RESUMEN
Several murine IgM monoclonal antibodies (mAbs) promoting remyelination in mice were shown to be germline gene-encoded natural autoantibodies that react with oligodendrocytes and intracellular antigens. Here, we show that human oligodendrocyte-reactive IgM mAb DS1F8 derived from a patient with multiple sclerosis targets microtubule-like structures similar to the murine mAbs. Sequencing of the cDNAs of the variable regions revealed that the antigen-binding domains are also encoded by germline genes. These similarities of mAb DS1F8 to the murine mAbs promoting remyelination suggest that this human mAb is a natural autoantibody. This may imply that the engineering of human autoantibodies for therapy of demyelinating diseases is feasible.
Asunto(s)
Anticuerpos Monoclonales/química , Autoanticuerpos/química , Enfermedades Autoinmunes/inmunología , Sitios de Unión de Anticuerpos , Inmunoglobulina M/química , Esclerosis Múltiple/inmunología , Oligodendroglía/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Autoanticuerpos/genética , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/terapia , Secuencia de Bases , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente Indirecta , Reordenamiento Génico de Linfocito B , Genes de Inmunoglobulinas , Células HeLa , Humanos , Inmunoglobulina M/genética , Inmunoglobulina M/inmunología , Región Variable de Inmunoglobulina/genética , Inmunoterapia , Datos de Secuencia Molecular , Esclerosis Múltiple/genética , Esclerosis Múltiple/terapia , Vaina de Mielina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de SecuenciaRESUMEN
Galactocerebroside (GalC) and sulfatide are major constituent lipids in vertebrate myelin. Their precise immunolocalization in electron microscopy so far has been hampered by the fact that lipids are not immobilized by chemical fixation and thus get extracted during dehydration with organic solvents. Here, we examined the suitability of cryotechniques for the preservation and immunohistochemical localization of myelin glycolipids in rat brain at the ultrastructural level. Native cerebral cortex tissue, obtained by fine-needle biopsy, was cryoimmobilized by high-pressure freezing and dehydrated by freeze-substitution before embedding in Epon. This procedure resulted in an excellent preservation of brain ultrastructure. Concomitantly, immunogold labeling of ultrathin sections with the well-defined monoclonal antibodies (mAbs) O1, O4, and R-mAb, which were shown to react with GalC and/or sulfatide and some structurally related glycolipids, revealed a good conservation of relevant epitopes. These data suggest that in adult rat cerebral cortex, the most relevant antigens recognized by R-mAb, O1, and O4, namely GalC and sulfatide, are exclusively expressed in myelin structures. Because these mAbs are common markers for the identification of developing oligodendrocytes, this "postembedding glycolipid-labeling technique" holds great potential for studying oligodendroglial differentiation in normal and pathological conditions at the ultrastructural level.
Asunto(s)
Encéfalo , Criopreservación/métodos , Substitución por Congelación/métodos , Glucolípidos/análisis , Vaina de Mielina/ultraestructura , Animales , Biopsia con Aguja , Encéfalo/citología , Encéfalo/ultraestructura , Ensayo de Inmunoadsorción Enzimática , Resinas Epoxi , Microscopía Electrónica , Microscopía Inmunoelectrónica/métodos , Presión , RatasRESUMEN
In a previous paper, we described the production of a sulfatide-reactive IgM antibody-secreting B cell line that was obtained by Epstein-Barr virus transformation of peripheral B cells from a patient with multiple sclerosis (MS) (Uhlig and Dernick, 1989). In the present study, we demonstrate that this human monoclonal antibody (humAb) DS1F8 selectively binds to the surface of living oligodendrocytes in mixed brain cell cultures of newborn rats. Since a mouse mAb reactive with sulfatide was shown to inhibit oligodendrocyte progenitor differentiation, autoantibodies with binding specificities similar to DS1F8 could play a role in the demyelinating process in the CNS.