RESUMO
Myasthenia gravis is a paralytic disorder with autoantibodies against acetylcholine receptors at the neuromuscular junction. A proportion of patients instead has antibodies against muscle-specific kinase, a protein essential for acetylcholine receptor clustering. These are generally of the immunoglobulin-G4 subclass and correlate with disease severity, suggesting specific myasthenogenic activity. However, immunoglobulin-G4 subclass antibodies are generally considered to be 'benign' and direct proof for their pathogenicity in muscle-specific kinase myasthenia gravis (or other immunoglobulin-G4-associated disorders) is lacking. Furthermore, the exact electrophysiological synaptic defects caused at neuromuscular junctions by human anti-muscle-specific kinase autoantibodies are hitherto unknown. We show that purified immunoglobulin-G4, but not immunoglobulin-G1-3, from patients with muscle-specific kinase myasthenia gravis binds to mouse neuromuscular junctions in vitro, and that injection into immunodeficient mice causes paralysis. Injected immunoglobulin-G4 caused reduced density and fragmented area of neuromuscular junction acetylcholine receptors. Detailed electrophysiological synaptic analyses revealed severe reduction of postsynaptic acetylcholine sensitivity, and exaggerated depression of presynaptic acetylcholine release during high-rate activity, together causing the (fatigable) muscle weakness. Intriguingly, compensatory transmitter release upregulation, which is the normal homeostatic response in acetylcholine receptor myasthenia gravis, was absent. This conveys extra vulnerability to neurotransmission at muscle-specific kinase myasthenia gravis neuromuscular junctions. Thus, we demonstrate that patient anti-muscle-specific kinase immunoglobulin-G4 is myasthenogenic, independent of additional immune system components, and have elucidated the underlying electrophysiological neuromuscular junction abnormalities.
Assuntos
Imunoglobulina G/efeitos adversos , Imunoglobulina G/sangue , Miastenia Gravis/sangue , Doenças da Junção Neuromuscular/complicações , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologia , Potenciais de Ação/efeitos dos fármacos , Adulto , Animais , Autoanticorpos/sangue , Modelos Animais de Doenças , Eletromiografia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Placa Motora/efeitos dos fármacos , Placa Motora/fisiopatologia , Contração Muscular/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Força Muscular/fisiologia , Miastenia Gravis/complicações , Miastenia Gravis/imunologia , Miastenia Gravis/terapia , Condução Nervosa/efeitos dos fármacos , Condução Nervosa/fisiologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Junção Neuromuscular/ultraestrutura , Doenças da Junção Neuromuscular/patologia , Plasmaferese/métodos , Adulto JovemRESUMO
Large scale, highly specific purification of valuable proteins from blood and removal of undesirable components promise to have wide therapeutic applications. Moreover, depletion of bulk proteins from blood is a prerequisite for clinical proteomics. Here we describe the development of specific, high affinity Camelid antibody fragments (VHH) derived from immune libraries for purification and depletion of the bulk protein HSA and IgG from human serum and plasma for therapeutic and research purposes. The anti-IgG VHH substantially improved depletion of IgGs from blood over the classical method based on protein A. To demonstrate the improved performance of VHH based IgG depletion, we analyzed the presence of auto-antibodies in human plasma before and after depletion from two groups of patients with auto-immune disease: Goodpasture syndrome (GP) and systemic lupus erythematosus (SLE). VHHs can be produced efficiently and cost effectively in Saccharomyces cerevisiae, a genetically regarded as safe (GRAS) microorganism. A good manufacturing process (GMP) for purification of these VHHs has also been developed. Moreover, as VHHs are single protein chains, they can be coupled relatively easily to solid matrices. These three factors are important for developing affinity purification medication.