C4-N fragment (95 amino acids) induced the most pronounced proliferative response irrespective of whether the lymphocyte culture was derived from immunized or MHV-JHM-infected rats. We established T cell lines specific for the truncated N protein fragments and tested their potential to mediate protection by transfer experiments. Only the T cell line C4-N and the T cell line specific for the full-length N protein were protective. By contrast, all truncated N protein fragments elicited a humoral immune response and contained antigenic sites recognized by antibodies from diseased rats.
Assuntos
Antígenos Virais/imunologia , Antígenos CD4/metabolismo , Capsídeo/imunologia , Infecções por Coronaviridae/imunologia , Coronaviridae/imunologia , Encefalomielite/imunologia , Linfócitos T/fisiologia , Vacinas Sintéticas/imunologia , Proteínas do Core Viral/imunologia , Animais , Animais Lactentes , Anticorpos Antivirais/sangue , Formação de Anticorpos , Western Blotting , Coronaviridae/patogenicidade , Infecções por Coronaviridae/prevenção & controle , Encefalomielite/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Ratos , Ratos Endogâmicos Lew , Linfócitos T Auxiliares-Indutores/fisiologia , Vacinas Virais/imunologiaRESUMO
The surface glycoprotein S (spike) of coronaviruses is believed to be an important determinant of virulence and displays extensive genetic polymorphism in cell culture isolates. This led us to consider whether the observed heterogeneity is reflected by a quasispecies distribution of mutated RNA molecules within the infected organ. Coronavirus infection of rodents is a useful model system for investigating the pathogenesis of virus-induced central nervous system (CNS) disease. Here, we investigated whether genetic changes in the S gene occurred during virus persistence in vivo. We analysed the variability of S gene sequences directly from the brain tissue of Lewis rats infected with the coronavirus mouse hepatitis virus (MHV) variant JHM-Pi using RT-PCR amplification methods. The S gene sequence displayed a remarkable genetic stability in vivo. No evidence for a quasispecies distribution was found by sequence analysis of amplified S gene fragments derived from the CNS of Lewis rats. Furthermore, the S gene also remained conserved under the selection pressure of a neutralizing antibody. Only a few mutations predicted to result in amino acid changes were detected in single clones. The changes were not represented in the consensus sequence. These results indicate that to retain functional proteins under the constraints of a persistent infection in vivo, conservation of sequence can be more important than heterogeneity.
Assuntos
Encefalopatias/virologia , Glicoproteínas de Membrana/genética , Vírus da Hepatite Murina/genética , Ratos Endogâmicos Lew/virologia , Proteínas do Envelope Viral/genética , Animais , Sequência de Bases , Sequência Conservada , Camundongos , Dados de Sequência Molecular , Mutação , Ratos , Análise de Sequência , Glicoproteína da Espícula de CoronavírusRESUMO
Increasing evidence points to a role of the mitogenic Ras/Raf/MEK/ERK signaling cascade in regulation of human immunodeficiency virus type 1 (HIV-1) gene expression. Stimulation of elements of this pathway leads to transactivation of the HIV-1 promoter. In particular, the NF-kappaB motif in the HIV long terminal repeat (LTR) represents a Raf-responsive element in fibroblasts. Regulation of the Raf kinase in T cells differs from findings with a variety of cell lines that the catalytic domain of Raf (Raf(delta26-303)) shows no activity. In this study, we restored the activity of the kinase in T cells by fusing its catalytic domain to the CAAX motif (-Cx) of Ras, thus targeting the enzyme to the plasma membrane. Constitutive activity of Raf was demonstrated by phosphorylation of mitogen-activated protein kinase kinase (MEK) and endogenous mitogen-activated protein kinase 1/2 (ERK1/2) in A3.01 T cells transfected with Raf(delta26-303)-Cx. Membrane-targeted Raf also stimulates NF-kappaB, as judged by kappaB-dependent reporter assays and enhanced NF-kappaB p65 binding on band shift analysis. Moreover, we found that active Raf transactivates the HIV(NL4-3) LTR in A3.01 T lymphocytes and that dominant negative Raf (C4) blocked 12-O-tetradecanoylphorbol-13-acetate induced transactivation. When cotransfected with infectious HIV(NL4-3) DNA, membrane-targeted Raf induces viral replication up to 10-fold over basal levels, as determined by the release of newly synthesized p24gag protein. Our study clearly demonstrates that the activity of the catalytic domain of Raf in A3.01 T cells is dependent on its cellular localization. The functional consequences of active Raf in T lymphocytes include not only NF-kappaB activation and transactivation of the HIV(NL4-3) LTR but also synthesis and release of HIV particles.
Assuntos
HIV-1/fisiologia , MAP Quinase Quinase Quinase 1 , Proteínas Quinases Ativadas por Mitógeno , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Linfócitos T/virologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Membrana Celular/metabolismo , Ativação Enzimática , Humanos , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Linfócitos T/metabolismo , Ativação Transcricional , Replicação ViralRESUMO
T cell activation leads via multiple intracellular signaling pathways to rapid induction of interleukin-2 (IL-2) expression, which can be mimicked by costimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin. We have identified a distal IL-2 enhancer regulated by the Raf-MEK-ERK signaling pathway, which can be induced by TPA/ionomycin treatment. It contains a dyad symmetry element (DSE) controlled by the Ets-like transcription factor GA-binding protein (GABP), a target of activated ERK. TPA/ionomycin treatment of T cells stimulates both mitogen-activated ERK, as well as the stress-activated mitogen-activated protein kinase family members JNK/SAPK and p38. In this study, we investigated the contribution of the stress-activated pathways to the induction of the distal IL-2 enhancer. We show that JNK- but not p38-activating pathways regulate the DSE activity. Furthermore, the JNK/SAPK signaling pathway cooperates with the Raf-MEK-ERK cascade in TPA/ionomycin-induced DSE activity. In T cells, overexpression of SPRK/MLK3, an activator of JNK/SAPK, strongly induces DSE-dependent transcription and dominant negative kinases of SEK and SAPK impair TPA/ionomycin-induced DSE activity. Blocking both ERK and JNK/SAPK pathways abolishes the DSE induction. The inducibility of the DSE is strongly dependent on the Ets-core motifs, which are bound by GABP. Both subunits of GABP are phosphorylated upon JNK activation in vivo and three different isoforms of JNK/SAPK, but not p38, in vitro. Our data suggest that GABP is targeted by signaling events from both ERK and JNK/SAPK pathways. GABP therefore is a candidate for signal integration and regulation of IL-2 transcription in T lymphocytes.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Interleucina-2/genética , MAP Quinase Quinase Quinases , Proteínas Quinases Ativadas por Mitógeno , Linfócitos T/enzimologia , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Ativação Enzimática , Fator de Transcrição de Proteínas de Ligação GA , Humanos , Ionomicina/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
The neurotropic mouse hepatitis virus MHV-JHM induces central nervous system (CNS) demyelination in Lewis rats that pathologically resembles CNS lesions in multiple sclerosis. The mechanisms of MHV-JHM-induced demyelination remain unclear and several studies have implicated the role of the immune response in this process. We have shown previously that protective immunity against MHV-JHM-induced encephalomyelitis was induced by immunization with a vaccinia virus (VV) recombinant expressing MHV-JHM S-protein (VV-S). Here, we present evidence that the time of MHV-JHM challenge after immunization with VV-S plays a critical role in protective immunity. The induction of virus-neutralizing S-protein-specific antibodies prior to the MHV-JHM challenge modulates the disease process and a subacute encephalomyelitis based on a persistent virus infection developed. Typical pathological alterations were lesions of inflammatory demyelination. In addition, the results indicate that after seroconversion, CD8+ T cells were no longer essential for virus elimination in contrast to their role in protection during acute encephalomyelitis.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/prevenção & controle , Encefalomielite/prevenção & controle , Glicoproteínas de Membrana/imunologia , Vírus da Hepatite Murina/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Doenças Desmielinizantes/imunologia , Encefalomielite/imunologia , Encefalomielite/patologia , Feminino , Imunização , Imunoglobulina G/sangue , Depleção Linfocítica , Camundongos , Vírus da Hepatite Murina/isolamento & purificação , Ratos , Ratos Endogâmicos Lew , Proteínas Recombinantes de Fusão/imunologia , Glicoproteína da Espícula de Coronavírus , Fatores de TempoRESUMO
The serine/threonine protein kinase Raf-1 is a component of a conserved intracellular signaling cascade that controls responses to various extracellular stimuli. Transcription from several promoters, including the oncogene-responsive element in the polyomavirus enhancer, the c-fos promoter, as well as other AP-1- and Ets-dependent promoters, can be induced by Raf-1 kinase. Previously, we have shown that activated Raf-1 kinase transactivates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and have identified the NF-kappaB binding motif as a Raf-1-responsive element (RafRE). We now report that Raf-1 kinase-induced transactivation from the HIV RafRE involves the purine-rich-repeat-binding protein (GABP), which is composed of two distinct subunits (alpha and beta). GABP alpha is an Ets oncogene-related DNA-binding protein, and GABP beta contains four ankyrin-like repeats that have been shown to be essential in protein-protein interactions. In electrophoretic mobility shift assays using nuclear extracts from human Jurkat T cells, a protein-DNA complex which was supershifted with antiserum against GABP alpha and GABP beta was observed. Purified recombinant GABP alpha and beta interact with the HIV RafRE as judged from DNA binding assays. Cotransfection experiments with GABP alpha and beta and Raf-1 kinase demonstrate synergistic transactivation of the HIV-1 promoter. Point mutations in the HIV RafRE abolished the Raf-1 kinase as well as GABP alpha- and beta-induced transactivation. The observed Raf-1-GABP synergism presumably involves phosphorylation of GABP subunits, as treatment of cells with Raf-1 kinase activators serum and 12-O-tetradecanoylphorbol-13-acetate increases phosphorylation of GABP in vivo. However, GABP is not a target of Raf-1 kinase; instead, it is a substrate of mitogen-activated protein kinase (MAPK/ERK), since in vitro phosphorylation of GABP alpha and beta was achieved by the reconstituted protein kinase cascade but not with purified Raf-1 or MEK. These results suggest that Raf-1 kinase- induced activation of the HIV-1 promoter is mediated by the classical cytoplasmic cascade resulting in MAPK/ERK-mediated phosphorylation of GABP alpha and beta. Because the HIV RafRE corresponds to a region within the promoter which is essential for regulation of HIV-1 expression, the data indicate that in addition to NK-kappaB, GABP transcription factors are important for induced expression of HIV.