RESUMEN
Current immunotherapy of myasthenia gravis (MG) is often effective, but entails risks of infection and neoplasia. The "Guided Missile" strategy described here is designed to target and eliminate the individual's unique AChR-specific T cell repertoire, without otherwise interfering with the immune system. We genetically engineered dendritic cells to present AChR epitopes and simultaneously express Fas ligand in an ongoing EAMG model. In both in vitro and in vivo experiments, these engineered cells specifically killed AChR-responsive T cells without otherwise damaging the immune system. AChR antibodies were markedly reduced in the treated mice. Translation of this method to treat human MG is possible.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/trasplante , Inmunoterapia/métodos , Miastenia Gravis Autoinmune Experimental/terapia , Animales , Anticuerpos/sangre , Células Cultivadas , Modelos Animales de Enfermedad , Epítopos/genética , Epítopos/inmunología , Proteína Ligando Fas/genética , Proteína Ligando Fas/inmunología , Femenino , Ingeniería Genética , Ratones , Ratones Endogámicos C57BL , Miastenia Gravis Autoinmune Experimental/sangre , Miastenia Gravis Autoinmune Experimental/inmunología , Receptores Colinérgicos/genética , Receptores Colinérgicos/inmunología , Linfocitos T/inmunologíaRESUMEN
PURPOSE OF RESEARCH: Although the pathogenesis of myasthenia gravis (MG) as an antibody mediated disorder of acetylcholine receptors (AChRs) at neuromuscular junctions is well understood, the origin of the autoimmune response is unclear. The thymus is intimately involved in initiation of the autoimmune response; the antigen, AChR, is present in the thymus, but how the autoimmune response is triggered is not known. Granzyme B (GrB), a proteolytic enzyme present in cytolytic T cells and natural killer (NK) cells, selectively cleaves many potential autoantigens (but few non-autoantigens), generating novel fragments that trigger autoreactive responses. This protease has been strongly implicated in the pathogenesis of several autoimmune diseases including lupus, rheumatoid arthritis, dermatomyositis, and others. In the studies described in this manuscript, we examined the ability of GrB to cleave the AChR subunits, and performed biochemical, immunohistochemical and molecular studies on thymus glands from myasthenic patients and controls to assess GrB expression. MAIN RESULTS: GrB efficiently and specifically cleaves subunits of AChR, especially the epsilon subunit. GrB is present in thymus glands from myasthenia patients, but is absent in control thymuses. CONCLUSIONS: Our results provide evidence supporting a potential role for GrB in the process of initiation of MG, and are consistent with the concept of an immunodominant epsilon epitope.
Asunto(s)
Granzimas/metabolismo , Granzimas/farmacología , Miastenia Gravis/patología , Timo/efectos de los fármacos , Timo/metabolismo , Autoinmunidad , Línea Celular , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Granzimas/genética , Humanos , Metionina/metabolismo , Receptores Colinérgicos/clasificación , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Receptores Nicotínicos , Isótopos de Azufre/metabolismo , TransfecciónRESUMEN
Although treatment of MG with general immunosuppressive agents is often effective, it has important drawbacks, including suppression of the immune system as a whole, with the risks of infection and neoplasia, and numerous other adverse side effects. Ideally, treatment of MG should eliminate the specific pathogenic autoimmune response to AChR, without otherwise suppressing the immune system or producing other adverse side effects. Although antibodies to AChR are directly responsible for the loss of AChRs at neuromuscular junctions in MG, the AChR antibody response is T cell-dependent, and immunotherapy directed at T cells can abrogate the autoantibody response, with resulting benefit. As in other autoimmune diseases, the T cell response in MG is highly heterogeneous. The design of specific immunotherapy must take this heterogeneity into account and target the entire repertoire of AChR-specific T cells. We describe our investigation of a novel strategy for specific immunotherapy of MG, involving gene transfer to convert antigen-presenting cells (APCs) to "guided missiles" that target AChR-specific T cells, and that induce apoptosis and elimination of those T cells. This strategy uses the ability of APCs from a given individual to present the entire spectrum of AChR epitopes unique for that individual, and thereby to target the entire repertoire of antigen-specific T cells of the same individual. Using viral vectors, we have genetically engineered the APCs to process and present the most important domain of the AChR molecule, and to express a "warhead" of Fas ligand (FasL) to eliminate the activated AChR-specific T cells with which they interact. Our results show that the APCs express the appropriate gene products, and effectively and specifically eliminate AChR-specific T cells by the Fas/FasL pathway, while sparing T cells of other specificities.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Ingeniería Genética/métodos , Inmunoterapia , Miastenia Gravis Autoinmune Experimental/terapia , Linfocitos T/inmunología , Animales , Muerte Celular , Línea Celular , Células Dendríticas , Humanos , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miastenia Gravis Autoinmune Experimental/inducido químicamente , Miastenia Gravis Autoinmune Experimental/veterinaria , Proteínas/metabolismo , Ratas , Ratas Endogámicas Lew , Receptores Colinérgicos/química , Receptores Colinérgicos/inmunología , Receptores Colinérgicos/metabolismo , Transducción de Señal , Bazo/citología , Bazo/inmunología , Linfocitos T/metabolismo , Factores de Tiempo , Receptor fas/metabolismoRESUMEN
We describe a strategy for specific immunotherapy of myasthenia gravis (MG) based on genetic engineering of antigen presenting cells (APCs) to present the autoantigen acetylcholine receptor (AChR) and express the "warhead" Fas ligand (FasL). For transduction of APCs we prepared recombinant attenuated vaccinia virus vectors carrying the following three gene constructs: (i) AChR fused to LAMP1 to present AChR and target AChR-specific T cells; (ii) FasL to eliminate the targeted T cells; and (iii) truncated FADD to protect APCs from self-destruction by FasL. The engineered APCs effectively expressed the genes of interest and killed AChR-specific T cells in culture by the Fas/FasL pathway. T cells specific for an unrelated antigen were spared. Our in vitro demonstration that engineered APCs target and kill antigen-specific T cells represents a promising novel strategy for specific immunotherapy of MG and other autoimmune diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Autoantígenos/inmunología , Proteínas Portadoras/inmunología , Glicoproteínas de Membrana/inmunología , Miastenia Gravis Autoinmune Experimental/inmunología , Receptores Colinérgicos/inmunología , Linfocitos T/inmunología , Receptor fas/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos CD/genética , Antígenos CD/inmunología , Autoantígenos/genética , Proteínas Portadoras/genética , Línea Celular , Proteína Ligando Fas , Proteína de Dominio de Muerte Asociada a Fas , Femenino , Expresión Génica , Vectores Genéticos , Inmunoterapia , Proteína 1 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Miastenia Gravis Autoinmune Experimental/terapia , Ratas , Ratas Endogámicas Lew , Receptores Colinérgicos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Células Tumorales Cultivadas , Virus VacciniaRESUMEN
We tested the hypothesis that APCs genetically engineered to present an Ag and to express Fas ligand (FasL) simultaneously can target and eliminate Ag-specific T cells. Transgenic T cells specific for influenza hemagglutinin (HA) were used as targets. We prepared recombinant vaccinia virus vectors (VVV) to transfer the gene constructs individually or simultaneously into APCs. We prevented unwanted viral replication by attenuating the VVVs with psoralen-UV light treatment. For presentation of the HA Ag, APCs were transduced with cDNA for HA flanked by sequences of the lysosome-associated membrane protein that direct efficient processing and presentation of the Ag by APCs. As a "warhead" for the APCs, we transduced them with the gene for FasL, which induces apoptosis of Fas-expressing activated T cells. To protect the transduced APCs from self-destruction by FasL, we transferred cDNA for a truncated form of Fas-associated death domain, which inhibits Fas-mediated cell death. Our results show that the engineered APCs effectively expressed the genes of interest. APCs transduced with VVV carrying all three gene constructs specifically killed HA-transgenic T cells in culture. Coculture with T cells specific for an unrelated Ag (OVA) had no significant effect. Our in vitro findings show that APCs can be genetically engineered to target and kill Ag-specific T cells and represent a promising novel strategy for the specific treatment of autoimmune diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Traslado Adoptivo/métodos , Células Presentadoras de Antígenos/trasplante , Ingeniería de Proteínas/métodos , Adyuvantes Inmunológicos/genética , Animales , Apoptosis/genética , Apoptosis/inmunología , Proteínas Portadoras/genética , Línea Celular , Citotoxicidad Inmunológica/genética , Proteína Ligando Fas , Proteína de Dominio de Muerte Asociada a Fas , Marcación de Gen/métodos , Técnicas de Transferencia de Gen , Vectores Genéticos/síntesis química , Vectores Genéticos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Interfase/genética , Interfase/inmunología , Ligandos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/toxicidad , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Recombinación Genética/inmunología , Linfocitos T/inmunología , Linfocitos T/patología , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Receptor fas/genética , Receptor fas/inmunologíaRESUMEN
The authors report the use mycophenolate mofetil (MM) in the treatment of neuromuscular diseases. Thirty-eight patients (32 with MG, three with inflammatory myopathy, and three with chronic acquired demyelinating neuropathy) were treated with MM for an average duration of 12 months. All patients tolerated MM without major side effects. Twenty-four patients improved either in their functional status or in their ability to reduce corticosteroid dose. Mean time to improvement was 5 months.
Asunto(s)
Inmunosupresores/administración & dosificación , Miastenia Gravis/tratamiento farmacológico , Ácido Micofenólico/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunosupresores/efectos adversos , Masculino , Persona de Mediana Edad , Ácido Micofenólico/efectos adversos , Ácido Micofenólico/análogos & derivados , Miositis/tratamiento farmacológico , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
The pathogenesis of motor neuron loss in amyotrophic lateral sclerosis (ALS) is thought to involve both glutamate-mediated excitotoxicity and oxidative damage due to the accumulation of free radicals and other toxic molecules. Cyclooxygenase-2 (COX-2) may play a key role in these processes by producing prostaglandins, which trigger astrocytic glutamate release, and by inducing free radical formation. We tested the effects of COX-2 inhibition in an organotypic spinal cord culture model of ALS. The COX-2 inhibitor (SC236) provided significant protection against loss of spinal motor neurons in this system, suggesting that it may be useful in the treatment of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Isoenzimas/metabolismo , Neuronas Motoras/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Ciclooxigenasa 2 , Modelos Animales de Enfermedad , Técnicas de Cultivo de Órganos , Pirazoles/metabolismo , Ratas , Médula Espinal/metabolismo , Sulfonamidas/metabolismoRESUMEN
We describe a strategy for specific immunotherapy of autoimmune disease based on targeting the antigen-specific T cells in an experimental model of myasthenia gravis. To address the problem of heterogeneity of the T cell repertoire, we have genetically engineered antigen presenting cells (APCs) to process and present epitopes of the autoantigen, acetylcholine receptor (AChR), to the entire spectrum of AChR-specific syngeneic T cells. APCs derived from BALB/c mice were stably transfected with cDNA for the key immunogenic domain of the AChR alpha-subunit, flanked by sequences of the lysosome-associated membrane protein (LAMP) that direct APCs to process and present the antigen via the MHC Class II pathway. Transfected APCs strongly stimulated AChR-specific T cells from BALB/c mice. Fas ligand, or antibody to Fas, abrogated the T cell response, by inducing apoptosis of the APC-stimulated T cells. The new results of this investigation are (1) that autoreactive T cells can be effectively targeted by autologous APCs that are engineered to present the relevant autoantigen, and (2) that these specifically targeted and activated T cells can be profoundly inhibited by agents that trigger the Fas-mediated apoptosis pathway. The present findings suggest that engineering APCs for simultaneous presentation of the autoantigen and delivery of FasL will provide a powerful strategy for the elimination of autoreactive T cells.
Asunto(s)
Células Presentadoras de Antígenos/fisiología , Enfermedades Autoinmunes/terapia , Epítopos , Marcación de Gen/métodos , Inmunoterapia/métodos , Linfocitos T/inmunología , Animales , Anticuerpos/farmacología , Proteína Ligando Fas , Femenino , Ingeniería Genética , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Linfocitos T/efectos de los fármacos , Receptor fas/inmunologíaRESUMEN
Large suppressive macrophages (LSM) were induced by restimulating spleen cells from rats with experimental autoimmune myasthenia gravis (EAMG) in vitro, with the autoantigen acetylcholine receptor (AChR) in the presence of cyclosporine A. LSM, purified from these cultures, are extremely potent suppressors of AChR-stimulated lymphoproliferative responses and antibody responses in vitro. In the present study, we have analyzed the factors that determine susceptibility of primed lymph node cells (pLNC) to suppression by LSM and examined the fate of these cells. We found three characteristics of pLNC that influenced their susceptibility to suppression. First, pLNC were required to be activated (by antigen in these experiments) in order for suppression to occur. Resting lymphocytes were not affected, even when they were present in cultures where antigen-activated lymphoblasts were being actively suppressed. Second, antigen specificity of the responder cells influenced their susceptibility to suppression by LSM. AChR-specific cells were relatively more susceptible to suppression by AChR-induced LSM than pLNC primed to an unrelated antigen, keyhole limpet hemocyanin. Third, T cell proliferation was suppressed by LSM to a far greater extent than antibody production by B cells. Using enriched T cell blasts generated from AChR-stimulated T cell lines, we found that LSM rapidly suppressed [3H]TdR uptake and induced DNA fragmentation assessed by the TUNEL assay (within 8 h of coculture) and induced morphological signs of apoptosis of T cells (within 24 h). Few, if any, blasts remained by 48 h of coculture. The ability to suppress an activated immune response permanently, without affecting nonactivated, bystander lymphocytes, holds promise that LSM, or their cellular products, could be used for immunotherapy of autoimmune diseases such as myasthenia gravis.
Asunto(s)
Apoptosis , Enfermedades Autoinmunes/terapia , Macrófagos/fisiología , Linfocitos T/inmunología , Animales , Recuento de Células , Citotoxicidad Inmunológica , Fragmentación del ADN , Epítopos , Femenino , Inmunoterapia , Activación de Linfocitos , Ratas , Ratas Endogámicas LewRESUMEN
We examined the effects of CTLA4Ig treatment in an experimental model of myasthenia gravis (EAMG) induced by immunizing Lewis rats with purified Torpedo acetylcholine receptor (AChR). During a primary response, CTLA4Ig treatment inhibited AChR antibody production profoundly, and induced a shift of AChR antibody isotypes from the normally predominant IgG2 isotype pattern toward an IgG1 response. Challenge of rats previously treated with CTLA4Ig produced markedly lower AChR antibody responses compared to untreated controls, persistent inhibition of the IgG2b isotype, and no development of EAMG. Treatment of a secondary AChR response with CTLA4Ig or with DAB389IL2 (which kills lymphocytes expressing IL2 receptors) inhibited AChR antibody responses, and clinical EAMG moderately. In contrast, combined treatment with CTLA4Ig plus DAB389IL2 strikingly inhibited AChR antibody levels, and completely prevented EAMG. Our results suggest that the therapeutic benefit of CTLA4Ig may be due to overall inhibition of AChR antibody production as well as a shift in the antibody isotype repertoire.
Asunto(s)
Antígenos de Diferenciación/uso terapéutico , Toxina Diftérica/uso terapéutico , Inmunoconjugados , Inmunosupresores/uso terapéutico , Inmunoterapia , Interleucina-2/uso terapéutico , Miastenia Gravis/terapia , Abatacept , Animales , Formación de Anticuerpos/efectos de los fármacos , Antígenos CD , Autoanticuerpos/inmunología , Antígeno CTLA-4 , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Inmunoglobulina G/análisis , Isotipos de Inmunoglobulinas/análisis , Ratas , Ratas Endogámicas Lew , Receptores Colinérgicos/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Valores de ReferenciaRESUMEN
Based on our current information, the robust differences in responses of B6 and bm12 mice after immunization with AChR are as follows: (1) The AChR-specific T cell repertoires are strikingly different. The epitope specificities, as well as the rearranged TCR alpha and beta chains and their CDR3 domains, are virtually nonoverlapping in the two strains of mice. (2) The AChR antibody responses are quantitatively different, both to Torpedo AChR and to the autoantigen--mouse AChR. (3) The isotype distribution of AChR antibodies favors IgG2b in B6 mice, but not in bm12 mice. (4) The clinical manifestations of EAMG are qualitatively and quantitatively different in the two strains. These considerations have led to the following scheme, illustrated diagrammatically in FIGURE 2, to explain the differences in EAMG in B6 and bm12 mice: (1) The MHC Class II of B6 mice binds the alpha 146-162 peptide of Torpedo AChR with high affinity, while the genetically altered MHC Class II of bm12 mice does not, as previously suggested (see FIGURE 2). (2) The alpha 146-162/MHC Class II complex occurs only in B6 mice and interacts with T cells having appropriate TCRs, resulting in their stimulation and expansion. Although T cells of appropriate specificity are also available in the bm12 strain, the relevant peptide/MHC Class II complex is not present. Therefore, very few T cells with specificity for alpha 146-162 are stimulated, and those that are stimulated have different TCRs. T cells with specificity for other AChR peptides are also present and expanded in both strains of mice, but they have less influence on the outcome of the immune response. (3) The alpha 146-162-specific T cells of B6 mice, in turn, interact strongly with AChR-specific B cells of B6 mice. These B cells present the same epitope/MHC Class II complex as the APCs and therefore interact well with the alpha 146-162-specific T cells (FIGURE 2). Thus, T cells of this specificity appear to provide more efficient help for AChR antibody production than T cells with specificity for other Torpedo AChR epitopes. This results in production of greater amounts of AChR antibodies, including a critical subset that cross-reacts with autologous mouse AChR. The higher autoantibody levels contribute to the greater susceptibility to EAMG and to the greater severity of manifestations in the B6 strain compared with the bm12 strain. (4) There is a bias in B6 mice toward the production of AChR antibodies of IgG2b isotype. We suggest that T cells specific for alpha 146-162 may contribute to this isotype bias. The IgG2b antibodies appear to have particularly potent "myasthenogenic" effects in rats and mice. (5) Finally, it should be emphasized that these differences in immunological and clinical aspects of EAMG in B6 and bm12 mice are relative rather than absolute. T cells that respond to AChR epitopes other than alpha 146-162 can also provide help for AChR antibody production, albeit less potent. In a sense, this model represents a special case of molecular mimicry. In this case, the source of the foreign antigenic molecule is injection rather than the more usual route of infection. The antigen (Torpedo AChR) is one that these mice would never naturally encounter, and the critical amino acid (lysine 155) of the key epitope (alpha 146-162) is present only in the AChR of electric organs of electric fish and not in the AChR of mice, chickens, cows, or humans. The important point is that a detail of the structure of the foreign antigen--that is, a particular peptide of Torpedo AChR--can determine the severity of an antibody-mediated autoimmune disease, depending on how it interacts with a detail of the structure of the MHC Class II molecule and, in turn, on how the peptide/MHC Class II complex interacts with the available T cell repertoire. (ABSTRACT TRUNCATED)
Asunto(s)
Miastenia Gravis/fisiopatología , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Humanos , Hibridomas/inmunología , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Miastenia Gravis/genética , Miastenia Gravis/inmunología , Ratas , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunologíaRESUMEN
Myasthenia gravis is an autoimmune disorder characterized by muscle weakness, due to an antibody-mediated deficit of acetylcholine receptors (AChRs) at neuromuscular junctions. We analyzed the factors that determine the severity of experimental myasthenia gravis (EAMG) induced by immunization with Torpedo AChR, in two congenic strains of mice--B6 mice, which are highly susceptible to EAMG; and bm12 mice, which are relatively resistant, and differ only in a change of three amino acids in MHC Class II. We prepared large numbers of AChR-specific T cell hybridomas from each strain and characterized their epitope specificities and T cell receptor (TCR) gene usage: Half the B6 hybridomas responded to a single AChR peptide (alpha 146-162), and their TCR genes encoded restricted V alpha and V beta chains and CDR3 motifs. bm12 hybridomas had different epitope specificities and different, less restricted TCR genes. APCs were able to present AChR or AChR-derived peptides virtually exclusively to hybridomas of their own strain. Levels of antibodies to Torpedo and autoantibodies to mouse AChR were higher in B6 mice, and were biased toward the IgG2b isotype. We conclude that the "better fit" of MHC II, peptide, and TCR in the B6 mice enhanced cognate interactions of APCs with T cells, and T cells with B cells, resulting in a more abundant and pathogenic AChR antibody response, and thus more severe EAMG.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Miastenia Gravis/inmunología , Receptores Colinérgicos/inmunología , Animales , Especificidad de Anticuerpos , Presentación de Antígeno , ADN Complementario/genética , Femenino , Reordenamiento Génico de Linfocito T , Hibridomas/inmunología , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Especificidad de la Especie , Torpedo/inmunologíaRESUMEN
OBJECTIVE: The authors evaluated the response to extended cervicomediastinal thymectomy as a component of the integrated management of patients with myasthenia gravis in a large series of patients from a single institution. The authors evaluated the response to therapy with respect to a graded, multivariate, ordinal scale chosen to reflect the full range of the disease's manifestations. SUMMARY BACKGROUND DATA: A number of series, of varying sizes, describe the response of patients with myasthenia gravis to thymectomy primarily with respect to the bivariate endpoint of the presence or absence of "remission." These studies fail to describe the full spectrum of postoperative disease severity and have been unable to quantify definitively the influence of putative prognostic variables, nor to assess definitively the statistical significance of apparent improvements over time. METHODS: The authors evaluated 202 consecutive patients who underwent trans-sternal thymectomy for symptomatic myasthenia gravis from 1969 through 1996 at the Johns Hopkins Hospital. The response to surgery, combined with postoperative medical therapy, was evaluated by a standardized scale reflecting the full spectrum of the disease. These data were analyzed by a novel mean multivariate regression analysis, which allowed the quantification of the statistical significance of apparent responses over time and the evaluation of the independent influence of each of nine putative prognostic indicators. RESULTS: There was no perioperative mortality and a 33% perioperative morbidity. There was a marked clinical response at 6 months to 1 year after surgery that was sustained for at least 10 years thereafter. The median increment of improvement was two (of five) classes. Eighty-six percent and 83% of the patients had improved by at least one class at 5 and 10 years, respectively. These changes were statistically significant (p < 0.001). Whereas the use of extended cervicomediastinal thymectomy was associated with a greater than twofold chance of improvement, compared to conventional trans-sternal thymectomy, neither the pathologic diagnosis (presence of thymoma) nor the age at surgery proved to be negative predictors of postoperative response. CONCLUSIONS: Extended cervicomediastinal thymectomy is the procedure of choice as a component of the integrated management of myasthenia gravis, with significant improvement seen in the group as a whole, as well as in subgroups of patients with thymoma and those older than 40 years of age.
Asunto(s)
Miastenia Gravis/cirugía , Timectomía/métodos , Adolescente , Adulto , Anciano , Niño , Intervalos de Confianza , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Miastenia Gravis/clasificación , Oportunidad Relativa , Pronóstico , Análisis de Regresión , Reoperación , Factores de Riesgo , Infección de la Herida Quirúrgica/etiología , Tasa de Supervivencia , Timectomía/efectos adversos , Timectomía/mortalidad , Resultado del TratamientoRESUMEN
Autoimmune mechanisms have recently been implicated in the pathogenesis of an increasing number of neuromuscular diseases. Many of these diseases can be treated with immunotherapeutic agents that are currently available--often with striking success. However, lack of specificity and adverse side effects impose limits on the effectiveness of these immunosuppressive treatments. This article reviews the basic principles of autoimmunity and immune tolerance, and outlines strategies that produce (a) generalized immunosuppression; (b) "selective" immunotherapy; and (c) "antigen-specific" immunotherapy. General immunosuppressive treatments, which are the ones most commonly used in current practice, down-regulate the immune system at multiple levels in "shotgun" fashion. The agents described here include: adrenal corticosteroids, azathioprine, cyclophosphamide, chlorambucil, methotrexate, total lymphoid irradiation, plasmapheresis, and intravenous immunoglobulin. "Selective" immunotherapeutic strategies are designed to interfere with mechanisms intrinsic to the immune system. Agents that are now being used clinically, or are in advanced stages of development include: cyclosporin A, which interferes with synthesis of the cytokine interleukin 2 (IL2); IL2 toxin, which binds to IL2 receptors on activated T cells, is endocytosed, and kills the cells; and CTLA4Ig, which blocks costimulatory molecules, thus preventing full activation of T cells. We have found that combinations of the selective agents may enhance their effectiveness. "Specific" strategies are designed to inactivate or suppress antigen-specific T cells. Oral administration of autoantigens has been shown to prevent experimental autoimmune diseases specifically, but the conditions required to suppress ongoing autoimmune diseases are capricious, and depend on many factors. Finally, we describe a method that is still in the experimental stage, which is designed to modify the individual's own antigen-presenting cells so that they will target and inactivate antigen-specific T cells, and thereby turn off the specific autoimmune response. Currently available immunosuppressive methods can now be used successfully to treat many autoimmune neuromuscular diseases, and the application of selective and specific immunotherapeutic strategies promises more precise and effective treatment in the future.
Asunto(s)
Inmunoterapia/tendencias , Enfermedades Neuromusculares/terapia , Autoinmunidad , Humanos , Tolerancia Inmunológica , Terapia de Inmunosupresión , Inmunosupresores/uso terapéutico , Inmunoterapia/métodosRESUMEN
The differentiation of thymocytes into mature post-thymic T cells requires rearrangement of T-cell antigen receptor genes from germline to a spectrum of spliced configurations encoding functional receptors. In the T-cell receptor beta chain locus, this process occurs via a hierarchical series of recombination events, linking "diversity" and "joining" segments, then "variable" and "diversity" segments. The authors report Southern blot analysis of the T-cell receptor beta locus in normal human thymic tissue after restriction digestion with Bam HI identifying rearranged DNA fragments in 5 of 6 samples, which probably represent an intermediate deletion joining D beta 1 to a J beta 2 segment without rearrangement of the upstream V region. Hybridization intensity was in the range of 5% to 30% of represented DNA. Reduction of signal from bands containing C beta 1 and J beta 2 sequences after Eco RI and Hind III digestion was consistent with this model. A probe specific for J beta 1 did not hybridize with the rearranged fragment while J beta 2 specific sequences did not hybridize with the rearranged fragment while J beta 2 specific sequences did, indicating a deletion of the region of J beta 1, but limited to sequences upstream of J beta 2. Most peripheral lymphoid specimens do not demonstrate similar rearranged DNA fragments, however T-cell-rich populations may do so. Analysis of 31 additional surgically resected thymic tissues and three polyclonal T-cell-rich peripheral lymphoid specimens revealed a similarly rearranged fragment in 13 of 14 thymuses with follicular hyperplasia; 6 of 9 thymomas; 5 of 6 malignant (invasive) thymomas; 0 of 2 thymic carcinomas; and, at very low abundance, in 3 of 3 peripheral T-cell populations. Because this nongermline T-cell receptor gene apparently reflects polyclonal incomplete rearrangement, rather than clonality, awareness of this phenomenon may help prevent erroneous diagnosis of T-cell lymphoma for mediastinal lymphoid lesions with an apparent "clonal" T-cell receptor gene rearrangement. If the intermediate deletion is derived from an ongoing process, the data suggest that in many cases neoplastic thymic epithelium may retain functions necessary not only to support a resident thymocyte population, but also to activate ongoing T-cell differentiation. Alternatively, the intermediate deletion may derive from abandoned non-productive rearrangements.
Asunto(s)
Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Linfocitos T/citología , Timoma/patología , Timo/citología , Neoplasias del Timo/patología , Southern Blotting , Diferenciación Celular , Células Clonales , Humanos , Mapeo Restrictivo , Linfocitos T/inmunología , Timoma/genética , Timoma/inmunología , Timo/inmunología , Timo/patología , Neoplasias del Timo/genética , Neoplasias del Timo/inmunologíaRESUMEN
Because of the antibody-mediated pathogenesis of MG, it is of particular interest to understand the effects of oral administration of the autoantigen AChR on the disease process. It is now clear that feeding AChR prior to immunization can prevent clinical manifestation of EAMG. It initially primed, then inhibited, antibody responses to foreign (Torpedo) AChR and self (rat) AChR, with a delayed onset. Cellular responses to AChR, evaluated by lymphocyte proliferation and IL-2 production, were markedly inhibited. The effects were dependent on the dose and purity of the fed antigen. Tolerance to an orally administered unrelated antigen, OVA, was more prompt in development and more profound, illustrating the influence of the nature of the antigen on tolerance. The tolerance induced was antigen specific. Oral administration of AChR after immunization resulted in inhibition of the clinical manifestation of EAMG, concomitant with a paradoxical enhancement of the AChR-antibody responses. Both the clinical benefit and the antibody response appear to be dependent on the feeding protocol. These findings suggest that a molecule with less immunogenic potential than native AChR may be required for safe and effective oral treatment of ongoing disease.
Asunto(s)
Autoantígenos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Tolerancia Inmunológica , Miastenia Gravis/inmunología , Miastenia Gravis/terapia , Administración Oral , Animales , Formación de Anticuerpos , Autoantígenos/administración & dosificación , Encefalomielitis Autoinmune Experimental/terapia , Humanos , Inmunoterapia , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Conejos , Ratas , Ratas Endogámicas Lew , Receptores Colinérgicos/inmunología , Linfocitos T/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
The pathogenic antibody response to acetylcholine receptor (AChR) in experimental autoimmune myasthenia gravis (EAMG) is T cell dependent. Therefore, it should be possible to design specific immunotherapeutic approaches to treat EAMG (and human MG) by interfering with AChR-specific helper T cells. Productive T cell activation by antigen requires at least two signals: one signal delivered through the T cell receptor by antigen and a second costimulatory signal delivered through the CD28 receptor via the B7 counterreceptor expressed on antigen-presenting cells. Here we show that interference with the B7 costimulatory signal, using a soluble CD28 analogue, CTLA4Ig, resulted in a profound decrease in IL2 production and significantly decreased lymphoproliferative responses and antibody responses by primed lymph node cells from rats with EAMG, when stimulated with AChR in vitro. Nonclonal AChR-specific T cell lines, when stimulated with AChR in the presence of CTLA4Ig, were also inhibited in their ability to proliferate and to produce the cytokines IL2 and IFN-gamma. They remained deficient in their ability to produce IL2 when restimulated with AChR plus fresh antigen-presenting cells and showed variable inhibition of proliferation. The induction of hyporesponsiveness was accompanied by the expression of functional IL2 receptors, as shown by vigorous proliferative responses to addition of exogenous IL2. These results indicate that specific antigen stimulation in the presence of CTLA4Ig can induce certain features typical of anergy. CTLA4Ig provides a promising approach for the immunomodulation of MG and other antibody-mediated autoimmune diseases.
Asunto(s)
Antígenos de Diferenciación/farmacología , Anergia Clonal/inmunología , Inmunoconjugados , Fragmentos Fc de Inmunoglobulinas/farmacología , Activación de Linfocitos , Miastenia Gravis/inmunología , Abatacept , Animales , Formación de Anticuerpos/efectos de los fármacos , Antígenos CD , Antígenos de Diferenciación/uso terapéutico , Antígeno CTLA-4 , Línea Celular , Femenino , Tolerancia Inmunológica , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Interleucina-2/farmacología , Ganglios Linfáticos/citología , Activación de Linfocitos/efectos de los fármacos , Miastenia Gravis/terapia , Ratas , Ratas Endogámicas Lew , Receptores Colinérgicos/fisiología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
The hypothesis that abnormal antibodies may be involved in the pathogenesis of ALS has been supported in part by IgG's being present within motoneurons of ALS patients more frequently than in motoneurons of controls. IgG, as well as other serum proteins, is also present in motoneurons of normal human and animal spinal cords. We attempted to determine whether the IgG found in motoneurons of ALS patients was localized by an immune-specific or nonspecific process. To address this question, we used immunocytochemistry to evaluate the presence and relative density of different serum proteins in spinal cords from nine patients with ALS. Both IgG and alpha 2-macroglobulin (alpha 2Mac) were present in motoneurons in all nine cases. More important, there was a close concordance between the IgG and alpha 2Mac immunolabeling of motoneurons. The presence of a nonimmune plasma protein--alpha 2Mac--in a similar distribution to IgG and with a similar intensity implies that the internalization of these proteins in motoneurons of patients with ALS is best explained by a nonselective mechanism of endocytosis of extracellular fluid.