RESUMEN
Antigen-specific immune responses are crucially involved in both multiple sclerosis (MS) and myasthenia gravis (MG). Teriflunomide is an immunomodulatory agent approved for treatment of MS through inhibition of lymphocyte proliferation. MG associated with muscle-specific tyrosine kinase (MuSK) antibodies often manifests with a severe disease course, prompting development of effective treatment methods. To evaluate whether teriflunomide treatment may ameliorate MuSK-autoimmunity, experimental autoimmune MG (EAMG) was induced by immunizing C57BL/6 (B6) mice three times with MuSK in complete Freund's adjuvant (CFA) (n = 17). MuSK-immunized mice were treated daily with teriflunomide (n = 8) or PBS (n = 9) starting from the third immunization (week 8) to termination (week 14). Clinical severity of EAMG was monitored. Immunological alterations were evaluated by measurement of anti-MuSK IgG, neuromuscular junction deposits, and flow cytometric analysis of lymph node cells. In MS patients under teriflunomide treatment, the peripheral blood B cell subset profile was analyzed. B6 mice treated with teriflunomide displayed relatively preserved body weight, lower EAMG prevalence, reduced average clinical grades, higher inverted screen scores, diminished anti-MuSK antibody and NMJ deposit levels. Amelioration of EAMG findings was associated with reduced memory B cell ratios in the lymph nodes. Similarly, MS patients under teriflunomide treatment showed reduced memory B cell, plasma cell, and plasmablast ratios. Teriflunomide treatment has effectively ameliorated MuSK-autoimmunity and thus may putatively be used in long-term management of MuSK-MG as an auxiliary treatment method. Teriflunomide appears to exert beneficial effects through inhibition of effector B cells.
Asunto(s)
Subgrupos de Linfocitos B/efectos de los fármacos , Crotonatos/administración & dosificación , Hidroxibutiratos/administración & dosificación , Esclerosis Múltiple/tratamiento farmacológico , Miastenia Gravis Autoinmune Experimental/tratamiento farmacológico , Nitrilos/administración & dosificación , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Colinérgicos/inmunología , Toluidinas/administración & dosificación , Adulto , Animales , Subgrupos de Linfocitos B/inmunología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/inmunología , Miastenia Gravis Autoinmune Experimental/sangre , Miastenia Gravis Autoinmune Experimental/diagnóstico , Miastenia Gravis Autoinmune Experimental/inmunología , Proteínas Tirosina Quinasas Receptoras/administración & dosificación , Receptores Colinérgicos/administración & dosificación , Resultado del TratamientoRESUMEN
Myasthenia gravis (MG) is an autoimmune disease affecting the neuromuscular junction. Approximately 9% of MG patients have autoantibodies targeting the muscle specific kinase (MuSK), and are challenging therapeutically, since they often present with more severe symptoms. A useful therapy is plasmapheresis, but it is highly non-specific. Antigen-specific immunoadsorption would only remove the pathogenic autoantibodies, minimizing the possible side effects and maximizing the benefit. We used rats with human MuSK-induced experimental autoimmune MG to perform antigen-specific immunoadsorptions, and found it very effective, resulting in a dramatic autoantibody titer decrease, while immunoadsorbed, but not mock-treated, animals showed an significant improvement of their clinical symptoms. Overall, the procedure was efficient, supporting its application for MG treatment.
Asunto(s)
Autoanticuerpos/administración & dosificación , Antígenos de Histocompatibilidad Clase II/administración & dosificación , Miastenia Gravis Autoinmune Experimental/tratamiento farmacológico , Plasmaféresis/métodos , Proteínas Tirosina Quinasas Receptoras/administración & dosificación , Receptores Colinérgicos/administración & dosificación , Animales , Autoanticuerpos/inmunología , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunización/métodos , Miastenia Gravis Autoinmune Experimental/inmunología , Ratas , Ratas Endogámicas Lew , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Colinérgicos/inmunología , Resultado del TratamientoRESUMEN
Experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis (MG), can be induced in C57BL/6 (B6, H-2 b) mice by 2-3 injections with Torpedo californica AChR (tAChR) in complete Freund's adjuvant. Some EAMG mice exhibit weight loss with muscle weakness. The loss in body weight, which is closely associated with bone structure, is particularly evident in EAMG mice with severe muscle weakness. However, the relationship between muscle weakness and bone loss in EAMG has not been studied before. Recent investigations on bone have shed light on association of bone health and immunological states. It is possible that muscle weakness in EAMG developed by anti-tAChR immune responses might accompany bone loss. We determined whether reduced muscle strength associates with decreased bone mineral density (BMD) in EAMG mice. EAMG was induced by two injections at 4-week interval of tAChR and adjuvants in two different age groups. The first tAChR injection was either at age 8 weeks or at 15 weeks. We measured BMD at three skeletal sites, including femur, tibia, and lumbar vertebrae, using dual energy X-ray absorptiometry. Among these bone areas, femur of EAMG mice in both age groups showed a significant decrease in BMD compared to control adjuvant-injected and to non-immunized mice. Reduction in BMD in induced EAMG at a later-age appears to parallel the severity of the disease. The results indicate that anti-tAChR autoimmune response alone can reduce bone density in EAMG mice. BMD reduction was also observed in adjuvant-injected mice in comparison to normal un-injected mice, suggesting that BMD decrease can occur even when muscle activity is normal. Decreased BMD observed in both tAChR-injected and adjuvant-injected mice groups were discussed in relation to innate immunity and bone-related immunology involving activated T cells and tumour necrosis factor-related cytokines that trigger osteoclastogenesis and bone loss.
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Densidad Ósea/inmunología , Resorción Ósea/patología , Debilidad Muscular/patología , Miastenia Gravis Autoinmune Experimental/patología , Absorciometría de Fotón , Factores de Edad , Animales , Resorción Ósea/inducido químicamente , Resorción Ósea/diagnóstico por imagen , Resorción Ósea/inmunología , Fémur/diagnóstico por imagen , Fémur/inmunología , Fémur/patología , Proteínas de Peces/administración & dosificación , Adyuvante de Freund/administración & dosificación , Vértebras Lumbares/diagnóstico por imagen , Vértebras Lumbares/inmunología , Vértebras Lumbares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Debilidad Muscular/inducido químicamente , Debilidad Muscular/diagnóstico por imagen , Debilidad Muscular/inmunología , Miastenia Gravis Autoinmune Experimental/inducido químicamente , Miastenia Gravis Autoinmune Experimental/diagnóstico por imagen , Miastenia Gravis Autoinmune Experimental/metabolismo , Receptores Colinérgicos/administración & dosificación , Índice de Severidad de la Enfermedad , Tibia/diagnóstico por imagen , Tibia/inmunología , Tibia/patología , Factores de Tiempo , Torpedo/metabolismoRESUMEN
An animal model of myasthenia gravis (MG), termed experimental autoimmune MG (EAMG), is an important tool for investigations of disease mechanisms and/or methods of treatment for this disease. EAMG can be induced in C57BL/6 (B6, H-2b) mice by 2-3 times injections at 4 weeks intervals with Torpedo californica (t) acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the protocol especially with a two-injection schedule occasionally produces a poor incidence of EAMG. We have investigated the efficacy of the additional adjuvant, inactive organisms of Bordetella pertussis (iBP), on the induction with a two-injection schedule. In a group immunized with tAChR in CFA + iBP, 76% of mice developed EAMG (average grade in exercise test, 1.02). Whereas, 46% of mice were found EAMG-positive (average grade, 0.73) in a group injected with tAChR/CFA alone. Thus, the combined use of CFA and iBP significantly increased both the occurrence and severity of clinical MG in the immunized mice. This was accompanied by higher antibody (Ab) and T-cell responses to tAChR. The effect on disease occurrence of the iBP use in a three-injection protocol was also described.
Asunto(s)
Bordetella pertussis/inmunología , Adyuvante de Freund/inmunología , Miastenia Gravis Autoinmune Experimental/inmunología , Receptores Colinérgicos/inmunología , Torpedo/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Fenómenos Electrofisiológicos , Femenino , Adyuvante de Freund/administración & dosificación , Inmunización , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Debilidad Muscular/inmunología , Debilidad Muscular/fisiopatología , Miastenia Gravis Autoinmune Experimental/diagnóstico , Fragmentos de Péptidos , Fenotipo , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/química , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ), characterized with muscle weakness. While MG develops due to acetylcholine receptor (AChR) antibodies in most patients, antibodies to muscle-specific receptor tyrosine kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4) may also be identified. Experimental autoimmune myasthenia gravis (EAMG) has been previously induced by both LRP4 immunization and passive transfer of LRP4 antibodies. OBJECTIVE: Our aim was to confirm previous results and to test the pathogenic effects of LRP4 immunization in a commonly used mouse strain C57BL/6 (B6) using a recombinantly expressed human LRP4 protein. METHODS: B6 mice were immunized with human LRP4 in CFA, Torpedo Californica AChR in CFA or only CFA. Clinical and pathogenic aspects of EAMG were compared among groups. RESULTS: LRP4- and AChR-immunized mice showed comparable EAMG clinical severity. LRP4-immunized mice displayed serum antibodies to LRP4 and NMJ IgG and complement factor C3 deposits. IgG2 was the dominant anti-LRP4 isotype. Cultured lymph node cells of LRP4- and AChR-immunized mice gave identical pro-inflammatory cytokine (IL-6, IFN-γ and IL-17) responses to LRP4 and AChR stimulation, respectively. CONCLUSION: Our results confirm the EAMG-inducing action of LRP4 immunization and identify B6 as a LRP4-EAMG-susceptible mouse strain. Demonstration of complement fixing anti-LRP4 antibodies in sera and complement/IgG deposits at the NMJ of LRP4-immunized mice indicates complement activation as a putative pathogenic mechanism. We have thus developed a practical LRP4-induced EAMG model using a non-conformational protein and a widely available mouse strain for future investigation of LRP4-related MG.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Inmunización/métodos , Inmunoglobulina G/biosíntesis , Proteínas Relacionadas con Receptor de LDL/administración & dosificación , Miastenia Gravis Autoinmune Experimental/inmunología , Receptores Colinérgicos/administración & dosificación , Animales , Complemento C3/metabolismo , Adyuvante de Freund/administración & dosificación , Humanos , Isotipos de Inmunoglobulinas/biosíntesis , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-17/biosíntesis , Interleucina-17/inmunología , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Proteínas Relacionadas con Receptor de LDL/inmunología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Linfocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Miastenia Gravis Autoinmune Experimental/inducido químicamente , Miastenia Gravis Autoinmune Experimental/patología , Cultivo Primario de Células , Receptores Colinérgicos/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Índice de Severidad de la Enfermedad , Torpedo/metabolismoRESUMEN
Myasthenia gravis (MG) is usually caused by antibodies against the muscle acetylcholine receptor (AChR). Experimental autoimmune MG (EAMG) is the animal model of MG, typically induced by immunization of rodents with AChR isolated from the electric organ of Torpedo californica. We have successfully induced EAMG in Lewis rats by immunization with the extracellular domains (ECDs) of the human AChR subunits (α, ß, γ, δ and ε) expressed in yeast. Analysis of the antibody titers revealed a robust antigenic response against all the peptides, but a marked difference in their pathogenicity; the α subunit ECD was the most pathogenic, resulting in the highest percentage of affected animals. Measurements of antibody titers, electromyographic tests and quantitation of the muscle AChR content, offered further support to these findings. The EAMG models presented herein, could be used for studying subunit-specific pathogenic mechanisms, and, more importantly, as tools for the evaluation of antigen-specific therapeutic approaches, which rely on the human AChR.
Asunto(s)
Modelos Animales de Enfermedad , Miastenia Gravis Autoinmune Experimental/inducido químicamente , Miastenia Gravis Autoinmune Experimental/inmunología , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/inmunología , Animales , Femenino , Humanos , Miastenia Gravis Autoinmune Experimental/metabolismo , Dominios Proteicos/fisiología , Subunidades de Proteína/inmunología , Subunidades de Proteína/toxicidad , Ratas , Ratas Endogámicas LewRESUMEN
BACKGROUND: Myasthenia gravis (MG) is an antibody-mediated autoimmune disease of the neuromuscular junction (NMJ), mostly associated with acetylcholine receptor (AChR) antibodies. Around 5-10 % of MG patients show antibodies to muscle-specific tyrosine kinase (MuSK). Mesenchymal stem cell (MSC) administration has been shown to ameliorate muscle weakness in the experimental autoimmune myasthenia gravis (EAMG) model induced by AChR immunization. METHODS: To investigate the efficacy of stem cell treatment in MuSK-related EAMG, clinical and immunological features of MuSK-immunized mice with or without dental follicle MSC (DFMSC) treatment were compared. RESULTS: MuSK-immunized mice intravenously treated with DFMSC after second and third immunizations showed significantly lower EAMG incidence and severity and reduced serum anti-MuSK antibody, NMJ IgG, and C3 deposit levels and CD11b+ lymph node cell ratios. Moreover, lymph node cells of DFMSC-administered mice showed reduced proliferation and IL-6 and IL-12 production responses to MuSK stimulation. By contrast, proportions of B and T cell populations and production of a wide variety of cytokines were not affected from DFMSC treatment. CONCLUSIONS: Our results suggest that DFMSC treatment shows its beneficial effects mostly through suppression of innate immune system, whereas other immune functions appear to be preserved. Stem cell treatment might thus constitute a specific and effective treatment method in MuSK-associated MG.
Asunto(s)
Saco Dental/trasplante , Inmunización/métodos , Trasplante de Células Madre Mesenquimatosas/métodos , Debilidad Muscular/terapia , Proteínas Tirosina Quinasas Receptoras/administración & dosificación , Receptores Colinérgicos/administración & dosificación , Animales , Células Cultivadas , Saco Dental/citología , Saco Dental/inmunología , Femenino , Humanos , Células Madre Mesenquimatosas/inmunología , Ratones , Ratones Endogámicos C57BL , Debilidad Muscular/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Colinérgicos/inmunologíaRESUMEN
Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are caused by Ab-mediated autoimmune responses to muscle nicotinic acetylcholine receptors (AChRs) that impair neuromuscular transmission, thereby causing muscle weakness. Previously, we discovered that i.p. injection of a therapeutic vaccine consisting of bacterially expressed cytoplasmic domains of human AChR subunits reduced the development of chronic EAMG in rats. In this article, we show that immunization with the therapeutic vaccine in adjuvants does not induce EAMG and, thus, is safe. The potency and efficacy of the therapeutic vaccine were greatly increased by s.c. administration of repeated low doses in IFA. Onset of chronic EAMG could be prevented. Established chronic EAMG could be rapidly reversed, modeling therapy of chronic MG. Therapy reduced pathological Abs assayed by immune precipitation of a main immunogenic region chimera. Successfully treated rats exhibited long-term resistance to reinduction of EAMG, suggesting a lasting cure of MG. A long-term effect of therapy was to change the isotype of the pathogenic Ab response from IgG2b, which fixes complement, to IgG1, which does not. Prevention and reversal of chronic EAMG was not caused by the isotype switch, but the isotype switch may contribute to resistance to reinduction of EAMG. Immunization with AChR cytoplasmic domains in adjuvant is promising as a safe, Ag-specific, potent, effective, rapidly acting, and long-lasting therapeutic approach to MG.
Asunto(s)
Miastenia Gravis Autoinmune Experimental/tratamiento farmacológico , Subunidades de Proteína/inmunología , Receptores Colinérgicos/inmunología , Vacunas/inmunología , Animales , Autoanticuerpos/biosíntesis , Femenino , Humanos , Cambio de Clase de Inmunoglobulina/efectos de los fármacos , Inmunoglobulina G/biosíntesis , Inyecciones Subcutáneas , Músculos/efectos de los fármacos , Músculos/inmunología , Músculos/patología , Miastenia Gravis Autoinmune Experimental/inducido químicamente , Miastenia Gravis Autoinmune Experimental/inmunología , Miastenia Gravis Autoinmune Experimental/patología , Subunidades de Proteína/administración & dosificación , Subunidades de Proteína/química , Ratas , Ratas Endogámicas Lew , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/química , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Torpedo , Vacunas/administración & dosificación , Vacunas/químicaRESUMEN
RATIONALE AND OBJECTIVES: In rats, 5-hydroxytryptamine(6) (5-HT(6)) receptor antagonists improve learning and memory, but the effects of agonists are poorly defined. This study investigated the effects of 5-HT(6) receptor agonists and antagonists on a rodent model of recognition memory. METHODS: Selective 5-HT(6) receptor agonists and antagonists were administered either alone, after a scopolamine-induced impairment, or combined with sub-effective doses of the acetylcholinesterase inhibitor, donepezil, or the glutamate NMDA receptor antagonist, memantine, in a novel object discrimination paradigm in adult rats. RESULTS: After a 4-h inter-trial delay to induce natural forgetting, vehicle-treated rats spent an equivalent time exploring novel and familiar objects during the choice trial. The 5-HT(6) receptor agonists, E-6801 (1.25-10 mg/kg i.p.) and EMD-386088 (5-10 mg/kg i.p.), and antagonists, SB-271046 and Ro 04-6790 (5 and 10 mg/kg), along with donepezil (0.1-3 mg/kg) and memantine (5-20 mg/kg) all produced significant and mostly dose-dependent increases in novel object exploration, indicative of memory enhancement. Furthermore, sub-effective doses of E-6801 (1 mg/kg) when co-administered with either SB-271046 (3 mg/kg), donepezil (0.1 mg/kg) or memantine (5 mg/kg), and EMD-386088 (2 mg/kg) co-administered with SB-271046 (3 mg/kg) also significantly enhanced object-recognition memory. Additionally, using a 1-min inter-trial delay, E-6801 (2.5 and 5 mg/kg) was as effective as donepezil (0.3 and 1 mg/kg) in reversing a scopolamine-induced (0.5 mg/kg) impairment in object recognition. CONCLUSIONS: This is the first study to demonstrate that E-6801, a potent 5-HT(6) receptor agonist, improves recognition memory by combined modulation of cholinergic and glutamatergic neurotransmission.
Asunto(s)
Indoles/farmacología , Memoria/efectos de los fármacos , Receptores de Serotonina/efectos de los fármacos , Agonistas de Receptores de Serotonina/farmacología , Sulfonamidas/farmacología , Tiazoles/farmacología , Animales , Relación Dosis-Respuesta a Droga , Indoles/administración & dosificación , Masculino , Ratas , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/administración & dosificación , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/administración & dosificación , Sulfonamidas/administración & dosificación , Tiazoles/administración & dosificaciónRESUMEN
Los factores genéticos pueden determinar susceptibilidad para la adicción a la nicotina. En los últimos años se ha señalado la importancia que la genética puede tener en el proceso de adicción al tabaco y en la difi cultad para su abandono, aún cuando se realicen intentos serios de abandono del tabaco. Se ha estudiado la relación de determinados genes en el consumo de tabaco, especialmente los que controlan el metabolismo hepático de la nicotina derivados del citocromo P-450, o los que regulan el proceso cerebral de liberación de dopamina. En estos estudios se ha observado que la existencia de ciertos polimorfi smos puede explicar hasta el 60% de la varianza. Cada vez existe un mayor conocimiento y una mayor comprensión de los factores genéticos involucrados en la dependencia nicotínica. Los receptores nicotínicos juegan un papel muy importante en la modulación de la adicción a la nicotina, habiéndose señalado el gen del receptor nicotínico CHRNA5 como un indicador fuertemente asociado a la dependencia de la nicotina. Existen determinadas variaciones genéticas en el gen del receptor CHRNA5 que son más frecuente en fumadores que en no fumadores, lo que podría condicionar la predisposición al consumo de tabaco y la mayor o menor difi cultad para el abandono del tabaco en los fumadores en general y en los que acuden a consultas de deshabituación en particular(AU)
No disponible
Asunto(s)
Humanos , Masculino , Femenino , Tabaquismo/tratamiento farmacológico , Tabaquismo/terapia , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos , Receptores Colinérgicos/uso terapéutico , Agonistas Nicotínicos/uso terapéutico , Fumar/genética , Salud Pública/métodos , Salud Pública/tendencias , Indicadores de Morbimortalidad , Cese del Hábito de Fumar/estadística & datos numéricos , Cese del Uso de Tabaco/métodosRESUMEN
Familial occurrence of myasthenia gravis is uncommon and reports of maternal transmission of muscle-specific tyrosine kinase (MuSK) antibody myasthenia are rarer still. We report two families with maternal transmission of MuSK antibody myasthenia gravis to the offspring by different mechanisms. The first family demonstrates transmission genetic susceptibility of inheriting myasthenia gravis from MuSK antibodies, whereas the second one demonstrates transplacental transmission of MuSK antibodies at birth.
Asunto(s)
Inmunidad Materno-Adquirida , Intercambio Materno-Fetal , Miastenia Gravis/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Colinérgicos/genética , Adulto , Autoanticuerpos/administración & dosificación , Autoanticuerpos/biosíntesis , Niño , Femenino , Humanos , Inmunidad Materno-Adquirida/genética , Recién Nacido , Masculino , Intercambio Materno-Fetal/genética , Miastenia Gravis/inmunología , Miastenia Gravis/metabolismo , Embarazo , Proteínas Tirosina Quinasas Receptoras/administración & dosificación , Receptores Colinérgicos/administración & dosificación , Adulto JovenRESUMEN
Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Previously we have revealed that oral treatment with the less native recombinant fragment of the extracellular domain of the human AChR (Halpha1-205) suppressed ongoing EAMG, whereas the more native recombinant Trx-Halpha1-210 exacerbated EAMG. In this study, we speculated on the role of B-cell epitopes in oral tolerogens for the induction of oral tolerance in EAMG. We developed a B-cell epitope-free AChR fragment (BF-AChR) by removing two major B-cell epitopes (67-76 and 129-145) from Trx-Halpha1-210. BF-AChR exhibited a poor response to EAMG sera and to AChR-specific B- and T-cells while its parent fragment, Trx-Halpha1-210, showed much higher reactivity. Oral administration of BF-AChR ameliorated the symptoms in ongoing myasthenic rats accompanied by a significant decrease in AChR-specific humoral and Th1 cellular responses. The underlying mechanism for BF-AChR-induced oral tolerance was mediated by a shift from Th1 to regulatory T-cell (IL-10(+), CD4(+) TGF-beta(+) or Foxp3(+)) responses. This shift was assessed by changes in the cytokine profile and a deviation in the anti-AChR IgG isotypes from IgG2a/IgG2b to IgG1. Our results suggest that the removal of pathogenic B-cell epitopes from AChR fragments increases tolerogenicity by reducing the activation and proliferation of autoreactive B- and T-cells. Collectively, careful consideration of the immunogenicity of a tolerogen is necessary to induce successful oral tolerance in autoimmune disorders.
Asunto(s)
Epítopos de Linfocito B/inmunología , Miastenia Gravis Autoinmune Experimental/tratamiento farmacológico , Miastenia Gravis Autoinmune Experimental/inmunología , Receptores Colinérgicos/inmunología , Proteínas Recombinantes/uso terapéutico , Administración Oral , Animales , Autoanticuerpos/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Citocinas/inmunología , Regulación hacia Abajo/efectos de los fármacos , Femenino , Inmunoglobulina G/inmunología , Inmunohistoquímica , Mediadores de Inflamación/inmunología , Miastenia Gravis Autoinmune Experimental/sangre , Ratas , Ratas Endogámicas Lew , Receptores Colinérgicos/administración & dosificación , Proteínas Recombinantes/farmacología , Eliminación de Secuencia , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Células TH1/efectos de los fármacos , Células TH1/inmunologíaRESUMEN
Immunization with Torpedo acetylcholine receptor (TAChR) induces experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 (B6) mice. EAMG development needs IL-12, which drives differentiation of Th1 cells. The role of IFN-gamma, an important Th1 effector, is not clear and that of IL-17, a proinflammatory cytokine produced by Th17 cells, is unknown. In this study, we examined the effect of simultaneous absence of IL-12 and IFN-gamma on EAMG susceptibility, using null mutant B6 mice for the genes of both the IL-12/IL-23 p40 subunit and IFN-gamma (dKO mice). Wild-type (WT) B6 mice served as control for EAMG induction. All mice were immunized with TAChR in Freund's adjuvant. dKO mice developed weaker anti-TAChR CD4(+)T cells and Ab responses than WT mice. Yet, they developed EAMG symptoms, anti-mouse acetylcholine receptor (AChR) Ab, and CD4(+) T cell responses against mouse AChR sequences similar to those of WT mice. dKO and WT mice had similarly reduced AChR content in their muscles, and IgG and complement at the neuromuscular junction. Naive dKO mice had significantly fewer NK, NKT, and CD4(+)CD25(+)Foxp3(+) T regulatory (Treg) cells than naive WT mice. Treg cells from TAChR-immunized dKO mice had significantly less suppressive activity in vitro than Treg cells from TAChR-immunized WT mice. In contrast, TAChR-specific CD4(+) T cells from TAChR-immunized dKO and WT mice secreted comparable amounts of IL-17 after stimulation in vitro with TAChR. The susceptibility of dKO mice to EAMG may be due to reduced Treg function, in the presence of a normal function of pathogenic Th17 cells.
Asunto(s)
Interferón gamma/deficiencia , Interferón gamma/genética , Interleucina-12/deficiencia , Interleucina-12/genética , Interleucina-23/deficiencia , Interleucina-23/genética , Miastenia Gravis Autoinmune Experimental/inmunología , Linfocitos T Reguladores/inmunología , Animales , Células Cultivadas , Femenino , Predisposición Genética a la Enfermedad , Inmunoglobulina G/análisis , Interleucina-17/metabolismo , Interleucina-17/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Miastenia Gravis Autoinmune Experimental/genética , Miastenia Gravis Autoinmune Experimental/patología , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/inmunología , Receptores Colinérgicos/fisiología , Índice de Severidad de la Enfermedad , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Células TH1/inmunología , Células TH1/metabolismoRESUMEN
Dendritic cells (DCs) have the potential to activate or tolerize T cells in an Ag-specific manner. Although the precise mechanism that determines whether DCs exhibit tolerogenic or immunogenic functions has not been precisely elucidated, growing evidence suggests that DC function is largely dependent on differentiation status, which can be manipulated using various growth factors. In this study, we investigated the effects of mobilization of specific DC subsets-using GM-CSF and fms-like tyrosine kinase receptor 3-ligand (Flt3-L)-on the susceptibility to induction of experimental autoimmune myasthenia gravis (EAMG). We administered GM-CSF or Flt3-L to C57BL/6 mice before immunization with acetylcholine receptor (AChR) and observed the effect on the frequency and severity of EAMG development. Compared with AChR-immunized controls, mice treated with Flt3-L before immunization developed EAMG at an accelerated pace initially, but disease frequency and severity was comparable at the end of the observation period. In contrast, GM-CSF administered before immunization exerted a sustained suppressive effect against the induction of EAMG. This suppression was associated with lowered serum autoantibody levels, reduced T cell proliferative responses to AChR, and an expansion in the population of FoxP3+ regulatory T cells. These results highlight the potential of manipulating DCs to expand regulatory T cells for the control of autoimmune diseases such as MG.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Factores de Transcripción Forkhead , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Miastenia Gravis Autoinmune Experimental/prevención & control , Linfocitos T Reguladores/citología , Animales , Enfermedades Autoinmunes/terapia , Comunicación Celular/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Inmunización , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos C57BL , Miastenia Gravis Autoinmune Experimental/inmunología , Miastenia Gravis Autoinmune Experimental/terapia , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/inmunologíaRESUMEN
UNLABELLED: Myasthenia gravis (MG) is mostly caused by anti-acetylcholine receptor (AChR) auto-antibodies (Abs). Such Abs are undetectable in 10-15% of MG patients, but many have anti-muscle-specific kinase (MuSK) Abs. We injected recombinant rat-MuSK extracellular domain in H-2(a), H-2(b), H-2(bm12) and H-2(d) mice. Certain strains exhibited exercise-induced fatigue, tremors, weight loss, and some died after 2-3 injections. Compound muscle action potentials showed decrement with low-frequency repetitive nerve stimulation. Miniature endplate potentials decreased, suggesting lower numbers of endplates functional AChRs. Myasthenic sera inhibited agrin-induced AChR aggregation in C2C12 myotubes. CONCLUSION: Anti-MuSK Abs induce MG, which might also result from blocking the agrin-signaling pathway.
Asunto(s)
Líquido Extracelular/enzimología , Miastenia Gravis/enzimología , Miastenia Gravis/inmunología , Proteínas Tirosina Quinasas Receptoras/administración & dosificación , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/inmunología , Potenciales de Acción/inmunología , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/sangre , Células Cultivadas , Cricetinae , Líquido Extracelular/inmunología , Femenino , Inmunización , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Esquelético/inmunología , Músculo Esquelético/metabolismo , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunologíaRESUMEN
In myasthenia gravis (MG), TNF and IL-1beta polymorphisms and high serum levels of these proinflammatory cytokines have been observed. Likewise, TNF and IL-1beta are critical for the activation of acetylcholine receptor (AChR)-specific T and B cells and for the development of experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. We tested the therapeutic effect of human recombinant IL-1 receptor antagonist (IL-1ra) in C57BL/6 mice with EAMG. Multiple daily injections of 0.01 mg of IL-1ra administered for 2 wk following two AChR immunizations decreased the incidence and severity of clinical EAMG. Furthermore, IL-1ra treatment of mice with ongoing clinical EAMG reduced the clinical symptoms of disease. The IL-1ra-mediated suppression of clinical disease was associated with suppressed serum IFN-gamma, TNF-alpha, IL-1beta, IL-2, IL-6, C3, and anti-AChR IgG1 without influencing total serum IgG. Therefore, IL-1ra could be used as a nonsteroidal drug for the treatment of MG.
Asunto(s)
Complemento C3/antagonistas & inhibidores , Citocinas/antagonistas & inhibidores , Inmunoglobulina G/sangre , Mediadores de Inflamación/antagonistas & inhibidores , Miastenia Gravis Autoinmune Experimental/prevención & control , Receptores Colinérgicos/inmunología , Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/uso terapéutico , Animales , Células Cultivadas , Complemento C3/fisiología , Citocinas/sangre , Inmunidad Celular , Epítopos Inmunodominantes/administración & dosificación , Epítopos Inmunodominantes/inmunología , Inmunoglobulina G/biosíntesis , Inmunosupresores/administración & dosificación , Inmunosupresores/uso terapéutico , Mediadores de Inflamación/sangre , Proteína Antagonista del Receptor de Interleucina 1 , Masculino , Ratones , Ratones Endogámicos C57BL , Miastenia Gravis Autoinmune Experimental/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Subunidades de Proteína/administración & dosificación , Subunidades de Proteína/inmunología , Receptores Colinérgicos/administración & dosificación , Índice de Severidad de la Enfermedad , Sialoglicoproteínas/administración & dosificación , Sialoglicoproteínas/fisiología , Torpedo/inmunologíaRESUMEN
Because presentation of acetylcholine receptor (AChR) peptides to T cells is critical to the development of myasthenia gravis, we examined the role of cathepsin S (Cat S) in experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. Compared with wild type, Cat S null mice were markedly resistant to the development of EAMG, and showed reduced T and B cell responses to AChR. Cat S null mice immunized with immunodominant AChR peptides showed weak responses, indicating failed peptide presentation accounted for autoimmune resistance. A Cat S inhibitor suppressed in vitro IFN-gamma production by lymph node cells from AChR-immunized, DR3-bearing transgenic mice. Because Cat S null mice are not severely immunocompromised, Cat S inhibitors could be tested for their therapeutic potential in EAMG.
Asunto(s)
Catepsinas/fisiología , Miastenia Gravis Autoinmune Experimental/enzimología , Miastenia Gravis Autoinmune Experimental/inmunología , Animales , Presentación de Antígeno/genética , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Autoanticuerpos/sangre , Linfocitos B/patología , Catepsinas/antagonistas & inhibidores , Catepsinas/deficiencia , Catepsinas/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Células Dendríticas/citología , Células Dendríticas/inmunología , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Epítopos de Linfocito T/inmunología , Femenino , Antígeno HLA-DR3/genética , Antígeno HLA-DR3/inmunología , Antígeno HLA-DR3/metabolismo , Humanos , Inmunidad Innata/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miastenia Gravis Autoinmune Experimental/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/inmunología , Receptores Colinérgicos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , TorpedoRESUMEN
Experimental autoimmune myasthenia gravis (EAMG) is severe in RIIIS/J mice, despite a significant B cell immunodeficiency and a massive TCR V beta gene deletion. Severity of EAMG in RIIIS/J mice is greater than MHC-identical (H-2(r)) B10.RIII mice, suggesting the influence of non-MHC genes as an EAMG-potentiating factor in this strain. To delineate the role of deleted TCR V beta genes in RIIIS/J mice, we obtained (RIIIS/J x B10.RIII)F(1) (V beta(b/c)) x RIIIS/J (V beta(c)) backcross mice using Mendelian genetic methods and immunized them with acetylcholine receptor. EAMG susceptibility was not elevated in mice with V beta(c) genotype having 70% V beta gene deletion. Next, we performed microarray analysis on 12,488 spleen cDNAs obtained from spleens of naive RIIIS/J and B10.RIII mice. In RIIIS/J mice, 263 cDNAs were overexpressed and 303 cDNAs were underexpressed greater than 2-fold, compared with B10.RIII mice. TCR gene expression was augmented, whereas NK receptor, C1q, and C3 gene expressions were diminished in RIIIS/J mice. RIIIS/J mice also had increased lymph node T cell counts, elevated serum anti-AChR Ab levels, and serum C3 and C1q-conjugated circulating immune complex levels. A direct correlation between increased serum C1q-conjugated circulating immune complex levels and disease severity was observed in RIIIS/J mice.
Asunto(s)
Adyuvantes Inmunológicos/sangre , Agammaglobulinemia/inmunología , Agammaglobulinemia/patología , Complejo Antígeno-Anticuerpo/sangre , Autoanticuerpos/fisiología , Miastenia Gravis Autoinmune Experimental/inmunología , Miastenia Gravis Autoinmune Experimental/patología , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/fisiología , Agammaglobulinemia/genética , Animales , Complejo Antígeno-Anticuerpo/biosíntesis , Complejo Antígeno-Anticuerpo/fisiología , Antígenos de Superficie/análisis , Autoanticuerpos/biosíntesis , Subgrupos de Linfocitos B/patología , Complemento C1q/biosíntesis , Complemento C3/biosíntesis , Eliminación de Gen , Centro Germinal/patología , Antígenos H-2/genética , Prueba de Histocompatibilidad , Inmunoglobulina G/sangre , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Recuento de Linfocitos , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Miastenia Gravis Autoinmune Experimental/epidemiología , Miastenia Gravis Autoinmune Experimental/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/inmunología , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Receptores KIR , Índice de Severidad de la Enfermedad , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Subgrupos de Linfocitos T/patologíaRESUMEN
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.
Asunto(s)
Citocinas/metabolismo , Tolerancia Inmunológica/fisiología , Terapia de Inmunosupresión , Miastenia Gravis/terapia , Animales , Modelos Animales de Enfermedad , Humanos , Inmunidad Mucosa , Interleucina-2/metabolismo , Miastenia Gravis/inmunología , Miastenia Gravis Autoinmune Experimental/inducido químicamente , Miastenia Gravis Autoinmune Experimental/prevención & control , Subunidades de Proteína , Ratas , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/inmunología , Proteínas Recombinantes , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.