RESUMO
Myasthenia gravis (MG) with antibodies to the muscle-specific receptor tyrosine kinase (MuSK) is a distinct sub-group of MG, affecting 5-8% of all MG patients. MuSK, a receptor tyrosine kinase, is expressed at the neuromuscular junctions (NMJs) from the earliest stages of synaptogenesis and plays a crucial role in the development and maintenance of the NMJ. MuSK-MG patients are more severely affected and more refractory to treatments currently used for MG. Most patients require long-term immunosuppression, stressing the need for improved treatments. Ideally, preferred treatments should specifically delete the antigen-specific autoimmune response, without affecting the entire immune system. Mucosal tolerance, induced by oral or nasal administration of an auto-antigen through the mucosal system, resulting in an antigen-specific immunological systemic hyporesponsiveness, might be considered as a treatment of choice for MuSK-MG. In the present study we have characterized several immunological parameters of murine MuSK-EAMG and have employed induction of oral tolerance in mouse MuSK-EAMG, by feeding with a recombinant MuSK protein one week before disease induction. Such a treatment has been shown to attenuate MuSK-EAMG. Both induction and progression of disease were ameliorated following oral treatment with the recombinant MuSK fragment, as indicated by lower clinical scores and lower anti-MuSK antibody titers.
Assuntos
Tolerância Imunológica/imunologia , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Administração Oral , Animais , Feminino , Camundongos , Receptores Proteína Tirosina Quinases/administração & dosagemRESUMO
Abnormal overexpression of CXCL13 is observed in many inflamed tissues and in particular in autoimmune diseases. Myasthenia gravis (MG) is a neuromuscular disease mainly mediated by anti-acetylcholine receptor autoantibodies. Thymic hyperplasia characterized by ectopic germinal centers (GCs) is a common feature in MG and is correlated with high levels of anti-AChR antibodies. We previously showed that the B-cell chemoattractant, CXCL13 is overexpressed by thymic epithelial cells in MG patients. We hypothesized that abnormal CXCL13 expression by the thymic epithelium triggered B-cell recruitment in MG. We therefore created a novel transgenic (Tg) mouse with a keratin 5 driven CXCL13 expression. The thymus of Tg mice overexpressed CXCL13 but did not trigger B-cell recruitment. However, in inflammatory conditions, induced by Poly(I:C), B cells strongly migrated to the thymus. Tg mice were also more susceptible to experimental autoimmune MG (EAMG) with stronger clinical signs, higher titers of anti-AChR antibodies, increased thymic B cells, and the development of germinal center-like structures. Consequently, this mouse model finally mimics the thymic pathology observed in human MG. Our data also demonstrated that inflammation is mandatory to reveal CXCL13 ability to recruit B cells and to induce tertiary lymphoid organ development.
Assuntos
Linfócitos B/patologia , Quimiocina CXCL13/fisiologia , Inflamação/complicações , Miastenia Gravis Autoimune Experimental/patologia , Hiperplasia do Timo/fisiopatologia , Animais , Linfócitos B/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Citometria de Fluxo , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Técnicas Imunoenzimáticas , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miastenia Gravis Autoimune Experimental/etiologia , Miastenia Gravis Autoimune Experimental/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
INTRODUCTION: Anti-acetylcholine receptor (AChR) autoantibodies target muscles in spontaneous human myasthenia gravis (MG) and its induced experimental autoimmune model MG (EAMG). The aim of this study was to identify novel functional mechanisms occurring in the muscle pathology of myasthenia. RESULTS: A transcriptome analysis performed on muscle tissue from MG patients (compared with healthy controls) and from EAMG rats (compared with control rats) revealed a deregulation of genes associated with the Interleukin-6 (IL-6) and Insulin-Like Growth Factor 1 (IGF-1) pathways in both humans and rats. The expression of IL-6 and its receptor IL-6R transcripts was found to be altered in muscles of EAMG rats and mice compared with control animals. In muscle biopsies from MG patients, IL-6 protein level was higher than in control muscles. Using cultures of human muscle cells, we evaluated the effects of anti-AChR antibodies on IL-6 production and on the phosphorylation of Protein Kinase B (PKB/Akt). Most MG sera and some monoclonal anti-AChR antibodies induced a significant increase in IL-6 production by human muscle cells. Furthermore, Akt phosphorylation in response to insulin was decreased in the presence of monoclonal anti-AChR antibodies. CONCLUSIONS: Anti-AChR antibodies alter IL-6 production by muscle cells, suggesting a putative novel functional mechanism of action for the anti-AChR antibodies. IL-6 is a myokine with known effects on signaling pathways such as Akt/mTOR (mammalian Target of Rapamycin). Since Akt plays a key role in multiple cellular processes, the reduced phosphorylation of Akt by the anti-AChR antibodies may have a significant impact on the muscle fatigability observed in MG patients.
Assuntos
Interleucina-6/metabolismo , Músculo Esquelético/metabolismo , Miastenia Gravis/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adolescente , Adulto , Animais , Anticorpos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Criança , Modelos Animais de Doenças , Feminino , Adjuvante de Freund/toxicidade , Perfilação da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-6/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Miastenia Gravis/etiologia , Miastenia Gravis/genética , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fosforilação , Ratos , Ratos Endogâmicos Lew , Receptores Colinérgicos/imunologia , Receptores de Interleucina-6/metabolismo , Adulto JovemRESUMO
Glucocorticoids (GC) display pleiotropic effects on the immune system. Macrophages are a major target for GC action. Here we show that dexamethasone (DEX), a synthetic GC, decreased viability of naïve bone marrow-derived macrophages (BMDM), involving an apoptotic mechanism. Administration of DEX together with lipopolysaccharide (LPS) protected BMDM against DEX-mediated cell death, suggesting that activated BMDM respond to DEX differently than naïve BMDM. An insight to the molecular basis of LPS actions was provided by a 7 fold increase in mRNA levels of glucocorticoid receptor beta (GRß), a GR dominant-negative splice variant which inhibits GRα's transcriptional activity. LPS did not inhibit all DEX-mediated effects on BMDM; DEX significantly reduced the percentage of BMDM expressing high levels of the cell surface markers F4/80 and CD11b and led to a decrease in macrophage inflammatory protein 1 alpha (MIP1-α) mRNA and protein levels. These two DEX-mediated effects were not prevented by LPS. Our finding that LPS did not reduce the DEX-induced elevation of glucocorticoid-induced leucine zipper (GILZ), a mediator of GCs anti-inflammatory actions, may provide an underlying mechanism. These findings enable a better understanding of clinical states, such as sepsis, in which macrophages are activated by endotoxins and treatment by GCs is considered.
Assuntos
Apoptose/efeitos dos fármacos , Dexametasona/toxicidade , Glucocorticoides/toxicidade , Animais , Antígenos de Diferenciação/metabolismo , Células da Medula Óssea/citologia , Antígeno CD11b/metabolismo , Quimiocina CCL3/genética , Quimiocina CCL3/metabolismo , Feminino , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Myasthenia gravis (MG) is an antibody mediated, T cell dependent autoimmune disease characterized by muscle fatigability in which autoantibodies directed to the acetylcholine receptor (AChR) impair neuromuscular transmission. The identification of CD4âºCD25âºFoxp3âºTreg cells as important regulators of tolerance opened a major area of investigation raising the possibility that a dysfunction in the Treg compartment is involved in the etiology and pathogenesis of autoimmune diseases, including MG. In this paper we summarize shortly Treg abnormalities that were reported in MG patients and report on our studies of Treg in experimental autoimmune MG (EAMG). Hopefully these studies would pave the way towards the development of novel Treg-based treatment modalities that will restore self-tolerance in MG and other autoimmune diseases. In our previous studies in EAMG we have shown that Treg cells transferred from healthy rat donors to myasthenic rats suppress EAMG. However, Treg cells from sick animals do not have the same in vivo suppressive activity as those from healthy donors. The objective of the present study was to further characterize quantitative and qualitative alterations in Treg cells of rats with EAMG. We found that the frequency of CD4âºCD25âºFoxp3âºTreg cells within the spleen and PBL was decreased in EAMG rats as compared to naïve and CFA-immunized healthy controls. Treg cells from myasthenic rats were less effective than Treg cells from controls in suppressing the proliferation of CD4âºT effector cells in response to ConA and of B cells in response to LPS. Moreover, CD4âºCD25⺠cells from EAMG rats exhibited an elevated extent of apoptosis and expressed upregulated levels of FAS and of Th17-associated cytokines. Since EAMG is an induced disease, these quantitative and qualitative alterations in Treg cells do not reflect predisposing impairments and seem to be associated with the specific autoimmune response resulting from AChR immunization.
Assuntos
Miastenia Gravis/imunologia , Linfócitos T Reguladores/imunologia , Animais , Humanos , Tolerância Imunológica , Miastenia Gravis/patologia , Miastenia Gravis/fisiopatologia , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/patologia , Ratos , Linfócitos T Reguladores/patologiaAssuntos
Miastenia Gravis/tratamento farmacológico , Humanos , Inflamação/imunologia , Inflamação/patologia , Miastenia Gravis/diagnóstico , Miastenia Gravis/imunologia , Miastenia Gravis/fisiopatologia , Receptores Colinérgicos/imunologia , Receptores Colinérgicos/metabolismo , Timo/imunologia , Timo/fisiopatologiaRESUMO
Establishment of tolerance in myasthenia gravis (MG) involves regulatory T (T(reg)) cells. Experimental autoimmune MG (EAMG) in rats is a suitable model for assessing the contribution of T(reg) cells to the immunopathology of the disease and for testing novel T(reg) cell-based treatment modalities. We have studied two immunotherapeutic approaches for targeting of T(reg) cells in myasthenia. By one approach we demonstrated that treatment of sick rats by ex vivo-generated exogenous T(reg) cells derived from healthy donors suppressed EAMG. By a different approach, we aimed at affecting the endogenous T(reg)/Th17 cell balance by targeting IL-6, which has a key role in controlling the equilibrium between pathogenic Th17 and suppressive T(reg) cells. We found that treatment of myasthenic rats by neutralizing anti-IL-6 antibodies shifted this equilibrium in favor of T(reg) cells and led to suppression of EAMG. Our results show that T(reg) cells could serve as potential targets in treating MG patients.
Assuntos
Imunoterapia/métodos , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/terapia , Linfócitos T Reguladores/metabolismo , Animais , Anticorpos Neutralizantes/uso terapêutico , Humanos , Tolerância Imunológica , Interleucina-6/imunologia , Ratos , Linfócitos T Reguladores/transplante , Células Th1/metabolismo , Células Th17/metabolismoRESUMO
Aire (autoimmune regulator) has a key role in the establishment of tolerance to autoantigens. Aire(-/-) mice present decreased thymic expression of AChR, significantly lower frequencies of regulatory T (T(reg)) cells, and higher expression of Th17 markers, compared to controls. We therefore predicted that Aire(-/-) mice would be more susceptible to induction of experimental autoimmune myasthenia gravis (EAMG). However, when EAMG was induced in young mice, Aire(-/-) mice presented a milder disease that wild-type (WT) controls. In contrast, when EAMG was induced in older mice, Aire(-/-) mice were more severely affected than WT mice. The relative resistance to EAMG in young Aire(-/-) mice correlated with increased numbers of T(reg) cells in their spleens compared to young controls. A similar age-related susceptibility was also observed when EAE was induced in Aire(-/-) mice, suggesting an age-related link among Aire, disease susceptibility, and peripheral T(reg) cells that may be a general feature of autoimmunity.
Assuntos
Miastenia Gravis/imunologia , Linfócitos T Reguladores/imunologia , Fatores de Transcrição/genética , Envelhecimento , Animais , Predisposição Genética para Doença , Camundongos , Camundongos Knockout , Proteína AIRERESUMO
Suppressive regulatory T cells (Treg) and pathogenic T helper 17 (Th17) cells are two lymphocyte subsets with opposing activities in autoimmune diseases. The proinflammatory cytokine IL-6 is a potent factor in switching immune responses in vivo from the induction of Treg to pathogenic Th17 cells. We studied the Treg and Th17 cell compartments in experimental autoimmune myasthenia gravis (EAMG) and healthy control rats in order to assess whether the equilibrium between Treg and Th17 cells is perturbed in the disease. We found that Th17 cell-related genes are upregulated and Treg-related genes are downregulated in EAMG. The shift in favor of Th17 cells in EAMG could be reversed by antibodies to IL-6. Administration of anti-IL-6 antibodies to myasthenic rats suppressed EAMG when treatment started at the acute or at the chronic phase of disease. Suppression of EAMG by anti-IL-6 antibodies was accompanied by a decrease in the overall rat anti-AChR antibody titer and by a reduced number of B cells as compared with control treatment. Administration of anti-IL-6 antibodies led to down-regulation of several Th17 related genes including IL-17, IL-17R, IL-23R and IL-21 but did not affect the number of Treg cells in the lymph nodes. These data identify IL-6 as an important target for modulation of autoimmune responses.
Assuntos
Interleucina-6/imunologia , Miastenia Gravis Autoimune Experimental/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Miastenia Gravis Autoimune Experimental/genética , Miastenia Gravis Autoimune Experimental/prevenção & controle , Ratos , Ratos Endogâmicos Lew , Receptores Colinérgicos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/metabolismoRESUMO
We have previously shown that several phosphodiesterase (PDE) subtypes are up-regulated in muscles and lymph node cells (LNC) of rats with experimental autoimmune myasthenia gravis (EAMG). In the present study we investigated PDE expression during the course of EAMG and experimental allergic encephalomyelitis (EAE) and found that the up-regulated expression of selected PDE subtypes in both experimental models is correlated with disease severity. In EAMG, PDE expression is correlated also with muscle damage. A similar up-regulation of PDE was also observed in the respective human diseases, MG and multiple sclerosis (MS). Our findings suggest that change in PDE expression levels is a general phenomenon in autoimmune diseases and may also be used as a marker for disease severity.
Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Esclerose Múltipla/enzimologia , Miastenia Gravis Autoimune Experimental/enzimologia , Miastenia Gravis/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Adolescente , Adulto , Animais , Biomarcadores/análise , Biomarcadores/sangue , Criança , Modelos Animais de Doenças , Progressão da Doença , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Feminino , Humanos , Isoenzimas/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/fisiopatologia , Atrofia Muscular/enzimologia , Atrofia Muscular/imunologia , Atrofia Muscular/fisiopatologia , Miastenia Gravis/imunologia , Miastenia Gravis/fisiopatologia , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/fisiopatologia , Valor Preditivo dos Testes , Ratos , Ratos Endogâmicos Lew , Índice de Gravidade de Doença , Regulação para Cima/imunologia , Adulto JovemRESUMO
Intravenous immunoglobulin (IVIG) treatment is beneficially used in autoimmune disorders including myasthenia gravis (MG) although its mode of action and active components are still not fully identified. In an attempt to isolate from IVIG a disease-specific suppressive fraction, IVIG was passed on columns of IgG from rats with experimental autoimmune MG (EAMG) or from MG patients. These chromatographies resulted in depletion of the suppressive activity of IVIG on rat EAMG whereas the minute amounts of IgG fractions eluted from the EAMG- or MG-specific columns retained the immunosuppressive activity of IVIG. These results demonstrate that a minor disease-specific immunoglobulin fraction present in IVIG is essential for its suppressive activity.
Assuntos
Anticorpos Anti-Idiotípicos/isolamento & purificação , Imunoglobulinas Intravenosas/uso terapêutico , Imunossupressores/uso terapêutico , Miastenia Gravis Autoimune Experimental/terapia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/uso terapêutico , Especificidade de Anticorpos , Autoanticorpos/imunologia , Cromatografia de Afinidade , Adjuvante de Freund/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulinas Intravenosas/química , Técnicas de Imunoadsorção , Imunossupressores/isolamento & purificação , Miastenia Gravis/imunologia , Miastenia Gravis Autoimune Experimental/imunologia , Ratos , Especificidade da EspécieRESUMO
DNA microarray technology was used to identify new potential drug targets for myasthenia gravis (MG), to delineate genes involved in the pathogenesis of the disease and to possibly target their protein products for immunotherapy. In this study we compared the gene expression in lymph node cells (LNC) and muscles of rats with experimental autoimmune MG (EAMG) to those of control, healthy rats. Of the genes that were found to be deregulated in EAMG, we chose to elaborate on two gene systems: (a) The chemokine IFN-gamma-inducible protein 10 (IP-10, CXCL10), and its receptor (CXCR3) and (b) phosphodiesterases.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/genética , Análise de Sequência com Séries de Oligonucleotídeos , Corticosteroides/uso terapêutico , Animais , Quimiocina CXCL10 , Quimiocinas CXC/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Miastenia Gravis/metabolismo , Miastenia Gravis/patologia , Inibidores de Fosfodiesterase/uso terapêutico , Receptores CXCR3 , Receptores de Quimiocinas/genéticaRESUMO
Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent antibody-mediated autoimmune disorders, in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. DNA microarray analysis revealed increased levels of several phosphodiesterase (PDE) subtypes in lymph node cells (LNC) and muscles of EAMG rats compared with healthy controls. Quantitative real-time PCR analysis indicated that EAMG is characterized by an increase of PDE subtypes 1, 3, 4, and 7 in LNC and of PDE subtypes 2, 3, 4, and 7 in muscles. Pentoxifylline (PTX), a general PDE inhibitor, inhibited the progression of EAMG when treatment started at either the acute or chronic stages of disease. This suppression was associated with down-regulation of humoral and cellular AChR-specific responses, as well as down-regulation of PDE4, TNF-alpha, IL-18, IL-12, and IL-10 in LNC and of PDEs 1, 4, 7, and TNF-alpha in muscles. The expression of Foxp3, a transcription factor essential for CD4+CD25+ regulatory T cell function, was increased in splenocytes although the number of these cells remained unchanged. PTX also reduced the expression of the endopeptidase cathepsin-l, a marker of muscle damage, in EAMG muscles. This study demonstrates the involvement of PDE regulation in EAMG pathogenesis and suggests that PDE inhibitors may be considered for immunotherapy of MG.
Assuntos
Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/enzimologia , Pentoxifilina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Catepsinas/metabolismo , Linhagem Celular , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Diester Fosfórico Hidrolases/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptores Colinérgicos/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
Myasthenia gravis (MG) is an autoimmune disease mediated by antibodies to nicotinic acetylcholine receptor (AChR) interfering with the neuromuscular transmission. Experimental autoimmune MG serves as an excellent animal model to study possible therapeutic modalities for MG. This review will focus on the different ways to turn off the autoimmune response to AChR, which results in suppression of myasthenia. This paper will describe the use of fragments or peptides derived from the AChR, antigen-presenting cells and anti-T cell receptor antibodies, and will discuss the underlying mechanisms of action. Finally, the authors propose new promising therapeutic prospects, including treatment based on the modulation of regulatory T cells, which have recently been found to be functionally defective in MG patients.
Assuntos
Miastenia Gravis/terapia , Vacinas/uso terapêutico , Animais , Anticorpos Monoclonais/uso terapêutico , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/transplante , Humanos , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Nicotínicos/imunologia , Linfócitos T/imunologiaRESUMO
Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are autoimmune disorders in which the acetylcholine receptor (AChR) is the major autoantigen. Microarray technology was used to identify new potential drug targets for treatment of myasthenia that would reduce the need for the currently used nonspecific immunosuppression. The chemokine IFN-gamma-inducible protein 10 (IP-10; CXCL10), a CXC chemokine, and its receptor, CXCR3, were found to be overexpressed in lymph node cells of EAMG rats. Quantitative real-time PCR confirmed these findings and revealed up-regulated mRNA levels of another chemoattractant that activates CXCR3, monokine induced by IFN-gamma (Mig; CXCL9). TNF-alpha and IL-1beta, which act synergistically with IFN-gamma to induce IP-10, were also up-regulated. These up-regulations were observed in immune response effector cells, namely, lymph node cells, and in the target organ of the autoimmune attack, the muscle of myasthenic rats, and were significantly reduced after suppression of EAMG by mucosal tolerance induction with an AChR fragment. The relevance of IP-10/CXCR3 signaling in myasthenia was validated by similar observations in MG patients. A significant increase in IP-10 and CXCR3 mRNA levels in both thymus and muscle was observed in myasthenic patients compared with age-matched controls. CXCR3 expression in PBMC of MG patients was markedly increased in CD4(+), but not in CD8(+), T cells or in CD19(+) B cells. Our results demonstrate a positive association of IP-10/CXCR3 signaling with the pathogenesis of EAMG in rats as well as in human MG patients.
Assuntos
Quimiocinas CXC/biossíntese , Quimiocinas CXC/genética , Perfilação da Expressão Gênica , Interferon gama/fisiologia , Miastenia Gravis Autoimune Experimental/imunologia , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Adolescente , Adulto , Idoso , Animais , Moléculas de Adesão Celular/biossíntese , Quimiocina CXCL10 , Quimiocinas CXC/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Linfonodos/imunologia , Linfonodos/metabolismo , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/imunologia , Músculo Esquelético/metabolismo , Miastenia Gravis/genética , Miastenia Gravis/imunologia , Miastenia Gravis/patologia , Miastenia Gravis Autoimune Experimental/genética , Miastenia Gravis Autoimune Experimental/prevenção & controle , Análise de Sequência com Séries de Oligonucleotídeos , Subunidades Proteicas/administração & dosagem , Subunidades Proteicas/imunologia , Ratos , Ratos Endogâmicos Lew , Receptores CXCR3 , Receptores de Quimiocinas/metabolismo , Receptores Nicotínicos/administração & dosagem , Receptores Nicotínicos/imunologia , Regulação para Cima/imunologiaRESUMO
A syngeneic rat recombinant fragment of the extracellular domain of the acetylcholine receptor (AChR) alpha-subunit (Ralpha1-205), administered orally, suppresses ongoing experimental autoimmune myasthenia gravis (EAMG) in rats. The underlying mechanism is a shift from Th1 to Th2 regulation as evidenced by downregulated mRNA expression levels of IFN-gamma and TNF-alpha, upregulated IL-10, changes in anti-AChR IgG isotypes and diminished Th1 signaling via CD28/CTLA-4:B7. Unlike the xenogeneic fragment, the syngeneic Ralpha1-205 does not induce elevation in TGF-beta and elicitation of autoregulatory cells. The ability to suppress EAMG by a non-immunogenic syngeneic fragment of AChR is encouraging and may in the future be applied for the treatment of myasthenia gravis in humans.
Assuntos
Terapia de Imunossupressão , Miastenia Gravis Autoimune Experimental/terapia , Receptores Nicotínicos/imunologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Tolerância Imunológica , Interferon gama/biossíntese , Interleucina-10/biossíntese , Miastenia Gravis Autoimune Experimental/imunologia , Peptídeos/imunologia , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th1/imunologia , Células Th2/imunologia , Transplante Heterólogo , Transplante Isogênico , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Evaluation of the activation state of protein kinase C (PKC) isozymes relies on analysis of subcellular translocation. A monoclonal antibody, 14E6, specific for the activated conformation of epsilonPKC, was raised using the first variable (V1) domain of epsilonPKC as the immunogen. 14E6 binding is specific for epsilonPKC and is greatly increased in the presence of PKC activators. Immunofluorescence staining by 14E6 of neonatal rat primary cardiac myocytes and the NG108-15 neuroblastoma glioma cell line, NG108-15/D2, increases rapidly following cell activation and is localized to new subcellular sites. However, staining of translocated epsilonPKC with 14E6 is transient, and the epitope disappears 30 min after activation of NG-108/15 cells by a D2 receptor agonist. In contrast, subcellular localization associated with activation, as determined by commercially available polyclonal antibodies, persists for at least 30 min. In vitro, epsilonRACK, the receptor for activated epsilonPKC, inhibits 14E6 binding to epsilonPKC, suggesting that the 14E6 epitope is lost or hidden when active epsilonPKC binds to its RACK. Therefore, the 14E6 antibody appears to identify a transient state of activated but non-anchored epsilonPKC. Moreover, binding of 14E6 to epsilonPKC only after activation suggests that lipid-dependent conformational changes associated with epsilonPKC activation precede binding of the activated isozyme to its specific RACK, epsilonRACK. Further, monoclonal antibody 14E6 should be a powerful tool to study the pathways that control rapid translocation of epsilonPKC from cytosolic to membrane localization on activation.
Assuntos
Anticorpos Monoclonais , Proteína Quinase C/metabolismo , Animais , Anticorpos Monoclonais/química , Sítios de Ligação , Encéfalo/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Insetos , Lipídeos/química , Microscopia de Fluorescência , Modelos Biológicos , Miócitos Cardíacos/metabolismo , Peptídeos/química , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Proteína Quinase C/química , Proteína Quinase C-épsilon , Estrutura Terciária de Proteína , Ratos , Receptores de Quinase C Ativada , Proteínas Recombinantes/químicaRESUMO
We have shown that mucosal administration of recombinant fragments corresponding to the human acetylcholine receptor (AChR) alpha subunit suppresses chronic ongoing experimental autoimmune myasthenia gravis (EAMG) in rats. Treated animals exhibit a Th1 to Th2/Th3 shift in their cytokine profile and downregulation of costimulatory factors. However, application of a xenogeneic recombinant fragment may have limitations when considered as a possible approach for the treatment of MG in humans. We therefore tested the potential of a syngeneic fragment and of long synthetic peptides to suppress EAMG. We found that a syngeneic fragment corresponding to the extracellular region of the rat AChR alpha subunit was as effective as the formerly described human xenogeneic fragment in suppressing ongoing EAMG. This is encouraging in view of the potential use of mucosally administered recombinant AChR fragments for the treatment of MG in humans. However, in severely affected individuals, this antigen-specific approach may need to be supported by direct modulation of cytokines and costimulatory factors known to be involved in the pathogenesis of EAMG. To test the potential of this approach, myasthenic rats were injected by antibodies either to the proinflammatory cytokine IL-18 or to the costimulatory factor CD40L. These treatments act via different mechanisms, but both lead to the alleviation of clinical symptoms even when given at the chronic phase of EAMG. We suggest that antagonists to key cytokines and/or costimulatory factors be used to augment antigen-specific treatments of myasthenia such as mucosal administration of AChR recombinant fragments.