RESUMEN
The cell adhesion molecule N-cadherin is involved in several processes during central nervous system development, but also in certain pathologic conditions in the adult brain, including tumorigenesis and Alzheimer's disease. N-cadherin function in inflammatory demyelinating disease has so far not been investigated. In vitro studies suggest a role of N-cadherin in myelination; on the other hand N-cadherin has been implicated in the formation of the glial scar, which is believed to impede remyelination. The aim of our study was to investigate the expression pattern of N-cadherin immunoreactivity in experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein (MOG-EAE), an animal model closely mimicking multiple sclerosis. It allows a detailed evaluation of all stages of de- and remyelination during lesion development. Immunopathological evaluation was performed on paraffin-embedded CNS sections sampled at days 20 to 120 post immunization. We found a predominant expression of N-cadherin on oligodendrocytes in early remyelinating lesions, while in fully remyelinated shadow plaques there was no detectable immunoreactivity for N-cadherin. This expression pattern indicates a role of N-cadherin in the initiation of remyelination, most likely by providing a guidance between myelin lamellae and oligodendrocytes. Once the initial contact is made N-cadherin is then rapidly downregulated and virtually absent after completion of the repair process, analog to its known role in developmental myelination. Our results show that N-cadherin plays an important role in creating a remyelination-facilitating environment.
Asunto(s)
Cadherinas/biosíntesis , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Vaina de Mielina/fisiología , Animales , Cadherinas/genética , Cadherinas/fisiología , Encefalomielitis Autoinmune Experimental/genética , Femenino , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Proteínas de la Mielina/biosíntesis , Proteínas de la Mielina/genética , Glicoproteína Mielina-Oligodendrócito , Ratas , Factores de TiempoRESUMEN
OBJECTIVE: To study the longitudinal dynamics of anti-myelin oligodendrocyte glycoprotein (MOG) autoantibodies in childhood demyelinating diseases. METHODS: We addressed the kinetics of anti-MOG immunoglobulins in a prospective study comprising 77 pediatric patients. This was supplemented by a cross-sectional study analyzing 126 pediatric patients with acute demyelination and 62 adult patients with multiple sclerosis (MS). MOG-transfected cells were used for detection of antibodies by flow cytometry. RESULTS: Twenty-five children who were anti-MOG immunoglobulin (Ig) positive at disease onset were followed for up to 5 years. Anti-MOG antibodies rapidly and continuously declined in all 16 monophasic patients with acute disseminated encephalomyelitis and in one patient with clinically isolated syndrome. In contrast, in 6 of 8 patients (75%) eventually diagnosed with childhood MS, the antibodies to MOG persisted with fluctuations showing a second increase during an observation period of up to 5 years. Antibodies to MOG were mainly IgG 1 and their binding was largely blocked by pathogenic anti-MOG antibodies derived from a spontaneous animal model of autoimmune encephalitis. The cross-sectional part of our study elaborated that anti-MOG Ig was present in about 25% of children with acute demyelination, but in none of the pediatric or adult controls. Sera from 4/62 (6%) adult patients with MS had anti-MOG IgG at low levels. CONCLUSIONS: The persistence or disappearance of antibodies to MOG may have prognostic relevance for acute childhood demyelination.
Asunto(s)
Autoanticuerpos/análisis , Encefalomielitis Aguda Diseminada/inmunología , Glicoproteína Asociada a Mielina/inmunología , Adolescente , Adulto , Unión Competitiva , Línea Celular , Niño , Preescolar , Estudios de Cohortes , Estudios Transversales , Femenino , Citometría de Flujo , Humanos , Inmunoglobulina G/análisis , Inmunoglobulinas/análisis , Lactante , Cinética , Estudios Longitudinales , Masculino , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Proteínas de la Mielina , Glicoproteína Mielina-Oligodendrócito , Estudios Prospectivos , TransfecciónRESUMEN
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Recent studies suggest that, beside focal lesions, diffuse inflammatory and degenerative processes take place throughout the MS brain. Especially, molecular alterations in the so-called normal appearing white matter suggest the induction of neuroprotective mechanisms against oxidative stress preserving cellular homeostasis and function. In this study we investigated whether in an animal model for MS, namely in experimental autoimmune encephalomyelitis (EAE), similar changes occur. We isolated normal appearing white and grey matter from the corpus callosum and the above lying cerebral cortex from DA rats with rMOG-induced EAE and carried out a gene expression analysis. Examination of corpus callosum revealed only minor changes in EAE rats. In contrast, we identified a number of gene expression alterations in the cerebral cortex even though morphological and cellular alterations were not evident. One of the most striking observations was the downregulation of genes involved in mitochondrial function as well as a whole set of genes coding for different glutamate receptors. Our data imply that molecular alterations are present in neurons far distant to inflammatory demyelinating lesions. These alterations might reflect degenerative processes induced by lesion-mediated axonal injury in the spinal cord. Our results indicate that the MOG-induced EAE in DA rats is a valuable model to analyze neuronal alterations due to axonal impairment in an acute phase of a MS-like disease, and could be used for development of neuroprotective strategies.
Asunto(s)
Encéfalo/patología , Expresión Génica/fisiología , Esclerosis Múltiple/patología , Neuroglía/metabolismo , Médula Espinal/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/fisiología , Femenino , Perfilación de la Expresión Génica/métodos , Proteína Ácida Fibrilar de la Glía/metabolismo , Esclerosis Múltiple/inducido químicamente , Esclerosis Múltiple/fisiopatología , Proteínas de la Mielina , Proteína Proteolipídica de la Mielina/metabolismo , Glicoproteína Asociada a Mielina , Glicoproteína Mielina-Oligodendrócito , Neuroglía/patología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Ratas , Médula Espinal/patología , Estadísticas no ParamétricasRESUMEN
PURPOSE: To investigate the intravital visibility of CNS lesions in rats with experimental autoimmune encephalomyelitis (EAE), the animal correlate of multiple sclerosis, using a 3-Tesla (T) whole-body MR system. MATERIALS AND METHODS: Three healthy Dark Agouti (DA) rats and 16 DA rats with clinical signs of EAE were examined on a 3T whole body-system using a normal wrist coil. In total, 25 examinations were preformed using T2- and T1-weigthed images in transverse and sagittal orientation with a slice thickness of 2 mm or 1 mm (voxel size up to 0.2 x 0.2 x 1 mm). Sedation was achieved by intraperitoneal injection of ketamine and xylazine. In addition, T1-weighted images were obtained after the instillation of 1.0 ml of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) (0.5 mmol/ml) into the peritoneal cavity. RESULTS: T2- and T1-weighted images of the brain and spinal cord with high spatial and contrast resolution could be obtained in all animals. The anatomical details of the olfactory bulb glomeruli, cerebellum foliae, ventricles and corpus callosum were clearly visible. The EAE lesions presented as hyperintense areas in T2-weighted images and could be demonstrated in all clinically affected animals by MRI and histologically verified. In total, the 16 affected rats had 28 cerebral and 2 spinal cord lesions (range 1 to 4, median 2). Contrast enhancement was noted in 12 animals and ranked as severe in ten and moderate in two cases. No adverse effects were noted due to sedation or intraperitoneal contrast injection. CONCLUSIONS: The intravital demonstration of cerebral and spinal cord EAE lesions in rats is possible on a 3T whole-body MR scanner using a normal wrist coil. Intraperitoneal injection of ketamine/xylazine and contrast agent is an easy, safe and effective procedure in rats.
Asunto(s)
Enfermedades Desmielinizantes/diagnóstico , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/diagnóstico , Encefalomielitis Autoinmune Experimental/patología , Imagen por Resonancia Magnética/métodos , Agonistas alfa-Adrenérgicos/administración & dosificación , Anestésicos Disociativos/administración & dosificación , Animales , Encéfalo/patología , Tronco Encefálico/patología , Cerebelo/patología , Medios de Contraste/administración & dosificación , Gadolinio DTPA/administración & dosificación , Inyecciones Intraperitoneales , Ketamina/administración & dosificación , Ratas , Médula Espinal/patología , Xilazina/administración & dosificaciónRESUMEN
IgG, its subclasses and IgE concentrations were measured in cerebrospinal fluid (CSF) and serum of multiple sclerosis (MS) patients and matched controls as surrogate markers for type 1 and type 2 immunity. IgE indices were significantly reduced in MS patients compared to controls. In contrast, IgG1 was elevated in CSF of MS patients and elevated indices indicated intrathecal synthesis. Because isotype switching to IgE and IgG4 is driven by type 2 immunity and occurrence of IgG1 has previously been found in type 1 immunity-dominated diseases, the results underscore a role of type 1 immunity in MS.
Asunto(s)
Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Esclerosis Múltiple/inmunología , Células TH1/inmunología , Adulto , Biomarcadores , Estudios de Casos y Controles , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/líquido cefalorraquídeo , Inmunoglobulina G/sangre , Inmunoglobulina G/líquido cefalorraquídeo , Masculino , Células Th2RESUMEN
Myelin oligodendrocyte glycoprotein (MOG) is an Ag present in the myelin sheath of the CNS thought to be targeted by the autoimmune T cell response in multiple sclerosis (MS). In this study, we have for the first time characterized the T cell epitopes of human MOG restricted by HLA-DR4 (DRB1*0401), an MHC class II allele associated with MS in a subpopulation of patients. Using MHC binding algorithms, we have predicted MOG peptide binding to HLA-DR4 (DRB1*0401) and subsequently defined the in vivo T cell reactivity to overlapping MOG peptides by testing HLA-DR4 (DRB1*0401) transgenic mice immunized with recombinant human (rh)MOG. The data indicated that MOG peptide 97-108 (core 99-107, FFRDHSYQE) was the immunodominant HLA-DR4-restricted T cell epitope in vivo. This peptide has a high in vitro binding affinity for HLA-DR4 (DRB1*0401) and upon immunization induced severe experimental autoimmune encephalomyelitis in the HLA-DR4 transgenic mice. Interestingly, the same peptide was presented by human B cells expressing HLA-DR4 (DRB1*0401), suggesting a role for the identified MOG epitopes in the pathogenesis of human MS.
Asunto(s)
Presentación de Antígeno , Linfocitos B/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/genética , Esclerosis Múltiple/inmunología , Glicoproteína Asociada a Mielina/inmunología , Secuencia de Aminoácidos , Animales , Autoantígenos/química , Autoantígenos/inmunología , Autoantígenos/metabolismo , Unión Competitiva , Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/patología , Mapeo Epitopo , Epítopos de Linfocito T/metabolismo , Antígenos HLA-DR/metabolismo , Cadenas HLA-DRB1 , Humanos , Epítopos Inmunodominantes , Cinética , Ratones , Ratones Transgénicos , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/química , Glicoproteína Asociada a Mielina/metabolismo , Glicoproteína Asociada a Mielina/farmacología , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacologíaRESUMEN
Experimental autoimmune neuritis (EAN) is the principal animal model for Guillain-Barré syndrome (GBS), an inflammatory disease of the peripheral nervous system. Little is known on the genetic regulation of these diseases. We provide the first genetic linkage analysis of EAN. Susceptibility to EAN in a rat F2 population segregated with high levels of anti-PNM IgG, as well as IgG2b and IgG2c isotype levels, which support that disease genes regulate preferential Th1/Th2 differentiation. Linkage analysis demonstrated co-localization of EAN loci with reported susceptibility loci for experimental arthritis and/or encephalomyelitis and a new region on chromosome 17. Further dissection of these loci may disclose disease pathways in GBS.
Asunto(s)
Ligamiento Genético , Neuritis Autoinmune Experimental/genética , Neuritis Autoinmune Experimental/inmunología , Animales , Cromosomas , Modelos Animales de Enfermedad , Femenino , Genotipo , Inmunoglobulina G/inmunología , Masculino , Sistema Nervioso Periférico/inmunología , Ratas , Ratas Endogámicas ACI , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Demyelination caused by inflammation of the CNS has been considered to be a major hallmark of multiple sclerosis (MS). Using experimental autoimmune encephalomyelitis, a model of MS, we demonstrate that an immune-mediated attack of the optic nerve is accompanied by an early degeneration of retinal ganglion cells (RGCs). The decrease of neuronal cell density was correlated with functional disabilities as assessed by visual evoked cortical potentials and electroretinogram. Visual acuity was significantly reduced. DNA degradation and activation of caspase-3 in RGCs indicate that cell death of RGCs is apoptotic. These findings show for the first time that an inflammatory attack against myelin components can lead to acute neuronal cell loss by apoptosis.
Asunto(s)
Apoptosis , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Neuronas/patología , Enfermedad Aguda , Animales , Caspasa 3 , Caspasas/metabolismo , Recuento de Células , Fragmentación del ADN , Enfermedades Desmielinizantes/patología , Electrorretinografía , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/fisiopatología , Potenciales Evocados Visuales , Femenino , Adyuvante de Freund , Inflamación/patología , Proteínas de la Mielina , Glicoproteína Asociada a Mielina , Glicoproteína Mielina-Oligodendrócito , Nervio Óptico/patología , Estimulación Luminosa , Ratas , Ratas Endogámicas BN , Células Ganglionares de la Retina/patología , Agudeza Visual , Corteza Visual/fisiopatologíaRESUMEN
Myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in rats closely mimics the human disease multiple sclerosis (MS). As in MS, genetic predisposition to MOG-EAE is regulated by both MHC and non-MHC genes. Based on disease regulatory influences on MOG-EAE on chromosome 10 in an F2 cross between susceptible DA and resistant ACI rats, we have now isolated this locus in a congenic rat strain to enable further dissection of disease mechanisms. This region is of particular interest, since it is homologous to human 17q for which human whole-genome scans have indicated harbors genes regulating susceptibility to MS. Phenotypic comparison between DA and the congenic DA.ACI-D10Rat2-D10Rat29 strain confirms that the chromosomal segment harbors gene(s) conferring strong protection against MOG-EAE. Furthermore, resistance to EAE in this congenic strain is associated with absence or a low level of inflammation and demyelination in the central nervous system. Levels of anti-MOG antibody isotypes did not differ between parental and congenic rats, thus an action on Th1/Th2 differentiation is unlikely. In conclusion, this is the first example of an EAE-regulating locus isolated in a congenic rat strain with retained phenotype. The mechanism by which gene(s) in the region act is still unclear and will require further studies with this congenic rat strain as a tool.
Asunto(s)
Encefalomielitis Autoinmune Experimental/genética , Glicoproteína Asociada a Mielina/inmunología , Animales , Autoanticuerpos/sangre , Encéfalo/patología , Mapeo Cromosómico , Predisposición Genética a la Enfermedad , Masculino , Proteínas de la Mielina , Glicoproteína Mielina-Oligodendrócito , Ratas , Ratas Endogámicas , Médula Espinal/patologíaRESUMEN
We dissected the requirements for disease induction of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis in MHC (RT1 in rat) congenic rats with overlapping MOG peptides. Immunodominance with regard to peptide-specific T cell responses was purely MHC class II dependent, varied between different MHC haplotypes, and was linked to encephalitogenicity only in RT1.B(a)/D(a) rats. Peptides derived from the MOG sequence 91-114 were able to induce overt clinical signs of disease accompanied by demyelinated CNS lesions in the RT1.B(a)/D(a) and RT1(n) haplotypes. Notably, there was no detectable T cell response against this encephalitogenic MOG sequence in the RT1(n) haplotype in peripheral lymphoid tissue. However, CNS-infiltrating lymphoid cells displayed high IFN-gamma, TNF-alpha, and IL-4 mRNA expression suggesting a localization of peptide-specific reactivated T cells in this compartment. Despite the presence of MOG-specific T and B cell responses, no disease could be induced in resistant RT1(l) and RT1(u) haplotypes. Comparison of the number of different MOG peptides binding to MHC class II molecules from the different RT1 haplotypes suggested that susceptibility to MOG-experimental autoimmune encephalomyelitis correlated with promiscuous peptide binding to RT1.B and RT1.D molecules. This may suggest possibilities for a broader repertoire of peptide-specific T cells to participate in disease induction. We demonstrate a powerful MHC class II regulation of autoaggression in which MHC class II peptide binding and peripheral T cell immunodominance fail to predict autoantigenic peptides relevant for an autoaggressive response. Instead, target organ responses may be decisive and should be further explored.
Asunto(s)
Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Antígenos de Histocompatibilidad Clase II/fisiología , Tejido Linfoide/inmunología , Glicoproteína Asociada a Mielina/inmunología , Linfocitos T/inmunología , Alelos , Secuencia de Aminoácidos , Animales , Animales Congénicos , Autoantígenos/análisis , Autoantígenos/inmunología , Linfocitos B/inmunología , Células Cultivadas , Sistema Nervioso Central/patología , Cromatografía de Afinidad , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Epítopos de Linfocito B/análisis , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/análisis , Epítopos de Linfocito T/inmunología , Femenino , Genes MHC Clase I , Genes MHC Clase II , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/aislamiento & purificación , Antígenos de Histocompatibilidad Clase II/metabolismo , Tolerancia Inmunológica , Tejido Linfoide/patología , Datos de Secuencia Molecular , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/administración & dosificación , Glicoproteína Asociada a Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Mucosa Nasal/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Unión Proteica/genética , Unión Proteica/inmunología , Ratas , Ratas Endogámicas ACI , Ratas Endogámicas Lew , Especificidad de la Especie , VacunaciónRESUMEN
Multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are immune-mediated diseases of the CNS. They are characterized by widespread inflammation, demyelination and a variable degree of axonal loss. Recent magnetic resonance spectroscopy studies have indicated that axonal damage and loss are a reliable correlate of permanent clinical disability. Accordingly, neuropathological studies have confirmed the presence and timing of axonal injury in multiple sclerosis lesions. The mechanisms of axonal degeneration, however, are unclear. Since calcium influx may mediate axonal damage, we have studied the distribution of the pore-forming subunit of neuronal (N)-type voltage-gated calcium channels in the lesions of multiple sclerosis and EAE. We found that alpha(1B), the pore-forming subunit of N-type calcium channels, was accumulated within axons and axonal spheroids of actively demyelinating lesions. The axonal staining pattern of alpha(1B) was comparable with that of beta-amyloid precursor protein, which is an early and sensitive marker for disturbance of axonal transport. Importantly, within these injured axons, alpha(1B) was not only accumulated, but also integrated in the axoplasmic membrane, as shown by immune electron microscopy on the EAE material. This ectopic distribution of calcium channels in the axonal membrane may result in increased calcium influx, contributing to axonal degeneration, possibly via the activation of neutral proteases. Our data suggest that calcium influx through voltage-dependent calcium channels is one possible candidate mechanism for axonal degeneration in inflammatory demyelinating disorders.
Asunto(s)
Axones/patología , Canales de Calcio Tipo N/análisis , Encefalomielitis Autoinmune Experimental/patología , Glicoproteínas , Inmunoglobulinas , Esclerosis Múltiple/patología , Médula Espinal/patología , Adulto , Animales , Axones/ultraestructura , Proteínas Sanguíneas/análisis , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Humanos , Inmunohistoquímica , Masculino , Microscopía Confocal , Microscopía Electrónica , Persona de Mediana Edad , Esclerosis Múltiple/fisiopatología , Ratas , Médula Espinal/fisiopatologíaRESUMEN
Proton magnetic resonance spectroscopy has shown elevated signals in the spectral region of lipids in acute multiple sclerosis lesions. The metabolite-nulling technique allows the separation of macromolecules from other metabolites, such as lactate, N-acetyl-aspartate, creatine, choline and myo-inositol. Using this technique in studies on multiple sclerosis patients, we were able to differentiate macromolecules biochemically in acute and chronic multiple sclerosis lesions. Ten patients with acute, contrast-enhancing multiple sclerosis lesions, 10 patients with chronic lesions and 10 healthy control subjects were investigated with a 1.5 T whole body system, using a stimulated echo acquisition mode (STEAM) sequence with metabolite-nulling and outer volume saturation. Metabolites and macromolecules were quantitated absolutely. The 0.9 and 1.3 parts per million (p.p.m.) resonances of the macromolecules were significantly elevated in acute lesions compared with chronic lesions and healthy controls (P < 0.001 for 0.9 p.p.m., P < 0.05 for 1.3 p.p.m.). The macromolecular resonances at 2.1 and 3.0 p.p.m. in acute and chronic lesions were normal. N-acetyl-aspartate was significantly reduced in acute and chronic lesions compared with controls (P < 0.05 and P < 0.01, respectively). Choline was significantly elevated in acute lesions compared with controls (P < 0.05). Up to now, elevated resonances at 0.9 and 1.3 p.p.m. in acute lesions have been interpreted as lipids. In metabolite-nulled spectra, the macromolecular resonances did not fit those of lipids and might have been due to proteins or polypeptides containing the amino acids alanine, threonine, valine, leucine and isoleucine. These account for approximately 40% of the amino acids of myelin proteolipid protein and for approximately 20% of myelin basic protein. The increased macromolecular resonances at 0.9 and 1.3 p.p.m. may be interpreted as biochemical markers of myelin fragments and may be used as reliable markers of acute multiple sclerosis lesions as they provide clear discrimination among acute and chronic lesions and controls.
Asunto(s)
Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Lipoma/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Esclerosis Múltiple/metabolismo , Enfermedad Aguda , Corticoesteroides/administración & dosificación , Adulto , Alanina/metabolismo , Aminoácidos/metabolismo , Biomarcadores/análisis , Colina/metabolismo , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Femenino , Humanos , Isoleucina/metabolismo , Leucina/metabolismo , Lipoma/complicaciones , Lipoma/diagnóstico , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/tratamiento farmacológico , Proteínas/metabolismo , Treonina/metabolismo , Valina/metabolismoRESUMEN
A causal role of IL-4 (Th2) production for recovery in experimental allergic neuritis (EAN) was indicated by experiments where Th1-like autoreactive cell populations, taken from the induction phase of the disease, were deviated to extensive secretion of IL-4 in a selective fashion, by ex vivo stimulation with autoantigen in the presence of IL-4. The deviated cells were adoptively transferred to EAN rats at a time just prior to the onset of clinical signs. This treatment ameliorated EAN compared with sham treatment. This therapeutic approach, with generation of autoreactive IL-4-secreting cells ex vivo followed by subsequent adoptive transfer, may become a new selective treatment of organ-specific autoimmune diseases since, in contrast to previous attempts, it is done in a physiological and technically easy way.
Asunto(s)
Traslado Adoptivo/métodos , Epítopos de Linfocito T/inmunología , Interleucina-4/biosíntesis , Proteína P2 de Mielina/inmunología , Neuritis Autoinmune Experimental/inmunología , Neuritis Autoinmune Experimental/prevención & control , Fragmentos de Péptidos/inmunología , Células Th2/trasplante , Animales , Citocinas/metabolismo , Epítopos de Linfocito T/toxicidad , Interleucina-4/genética , Interleucina-4/farmacología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Transfusión de Linfocitos , Masculino , Proteína P2 de Mielina/toxicidad , Neuritis Autoinmune Experimental/metabolismo , Fragmentos de Péptidos/toxicidad , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/farmacología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/trasplante , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Identification and quantitation of autoreactive T lymphocytes is crucial in order to understand the pathogenesis of autoimmune diseases. We used flow cytometry to analyze autoantigen-specific T cellular responses in the well characterized rat experimental autoimmune encephalomyelitis (EAE) model. Cells isolated from both the central nervous system (CNS) tissue and peripheral lymph nodes were analyzed directly ex vivo or after short term in vitro culture with specific autoantigen. CNS infiltrating T lymphocytes displaying an interferon-gamma response to selected encephalitogenic myelin protein epitopes were measured kinetically during an individual disease episode and also between relapses in a chronic rat EAE model. One of the EAE models used displays a restriction towards TCRBV8S2 chain usage by the encephalitogenic T cells. In this model, in vitro production of intracellular interferon-gamma was selectively detected within this T cell subset derived from both the CNS and peripheral lymph nodes. Furthermore, antigen-specific cells infiltrating the CNS in this model produced several-fold higher amounts of interferon-gamma upon antigen stimulation and displayed a significantly increased in vivo proliferation compared with peripheral lymphocytes. These data thus directly demonstrates that T cells stimulated by a specific autoantigen in the periphery primarily acquire effector functions in the cellular environment of the target organ of the autoantigen.
Asunto(s)
Sistema Nervioso Central/fisiopatología , Encefalomielitis Autoinmune Experimental/fisiopatología , Epítopos , Linfocitos T/fisiología , Animales , Formación de Anticuerpos , Autoantígenos/inmunología , Encéfalo/inmunología , Encéfalo/metabolismo , División Celular , Células Cultivadas , Sistema Nervioso Central/patología , Enfermedad Crónica , Citocinas/biosíntesis , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Interferón gamma/biosíntesis , Membranas Intracelulares/metabolismo , Cinética , Ratas , Ratas Endogámicas Lew , Recurrencia , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
Recent magnetic resonance (MR) studies of multiple sclerosis lesions indicate that axonal injury is a major correlate of permanent clinical deficit. In the present study we systematically quantified acute axonal injury, defined by immunoreactivity for beta-amyloid-precursor-protein in dystrophic neurites, in the central nervous system of 22 multiple sclerosis patients and 18 rats with myelin-oligodendrocyte glycoprotein (MOG)-induced chronic autoimmune encephalomyelitis (EAE). The highest incidence of acute axonal injury was found during active demyelination, which was associated with axonal damage in periplaque and in the normal appearing white matter of actively demyelinating cases. In addition, low but significant axonal injury was also observed in inactive demyelinated plaques. In contrast, no significant axonal damage was found in remyelinated shadow plaques. The patterns of axonal pathology in chronic active EAE were qualitatively and quantitatively similar to those found in multiple sclerosis. Our studies confirm previous observations of axonal destruction in multiple sclerosis lesions during active demyelination, but also indicate that ongoing axonal damage in inactive lesions may significantly contribute to the clinical progression of the disease. The results further emphasize that MOG-induced EAE may serve as a suitable model for testing axon-protective therapies in inflammatory demyelinating conditions.
Asunto(s)
Axones/patología , Encefalomielitis Autoinmune Experimental/patología , Esclerosis Múltiple/patología , Vaina de Mielina/patología , Enfermedad Aguda , Adulto , Anciano , Precursor de Proteína beta-Amiloide/análisis , Animales , Axones/química , Encéfalo/patología , Química Encefálica , Enfermedad Crónica , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Humanos , Inmunohistoquímica , Inflamación/metabolismo , Inflamación/patología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Vaina de Mielina/química , Ratas , Ratas Endogámicas LewRESUMEN
The definition of genes regulating the pathogenetic pathways of autoimmune neuroinflammation, may provide targets for new therapeutic strategies. This is not easily accomplished in human disease. Such genetic dissection can more readily be done by the use of inbred rodent strains. With these, genetic heterogeneity is avoided and variation in the environmental influences is minimized. Close mimicking of the human disease characteristics is desirable in such endeavors. Chronic relapsing experimental autoimmune encephalomyelitis (EAE) with MS-like histopathology is achieved after immunization of certain rat strains with myelin oligodendrocyte glycoprotein (MOG) or spinal cord homogenate. The major histocompatibility complex (MHC) regulate the ease by which the environmental trigger in the form of immunisation induces disease. Use of intra-MHC recombinant strains demonstrated major influences from the MHC class II genome region, but additional influences from both the MHC class I and III regions. These findings now provide a basis for studies of the mechanisms for MHC-controlled autoimmune pathogenicity leading to MS-like disease. Gene mapping of F2 crosses between susceptible and resistant rat strains demonstrated nine genome regions outside the MHC which regulate different phenotypes of rat EAE. Many of these co-localize with genome regions regulating other organ-specific disease such experimental arthritis, suggesting a sharing of disease pathways. Further finemapping can lead to the exact identification of disease regulating genes. Interestingly, we have also demonstrated a non-MHC gene control of the inflammatory response, in the form of glial cell activation, and neuronal degeneration, subsequent to anterior nerve root avulsion in rats. The genetic dissection of these influences may unravel pathways controlling CNS vulnerability.
Asunto(s)
Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Neuroinmunomodulación/genética , Animales , Modelos Animales de Enfermedad , Humanos , Ratas , Ratas EndogámicasRESUMEN
DNA vaccines that encode encephalitogenic sequences in tandem can protect from subsequent experimental autoimmune encephalomyelitis induced with the corresponding peptide. The mechanism for this protection and, in particular, if it is specific for the amino acid sequence encoding the vaccine are not known. We show here that a single amino acid exchange in position 79 from serine (nonself) to threonine (self) in myelin basic protein peptide MBP68-85, which is a major encephalitogenic determinant for Lewis rats, dramatically alters the protection. Moreover, vaccines encoding the encephalitogenic sequence MBP68-85 do not protect against the second encephalitogenic sequence MBP89-101 in Lewis rats and vice versa. Thus, protective immunity conferred by DNA vaccination exquisitely discriminates between peptide target autoantigens. No bystander suppression was observed. The exact underlying mechanisms remain elusive because no simple correlation between impact on ex vivo responses and protection against disease were noted.
Asunto(s)
Autoantígenos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/inmunología , Vacunas de ADN/inmunología , Animales , División Celular/inmunología , ADN/genética , ADN/inmunología , Encefalomielitis Autoinmune Experimental/genética , Epítopos , Inmunización , Datos de Secuencia Molecular , Proteína Básica de Mielina/genética , Fragmentos de Péptidos/química , Ratas , Ratas Endogámicas Lew , Bazo/inmunología , Linfocitos T/inmunología , Vacunas de ADN/genéticaRESUMEN
The impact of the strength and promiscuity of the self peptide-MHC class II interaction on susceptibility to autoimmune disease is uncertain. Here we studied allelic differences in the affinity of rat MHC class II molecules for myelin basic protein (MBP) peptides spanning from position 63 to 106. Predominantly peptides from this region are immunogenic in the rat and the MHC class II region determines if the response is disease promoting or disease protective. Strikingly, RT1.B (DQ-like) molecules showed much more allelic variation of MBP peptide binding than RT1.D (DR-like) molecules. Moderate to strong binding of particular MBP peptides correlated with their previously documented encephalitogenicity. Moreover, the differences in disease susceptibility to certain MBP peptides observed in the different rat strains were clearly reflected in the allelic diversity of the peptide binding profiles. In conclusion our findings demonstrate that disease-inducing stretches of MBP generally comprise good binding peptides.
Asunto(s)
Alelos , Encefalomielitis Autoinmune Experimental/etiología , Antígenos de Histocompatibilidad Clase II/genética , Proteína Básica de Mielina/inmunología , Secuencia de Aminoácidos , Animales , Susceptibilidad a Enfermedades , Haplotipos , Antígenos de Histocompatibilidad Clase II/metabolismo , Datos de Secuencia Molecular , Proteína Básica de Mielina/metabolismo , Fragmentos de Péptidos/inmunología , Ratas , Ratas Endogámicas LewRESUMEN
Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS) with a complex etiology comprising a genetically determined predisposition and a suspected auto- immune pathogenesis. Experimental autoimmune encephalomyelitis (EAE) is an animal model for MS, which can be used to define susceptibility loci for autoimmune neuroinflammation. We have recently established a chronic relapsing EAE model characterized by inflammation and focal demyelination in the CNS by immunizing a variety of rat strains with the CNS-specific myelin oligodendrocyte glycoprotein (MOG). This model is more MS-like than any other rodent EAE model described up to now. Here we present the first systematic genome search for chromosomal regions linked to phenotypes of MOG-induced EAE in a (DA x ACI) F(2)intercross. A genome-wide significant susceptibility locus linked to demyelination was identified on chromosome 18. This region has not been described in inflammatory diseases affecting other organs and the responsible gene or genes may thus be nervous system specific. Other chromosomal regions showing suggestive linkage to phenotypes of MOG-induced EAE were identified on chromosomes 10, 12 and 13. The chromosome 10 and 12 regions have previously been linked to arthritis in DA rats, suggesting that they harbour immunoregulatory genes controlling general susceptibility to autoimmune diseases. We conclude that identification of susceptibility genes for MOG-induced EAE on rat chromosomes 10, 12, 13 and 18 may disclose important disease pathways for chronic inflammatory demyelinating diseases of the CNS such as MS.
Asunto(s)
Mapeo Cromosómico , Encefalomielitis Autoinmune Experimental/genética , Ligamiento Genético , Glicoproteína Asociada a Mielina/genética , Animales , Femenino , Masculino , Proteínas de la Mielina , Glicoproteína Mielina-Oligodendrócito , RatasRESUMEN
We here study the adjuvant properties of immunostimulatory DNA sequences (ISS) and coinjected cytokine-coding cDNA in suppressive vaccination with DNA encoding an autoantigenic peptide, myelin basic protein peptide 68-85, against Lewis rat experimental autoimmune encephalomyelitis (EAE). EAE is an autoaggressive, T1-mediated disease of the CNS. ISS are unmethylated CpG motifs found in bacterial DNA, which can induce production of type 1 cytokines in vertebrates through the innate immune system. Because ISS in the plasmid backbone are necessary for efficient DNA vaccination, we studied the effect of one such ISS, the 5'-AACGTT-3' motif, in our system. Treatment with a DNA vaccine encoding myelin basic protein peptide 68-85 and containing three ISS of 5'-AACGTT-3' sequence suppressed clinical signs of EAE, while a corresponding DNA vaccine without such ISS had no effect. We further observed reduced proliferative T cell responses in rats treated with the ISS-containing DNA vaccine, compared with controls. We also studied the possible impact of coinjection of plasmid DNA encoding rat cytokines IL-4, IL-10, GM-CSF, and TNF-alpha with the ISS-containing DNA vaccine. Coinjection of IL-4-, IL-10-, or TNF-alpha-coding cDNA inhibited the suppressive effect of the DNA vaccine on EAE, whereas GM-CSF-coding cDNA had no effect. Coinjection of cytokine-coding cDNA with the ISS-deficient DNA vaccine failed to alter clinical signs of EAE. We conclude that the presence of ISS and induction of a local T1 cytokine milieu is decisive for specific protective DNA vaccination in EAE.