RESUMO
OBJECTIVE: Impairment of glycinergic neurotransmission leads to complex movement and behavioral disorders. Patients harboring glycine receptor autoantibodies suffer from stiff-person syndrome or its severe variant progressive encephalomyelitis with rigidity and myoclonus. Enhanced receptor internalization was proposed as the common molecular mechanism upon autoantibody binding. Although functional impairment of glycine receptors following autoantibody binding has recently been investigated, it is still incompletely understood. METHODS: A cell-based assay was used for positive sample evaluation. Glycine receptor function was assessed by electrophysiological recordings and radioligand binding assays. The in vivo passive transfer of patient autoantibodies was done using the zebrafish animal model. RESULTS: Glycine receptor function as assessed by glycine dose-response curves showed significantly decreased glycine potency in the presence of patient sera. Upon binding of autoantibodies from 2 patients, a decreased fraction of desensitized receptors was observed, whereas closing of the ion channel remained fast. The glycine receptor N-terminal residues 29 A to 62 G were mapped as a common epitope of glycine receptor autoantibodies. An in vivo transfer into the zebrafish animal model generated a phenotype with disturbed escape behavior accompanied by a reduced number of glycine receptor clusters in the spinal cord of affected animals. INTERPRETATION: Autoantibodies against the extracellular domain mediate alterations of glycine receptor physiology. Moreover, our in vivo data demonstrate that the autoantibodies are a direct cause of the disease, because the transfer of human glycine receptor autoantibodies to zebrafish larvae generated impaired escape behavior in the animal model compatible with abnormal startle response in stiff-person syndrome or progressive encephalitis with rigidity and myoclonus patients. ANN NEUROL 2020;88:544-561.
Assuntos
Autoanticorpos/imunologia , Encefalomielite/imunologia , Rigidez Muscular/imunologia , Receptores de Glicina/metabolismo , Rigidez Muscular Espasmódica/imunologia , Adulto , Idoso , Animais , Autoanticorpos/farmacologia , Autoantígenos/imunologia , Comportamento Animal/efeitos dos fármacos , Encefalomielite/metabolismo , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Rigidez Muscular/metabolismo , Receptores de Glicina/imunologia , Rigidez Muscular Espasmódica/metabolismo , Peixe-ZebraRESUMO
BACKGROUND: Autoantibodies against the paranodal protein contactin-1 have recently been described in patients with severe acute-onset autoimmune neuropathies and mainly belong to the IgG4 subclass that does not activate complement. IgG3 anti-contactin-1 autoantibodies are rare, but have been detected during the acute onset of disease in some cases. There is evidence that anti-contactin-1 prevents adhesive interaction, and chronic exposure to anti-contactin-1 IgG4 leads to structural changes at the nodes accompanied by neuropathic symptoms. However, the pathomechanism of acute onset of disease and the pathogenic role of IgG3 anti-contactin-1 is largely unknown. METHODS: In the present study, we aimed to model acute autoantibody exposure by intraneural injection of IgG of patients with anti-contacin-1 autoantibodies to Lewis rats. Patient IgG obtained during acute onset of disease (IgG3 predominant) and IgG from the chronic phase of disease (IgG4 predominant) were studied in comparison. RESULTS: Conduction blocks were measured in rats injected with the "acute" IgG more often than after injection of "chronic" IgG (83.3% versus 35%) and proved to be reversible within a week after injection. Impaired nerve conduction was accompanied by motor deficits in rats after injection of the "acute" IgG but only minor structural changes of the nodes. Paranodal complement deposition was detected after injection of the "acute IgG". We did not detect any inflammatory infiltrates, arguing against an inflammatory cascade as cause of damage to the nerve. We also did not observe dispersion of paranodal proteins or sodium channels to the juxtaparanodes as seen in patients after chronic exposure to anti-contactin-1. CONCLUSIONS: Our data suggest that anti-contactin-1 IgG3 induces an acute conduction block that is most probably mediated by autoantibody binding and subsequent complement deposition and may account for acute onset of disease in these patients. This supports the notion of anti-contactin-1-associated neuropathy as a paranodopathy with the nodes of Ranvier as the site of pathogenesis.
Assuntos
Contactina 1/imunologia , Síndrome de Guillain-Barré/complicações , Imunização Passiva/métodos , Imunoglobulina G/farmacologia , Transtornos Motores/fisiopatologia , Transtornos Motores/cirurgia , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Complemento C1q/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Síndrome de Guillain-Barré/etiologia , Humanos , Transtornos Motores/induzido quimicamente , Condução Nervosa/efeitos dos fármacos , Neurite Óptica/sangue , Neurite Óptica/imunologia , Nós Neurofibrosos/efeitos dos fármacos , Nós Neurofibrosos/metabolismo , Ratos , Ratos Endogâmicos Lew , Tempo de Reação/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Teste de Desempenho do Rota-Rod , Estatísticas não ParamétricasRESUMO
Stiff-person syndrome is the prototype of a central nervous system disorder with autoantibodies targeting presynaptic antigens. Patients with paraneoplastic stiff-person syndrome may harbour autoantibodies to the BAR (Bin/Amphiphysin/Rvs) domain protein amphiphysin, which target its SH3 domain. These patients have neurophysiological signs of compromised central inhibition and respond to symptomatic treatment with medication enhancing GABAergic transmission. High frequency neurotransmission as observed in tonic GABAergic interneurons relies on fast exocytosis of neurotransmitters based on compensatory endocytosis. As amphiphysin is involved in clathrin-mediated endocytosis, patient autoantibodies are supposed to interfere with this function, leading to disinhibition by reduction of GABAergic neurotransmission. We here investigated the effects of human anti-amphiphysin autoantibodies on structural components of presynaptic boutons ex vivo and in vitro using electron microscopy and super-resolution direct stochastic optical reconstruction microscopy. Ultrastructural analysis of spinal cord presynaptic boutons was performed after in vivo intrathecal passive transfer of affinity-purified human anti-amphiphysin autoantibodies in rats and revealed signs of markedly disabled clathrin-mediated endocytosis. This was unmasked at high synaptic activity and characterized by a reduction of the presynaptic vesicle pool, clathrin coated intermediates, and endosome-like structures. Super-resolution microscopy of inhibitory GABAergic presynaptic boutons in primary neurons revealed that specific human anti-amphiphysin immunoglobulin G induced an increase of the essential vesicular protein synaptobrevin 2 and a reduction of synaptobrevin 7. This constellation suggests depletion of resting pool vesicles and trapping of releasable pool vesicular proteins at the plasma membrane. Similar effects were found in amphiphysin-deficient neurons from knockout mice. Application of specific patient antibodies did not show additional effects. Blocking alternative pathways of clathrin-independent endocytosis with brefeldin A reversed the autoantibody induced effects on molecular vesicle composition. Endophilin as an interaction partner of amphiphysin showed reduced clustering within presynaptic terminals. Collectively, these results point towards an autoantibody-induced structural disorganization in GABAergic synapses with profound changes in presynaptic vesicle pools, activation of alternative endocytic pathways, and potentially compensatory rearrangement of proteins involved in clathrin-mediated endocytosis. Our findings provide novel insights into synaptic pathomechanisms in a prototypic antibody-mediated central nervous system disease, which may serve as a proof-of-principle example in this evolving group of autoimmune disorders associated with autoantibodies to synaptic antigens.
Assuntos
Autoanticorpos/administração & dosagem , Proteínas do Tecido Nervoso/administração & dosagem , Terminações Pré-Sinápticas/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Animais , Autoanticorpos/sangue , Células Cultivadas , Feminino , Humanos , Injeções Espinhais , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/sangue , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Gravidez , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Ratos , Ratos Endogâmicos Lew , Rigidez Muscular Espasmódica/sangue , Rigidez Muscular Espasmódica/diagnóstico , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismoRESUMO
BACKGROUND: We could previously identify components of both the innate and the adaptive immune system as disease modifiers in the pathogenesis of models for Charcot-Marie-Tooth (CMT) neuropathies type 1B and 1X. As part of the adaptive immune system, here we investigated the role of antibodies in a model for CMT1B. METHODS: Antibodies were localized and characterized in peripheral nerves of the CMT1B model by immunohistochemistry and Western blot analysis. Experimental ablation of antibodies was performed by cross breeding the CMT1B models with mutants deficient in B-lymphocytes (JHD-/- mutants). Ameliorated demyelination by antibody deficiency was reverted by intravenous injection of mouse IgG fractions. Histopathological analysis was performed by immunocytochemistry and light and quantitative electron microscopy. RESULTS: We demonstrate that in peripheral nerves of a mouse model for CMT1B, endogenous antibodies strongly decorate endoneurial tubes of peripheral nerves. These antibodies comprise IgG and IgM subtypes and are preferentially, but not exclusively, associated with nerve fiber aspects nearby the nodes of Ranvier. In the absence of antibodies, the early demyelinating phenotype is substantially ameliorated. Reverting the neuropathy by reconstitution with murine IgG fractions identified accumulating antibodies as potentially pathogenic at this early stage of disease. CONCLUSIONS: Our study demonstrates that in a mouse model for CMT1B, endogenous antibodies contribute to early macrophage-mediated demyelination and disease progression. Thus, both the innate and adaptive immune system are mutually interconnected in a genetic model for demyelination. Since in Wallerian degeneration antibodies have also been shown to be involved in myelin phagocytosis, our study supports our view that inherited demyelination and Wallerian degeneration share common mechanisms, which are detrimental when activated under nonlesion conditions.
Assuntos
Anticorpos/metabolismo , Doença de Charcot-Marie-Tooth/complicações , Doenças Desmielinizantes/etiologia , Análise de Variância , Animais , Antígenos de Diferenciação/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doenças Desmielinizantes/imunologia , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína P0 da Mielina/deficiência , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/imunologia , Neuropatia Ciática/metabolismo , Neuropatia Ciática/patologia , Transdução de Sinais/imunologiaRESUMO
OBJECTIVE: Autoantibodies against paranodal proteins have been described in patients with inflammatory neuropathies, but their association with pathology of nodes of Ranvier is unclear. We describe the clinical phenotype and histopathological changes of paranodal architecture of patients with autoantibodies against contactin-1, identified from a cohort with chronic inflammatory demyelinating polyradiculoneuropathy (n=53) and Guillain-Barré syndrome (n=21). METHODS: We used ELISA to detect autoantibodies against contactin-1. Specificity of the autoantibodies was confirmed by immunoblot assay, binding to contactin-1-transfected human embryonic kidney cells, binding to paranodes of murine teased fibres and preabsorption experiments. Paranodal pathology was investigated by immunofluorescence labelling of dermal myelinated fibres. RESULTS: High reactivity to contactin-1 by ELISA was found in four patients with chronic inflammatory demyelinating polyradiculoneuropathy and in none of the patients with Guillain-Barré syndrome, which was confirmed by cell binding assays in all four patients. The four patients presented with a typical clinical picture, namely acute onset of disease and severe motor symptoms, with three patients manifesting action tremor. Immunofluorescence-labelling of paranodal proteins of dermal myelinated fibres revealed disruption of paranodal architecture. Semithin sections showed axonal damage but no classical signs of demyelination. INTERPRETATION: We conclude that anti-contactin-1-related neuropathy constitutes a presumably autoantibody-mediated form of inflammatory neuropathy with distinct clinical symptoms and disruption of paranodal architecture as a pathological correlate. Anti-contactin-1-associated neuropathy does not meet morphological criteria of demyelinating neuropathy and therefore, might rather be termed a 'paranodopathy' rather than a subtype of demyelinating inflammatory neuropathy.
Assuntos
Autoanticorpos/imunologia , Contactina 1/imunologia , Síndrome de Guillain-Barré/imunologia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/imunologia , Nós Neurofibrosos/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Células HEK293/imunologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Polirradiculoneuropatia/imunologia , Adulto JovemRESUMO
The majority of patients with stiff person-syndrome (SPS) are characterized by autoantibodies to glutamate decarboxylase 65 (GAD65). In previous passive-transfer studies, SPS immunoglobulin G (IgG) induced SPS core symptoms. We here provide evidence that SPS-IgG causes a higher frequency of spontaneous vesicle fusions. Sustained GABAergic transmission and presynaptic GABAergic vesicle pool size remained unchanged. Since these findings cannot be attributed to anti-GAD65 autoantibodies alone, we propose that additional autoantibodies with so far undefined antigen specificity might affect presynaptic release mechanisms.
Assuntos
Neurônios GABAérgicos/citologia , Glutamato Descarboxilase/imunologia , Imunoglobulina G/farmacologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos dos fármacos , Rigidez Muscular Espasmódica/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Embrião de Mamíferos , Feminino , Neurônios GABAérgicos/efeitos dos fármacos , Hipocampo/citologia , Humanos , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Estatísticas não Paramétricas , Rigidez Muscular Espasmódica/imunologia , Sinaptofisina/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
Stiff person syndrome with auto-antibodies against amphiphysin is characterized by muscular stiffness, spasms, and anxiety which is a less appreciated core symptom. Here, we report that intrathecal application of purified immunoglobulin G-antibodies against amphiphysin from one patient induce anxiety behavior in rats. Immunostaining demonstrated binding of anti-amphiphysin antibodies to brain structures which are associated with anxiety disorders, such as the amygdala. We propose that antibody-mediated amphiphysin deficiency may account for anxiety behavior in stiff person syndrome via presynaptic dysregulation of GABAergic pathways.
Assuntos
Ansiedade/imunologia , Autoanticorpos/administração & dosagem , Imunoglobulina G/administração & dosagem , Proteínas do Tecido Nervoso/deficiência , Rigidez Muscular Espasmódica/psicologia , Animais , Autoantígenos/imunologia , Comportamento Animal , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Injeções Espinhais , Proteínas do Tecido Nervoso/imunologia , Ratos , Ratos Endogâmicos Lew , Rigidez Muscular Espasmódica/imunologiaRESUMO
Glatiramer acetate (GA) is a synthetic, random, basic copolymer capable of modulating adaptive T cell responses. In animal models of various inflammatory and degenerative central nervous system disorders, GA-induced T cells cross the blood-brain barrier, secrete high levels of anti-inflammatory cytokines and neurotrophins, and thus both reduce neuronal damage and promote neurogenesis. Recently, it has been suggested that GA itself may permeate the (impaired) blood-brain-barrier and directly protect neurons under conditions of inflammation-mediated neurodegeneration. To test this hypothesis, we examined the direct effects of GA on neuronal functionality and T cell-mediated neuronal apoptosis in culture, acute brain slices, and focal experimental autoimmune encephalomyelitis. GA caused a depolarization of the resting membrane potential and led to an immediate impairment of action potential generation in neurons. Moreover, GA-incubated neurons underwent dose-dependent apoptosis. Apoptosis of ovalbumin peptide-loaded major histocompatibility complex class I-expressing neurons induced by ovalbumin-specific effector T cells could be reduced by pre-incubation of T cells, but not neurons with GA. Similar results could be found using acute brain slices. In focal experimental autoimmune encephalomyelitis, lesion size and neuronal apoptosis could be limited by pretreating rats with GA, whereas intracerebral GA application into the inflammatory lesion had no effect on neuronal survival. Our data suggest that GA attenuates adaptive pro-inflammatory T cell responses, but does not exert direct neuroprotective effects.
Assuntos
Inflamação/patologia , Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Células Cultivadas , Citoproteção/efeitos dos fármacos , Citoproteção/imunologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Avaliação Pré-Clínica de Medicamentos , Embrião de Mamíferos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Acetato de Glatiramer , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Transgênicos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Synaptic inhibition is a central factor in the fine tuning of neuronal activity in the central nervous system. Symptoms consistent with reduced inhibition such as stiffness, spasms and anxiety occur in paraneoplastic stiff person syndrome with autoantibodies against the intracellular synaptic protein amphiphysin. Here we show that intrathecal application of purified anti-amphiphysin immunoglobulin G antibodies induces stiff person syndrome-like symptoms in rats, including stiffness and muscle spasms. Using in vivo recordings of Hoffmann reflexes and dorsal root potentials, we identified reduced presynaptic GABAergic inhibition as an underlying mechanism. Anti-amphiphysin immunoglobulin G was internalized into neurons by an epitope-specific mechanism and colocalized in vivo with presynaptic vesicular proteins, as shown by stimulation emission depletion microscopy. Neurons from amphiphysin deficient mice that did not internalize the immunoglobulin provided additional evidence of the specificity in antibody uptake. GABAergic synapses appeared more vulnerable than glutamatergic synapses to defective endocytosis induced by anti-amphiphysin immunoglobulin G, as shown by increased clustering of the endocytic protein AP180 and by defective loading of FM 1-43, a styryl dye used to label cell membranes. Incubation of cultured neurons with anti-amphiphysin immunoglobulin G reduced basal and stimulated release of γ-aminobutyric acid substantially more than that of glutamate. By whole-cell patch-clamp analysis of GABAergic inhibitory transmission in hippocampus granule cells we showed a faster, activity-dependent decrease of the amplitude of evoked inhibitory postsynaptic currents in brain slices treated with antibodies against amphiphysin. We suggest that these findings may explain the pathophysiology of the core signs of stiff person syndrome at the molecular level and show that autoantibodies can alter the function of inhibitory synapses in vivo upon binding to an intraneuronal key protein by disturbing vesicular endocytosis.
Assuntos
Autoanticorpos/uso terapêutico , Proteínas do Tecido Nervoso/imunologia , Inibição Neural/imunologia , Rigidez Muscular Espasmódica/imunologia , Rigidez Muscular Espasmódica/terapia , Ácido gama-Aminobutírico/metabolismo , Idoso , Animais , Autoanticorpos/administração & dosagem , Autoanticorpos/fisiologia , Células Cultivadas , Endocitose/imunologia , Feminino , Humanos , Imunização Passiva/métodos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/fisiologia , Imunoglobulina G/uso terapêutico , Potenciais Pós-Sinápticos Inibidores/fisiologia , Injeções Espinhais , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Ratos , Ratos Endogâmicos Lew , Rigidez Muscular Espasmódica/patologia , Ácido gama-Aminobutírico/deficiênciaRESUMO
Autoimmune responses in the CNS can be induced by adoptive transfer of CD4(+) T effector cells after antigen-restimulation and expansion of clonal cell lines in vitro. However, pathogenic factors remain partially elusive due to the lack of appropriate methods to achieve gene inactivation. Here we describe a protocol for stable gene silencing in differentiated rat T cells by retroviral transfer of small hairpin RNAs. Through the combination of an expression cassette containing the green fluorescent protein with a puromycin selection cassette this allows for the generation of pure knockdown cell lines suitable for tracking in animals. Exemplified for the glucocorticoid receptor, we demonstrate that gene silencing renders T effector cells unresponsive to ligand-induced apoptosis and gene regulation without affecting their ability to induce EAE in rats. Interestingly, glucocorticoid administration remains effective in the treatment of EAE despite strongly diminished glucocorticoid receptor expression in antigen-specific T cells. This highlights an important role of other cell types and bystander T cells as targets of glucocorticoid therapy. Collectively, our approach provides a simple tool for stable and efficient gene silencing in T effector cells, which should help to better understand brain autoimmune pathophysiology.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Encefalomielite Autoimune Experimental/imunologia , Glucocorticoides/uso terapêutico , Receptores de Glucocorticoides/genética , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Inativação Gênica , Glucocorticoides/farmacologia , Interferência de RNA/imunologia , Ratos , Ratos Endogâmicos Lew , RetroviridaeRESUMO
The blood-brain barrier (BBB) serves as a border limiting access of immunoglobulins from the circulation into the brain. This becomes relevant when studying the pathogenesis of antibody-mediated autoimmune CNS disorders. Here, we characterized the BBB dysfunction in a model of mild experimental adoptive transfer autoimmune encephalomyelitis (AT-EAE). We show that large molecules can readily penetrate the BBB between days 3 and 7 after EAE-induction. This model may be valuable for studying putative pathogenic effects of immunoglobulins in the central nervous system.
Assuntos
Barreira Hematoencefálica/fisiopatologia , Encefalomielite Autoimune Experimental/fisiopatologia , Albuminas/metabolismo , Animais , Peso Corporal , Permeabilidade Capilar , Córtex Cerebral/fisiopatologia , Ectodisplasinas/metabolismo , Azul Evans , Feminino , Humanos , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Atividade Motora , Ratos , Ratos Endogâmicos Lew , Índice de Gravidade de Doença , Medula Espinal/fisiopatologia , Fatores de TempoRESUMO
We provide evidence that TWIK-related acid-sensitive potassium channel 1 (TASK1), a member of the family of two-pore domain potassium channels relevant for setting the resting membrane potential and balancing neuronal excitability that is expressed on T cells and neurons, is a key modulator of T cell immunity and neurodegeneration in autoimmune central nervous system inflammation. After induction of experimental autoimmune encephalomyelitis, an experimental model mimicking multiple sclerosis, TASK1(-/-) mice showed a significantly reduced clinical severity and markedly reduced axonal degeneration compared with wild-type controls. T cells from TASK1(-/-) mice displayed impaired T cell proliferation and cytokine production, while the immune repertoire is otherwise normal. In addition to these effects on systemic T cell responses, TASK1 exhibits an independent neuroprotective effect which was demonstrated using both a model of acutely prepared brain slices cocultured with activated T cells as well as in vitro cultivation experiments with isolated optic nerves. Anandamide, an endogenous cannabinoid and inhibitor of TASK channels, reduced outward currents and inhibited effector functions of T cells (IFN-gamma production and proliferation); an effect completely abrogated in TASK1(-/-) mice. Accordingly, preventive blockade of TASK1 significantly ameliorated experimental autoimmune encephalomyelitis after immunization. Therapeutic application of anandamide significantly reduced disease severity and was capable of lowering progressive loss of brain parenchymal volume as assessed by magnetic resonance imaging. These data support the identification and characterization of TASK1 as potential molecular target for the therapy of inflammatory and degenerative central nervous system disorders.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Degeneração Neural/imunologia , Proteínas do Tecido Nervoso/imunologia , Canais de Potássio de Domínios Poros em Tandem/imunologia , Animais , Ácidos Araquidônicos/uso terapêutico , Axônios/patologia , Células Cultivadas , Técnicas de Cocultura , Progressão da Doença , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/prevenção & controle , Endocanabinoides , Feminino , Imunofenotipagem , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/imunologia , Degeneração Neural/patologia , Degeneração Neural/prevenção & controle , Proteínas do Tecido Nervoso/antagonistas & inibidores , Alcamidas Poli-Insaturadas/uso terapêutico , Bloqueadores dos Canais de Potássio/uso terapêutico , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Medula Espinal/patologia , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Técnicas de Cultura de TecidosRESUMO
Administration of high-dose myelin antigen induces massive T cell apoptosis in experimental autoimmune encephalomyelitis (EAE) but the nature of the target cells remains elusive. Here we have used a cell line established in eGFP-transgenic Lewis rats to distinguish between pathogenic and bystander T cells in adoptive transfer EAE. Intravenous application of gpMBP strongly reduced the amount of encephalitogenic cells in spinal cord and spleen while the number of the other T cells remained constant. This could be attributed to their differential sensitivity to apoptosis. Thus, antigen therapy selectively targets pathogenic T cells and should therefore limit potential adverse effects.
Assuntos
Antígenos/uso terapêutico , Apoptose/efeitos dos fármacos , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/terapia , Linfócitos T/efeitos dos fármacos , Transferência Adotiva/métodos , Animais , Caspase 3/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde , Camundongos , Camundongos Transgênicos , Proteína Básica da Mielina , Ratos , Ratos Endogâmicos Lew , Medula Espinal/patologia , Baço/metabolismo , Baço/patologiaRESUMO
Recruitment of naturally occurring CD4+ CD25+ regulatory T (T(reg)) cells is a highly promising approach for the treatment of experimental autoimmune encephalomyelitis (EAE), a widely used model of multiple sclerosis. Here, we studied the in vivo interaction of T(reg) cells, induced by the monoclonal anti-CD28 antibody JJ316, with encephalitogenic T cell lines established from eGFP-transgenic rats. By tracking these fluorescent cells using flow cytometry and confocal microscopy, we found that the activation and expansion of T(reg) cells inhibited infiltration of the CNS by pathogenic T cells. Interference with effector cell migration occured within the secondary lymphoid organs, since the early therapeutic effects were achieved despite the absence of T(reg) cells in the spinal cord. However, the delayed homing to the CNS seen after prophylactic JJ316 administration indicates that T(reg) cells may play an additional role within the target tissue. In addition, the blood-brain barrier remained largely intact after JJ316 treatment, the secretion of T(H)2 cytokines was augmented and the encephalitogenic T cells exhibited a reduced secretion of IFN-gamma. This in turn resulted in a reduced expression of the chemokine receptor CXCR-3 on effector T cells which may interfere with their capacity to infiltrate the CNS. Importantly, these effects were not achieved by direct action of JJ316 on the encephalitogenic cells. Our data rather suggest that polyclonal activation of T(reg) cells in the secondary lymphoid organs is instrumental in preventing the pathological transmigration of encephalitogenic T cells into the CNS. We anticipate that these results may help to better understand the role of T(reg) cells in controlling autoimmunity in the CNS.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD28/imunologia , Imunização Passiva/métodos , Esclerose Múltipla/terapia , Linfócitos T Reguladores/imunologia , Animais , Barreira Hematoencefálica , Movimento Celular , Proliferação de Células , Citometria de Fluxo , Imuno-Histoquímica/métodos , Interferon gama/imunologia , Ativação Linfocitária , Microscopia Confocal , Modelos Animais , Esclerose Múltipla/imunologia , Ratos , Ratos Endogâmicos Lew , Receptores CXCR3 , Receptores de Quimiocinas/imunologia , Medula Espinal/imunologia , Linfócitos T/imunologiaRESUMO
Inflammatory neuropathies associated with auto-antibodies against paranodal proteins like contactin-1 are reported to respond poorly to treatment with intravenous immunoglobulins (IVIG). A reason might be that IVIG interacts with the complement pathway and these auto-antibodies often belong to the IgG4 subclass that does not activate complement. However, some patients do show a response to IVIG, especially at the beginning of the disease. This corresponds with the finding of coexisting IgG subclasses IgG1, IgG2 and IgG3. We therefore aimed to investigate complement deposition and activation by samples of three patients with anti-contactin-1 IgG auto-antibodies of different subclasses as a potential predictor for response to IVIG. Complement deposition and activation was measured by cell binding and ELISA based assays, and the effect of IVIG on complement deposition was assessed by addition of different concentrations of IVIG. Binding of anti-contactin-1 auto-antibodies of all three patients induced complement deposition and activation with the strongest effect shown by the serum of a patient with predominance of IgG3 auto-antibodies. IVIG led to a reduction of complement deposition in a dose-dependent manner, but did not reduce binding of auto-antibodies to contactin-1. We conclude that complement deposition may contribute to the pathophysiology of anti-contactin-1 associated neuropathy, particularly in patients with predominance of the IgG3 subclass. The proportion of different auto-antibody subclasses may be a predictor for the response to IVIG in patients with auto-antibodies against paranodal proteins.
Assuntos
Autoanticorpos/metabolismo , Ativação do Complemento , Proteínas do Sistema Complemento/metabolismo , Contactina 1/imunologia , Imunoglobulinas Intravenosas/farmacologia , Fatores Imunológicos/farmacologia , Idoso , Animais , Doenças Autoimunes do Sistema Nervoso/sangue , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças Autoimunes do Sistema Nervoso/terapia , Proteínas do Sistema Complemento/classificação , Contactina 1/genética , Contactina 1/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Células HEK293 , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Masculino , Ligação Proteica/efeitos dos fármacos , Ratos , TransfecçãoRESUMO
Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease) caused by mutations in the CLN3 gene is the most prevalent inherited neurodegenerative disease in childhood resulting in widespread central nervous system dysfunction and premature death. The consequences of CLN3 mutation on the progression of the disease, on neuronal transmission, and on central nervous network dysfunction are poorly understood. We used Cln3 knockout (Cln3Δex1-6) mice and found increased anxiety-related behavior and impaired aversive learning as well as markedly affected motor function including disordered coordination. Patch-clamp and loose-patch recordings revealed severely affected inhibitory and excitatory synaptic transmission in the amygdala, hippocampus, and cerebellar networks. Changes in presynaptic release properties may result from dysfunction of CLN3 protein. Furthermore, loss of calbindin, neuropeptide Y, parvalbumin, and GAD65-positive interneurons in central networks collectively support the hypothesis that degeneration of GABAergic interneurons may be the cause of supraspinal GABAergic disinhibition.
Assuntos
Tonsila do Cerebelo/fisiopatologia , Cerebelo/fisiopatologia , Hipocampo/fisiopatologia , Lipofuscinoses Ceroides Neuronais/patologia , Lipofuscinoses Ceroides Neuronais/fisiopatologia , Transmissão Sináptica , Animais , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Chaperonas Moleculares , Rede Nervosa/fisiopatologia , Técnicas de Patch-ClampRESUMO
BACKGROUND: Stiff-person syndrome (SPS) with antibodies to amphiphysin is a paraneoplastic disorder of the central nervous system with a putative autoimmune pathogenesis. Proof of a causal role of the antibodies is still lacking for this and all other antibody-associated paraneoplastic syndromes of the central nervous system. METHODS: We obtained the plasma filtrate of a patient with breast cancer and SPS that responded to therapeutic plasmapheresis. The purified IgG fraction included high-titre antibodies to the synaptic protein amphiphysin. In a cotransfer design, this IgG fraction was injected intraperitoneally into female Lewis rats that had received encephalitogenic T-helper (Th) lymphocytes specific for myelin basic protein, to induce an immune-mediated leaky blood-brain barrier. The rats were followed up with behavioural tests, video photography, and electromyography. FINDINGS: The injection of the IgG fraction including antibodies to amphiphysin resulted in a dose-dependent stiffness with spasms resembling human SPS. Control IgG injected into rats that had received the same encephalitogenic Th cells had no effect. IgG binding was demonstrated in the central nervous system of rats that showed signs of the disorder. INTERPRETATION: These experiments support the hypothesis of a pathogenetic role of antibodies to amphiphysin, thus adding paraneoplastic SPS to the group of antibody-mediated autoimmune disorders. RELEVANCE TO PRACTICE: These findings provide a strong argument for a direct pathogenetic role of anti-amphiphysin in this type of SPS and support therapeutic attempts to eliminate these autoantibodies by plasmapheresis. The experimental approach used could help to elucidate the role of autoantibodies in other paraneoplastic syndromes, such as SPS with antibodies to glutamic acid decarboxylase, and others including anti-Hu-associated subacute cerebellar degeneration and limbic encephalitis.
Assuntos
Autoanticorpos/administração & dosagem , Imunização Passiva , Imunoglobulina G/administração & dosagem , Proteínas do Tecido Nervoso/imunologia , Síndromes Paraneoplásicas/imunologia , Rigidez Muscular Espasmódica/imunologia , Animais , Anticorpos/análise , Autoanticorpos/análise , Relação Dose-Resposta Imunológica , Feminino , Glutamato Descarboxilase/imunologia , Humanos , Imunoglobulina G/análise , Ratos , Ratos Endogâmicos Lew , Rigidez Muscular Espasmódica/diagnósticoRESUMO
BACKGROUND: Beside the determination of oligoclonal bands (OCBs) as a diagnostic biomarker in multiple sclerosis (MS), the presence of an intrathecal production of antibodies against the neurotropic viruses measles (M), rubella (R) and Varicella-Zoster (Z), the so called MRZ reaction (MRZR) is an even more specific diagnostic biomarker in MS. METHODS: We compared and validated the determination of the MRZR in 97 cerebrospinal fluid (CSF) and serum sample pairs of a bead-based multiplexing technique and a classical enzyme-linked immunosorbent assay (ELISA). RESULTS: Conformity of 94% (M), 94% (R), 94% (Z), 96% (H) and 97% for the interpretation of the MRZR was obtained. CONCLUSION: Based on our findings of high conformity between the multiplex technique and classical ELISA, as well as the time and cost savings multiplexing allows, we conclude that the multiplexing technique is applicable as a diagnostic tool for the determination of the MRZR.
Assuntos
Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Esclerose Múltipla/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/líquido cefalorraquidiano , Biomarcadores , Ensaio de Imunoadsorção Enzimática , Humanos , Esclerose Múltipla/virologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Oligoclonal immunoglobulin G bands (OCBs) restricted to the cerebrospinal fluid indicate intrathecal inflammation. Using isoelectric focusing and immunoblotting, they are detected in about 95 % of patients with clinically definite multiple sclerosis (MS). To elucidate whether in the remaining 5 % OCBs are truly absent or alternatively missed due to insufficient sensitivity of the routine measurement, we employed a new, highly sensitive nanoscale method for OCB detection. Capillary isoelectric focusing followed by immunological detection served to analyze OCBs in 33 well-characterized OCB-negative and 10 OCB-positive MS patients as well as in 100 OCB-negative control patients with non-inflammatory neurological diseases and 30 OCB-positive control patients with inflammatory neurological diseases. We detected intrathecal immunoglobulin G production in 10 out of 33 MS patients (30 %), initially diagnosed as being OCB-negative, and in all 10 OCB-positive MS patients, but in only 3 out of 100 non-inflammatory neurological controls (3 %) and in 29 of 30 inflammatory neurological controls (97 %). At least about one-third of MS patients without intrathecal immunoglobulin G synthesis according to standard methods are OCB-positive. Advanced methods for OCB detection may increase the analytical sensitivity for detecting OCB in patients with MS who are OCB-negative according to current routine methods.
Assuntos
Eletroforese Capilar/métodos , Imunoglobulina G/biossíntese , Imunoglobulina G/líquido cefalorraquidiano , Focalização Isoelétrica/métodos , Esclerose Múltipla/líquido cefalorraquidiano , Bandas Oligoclonais/líquido cefalorraquidiano , Adulto , Idoso , Estudos de Coortes , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Imunoensaio/métodos , Imunoglobulina G/sangue , Pessoa de Meia-Idade , Esclerose Múltipla/sangueRESUMO
MS is a highly prevalent neuroinflammatory disease of presumed autoimmune origin. Clinical observations and animal studies suggest that CD8(+) T cells play an important role in MS but their exact mechanisms are ill defined. When we actively induced EAE in CD8 knock-out DA rats, or adoptively transferred encephalitogenic CD4(+) T cells into CD8 knock-out DA rats, the disease course was indistinguishable from controls. Since our previous findings had revealed that the absence of CD8(+) T cells in Lewis rats ameliorated EAE, we compared antigen-induced T cell differentiation in both strains. Disease onset and the composition of the draining lymph nodes were similar but T cell activation in DA rats was much weaker. Moreover, oligoclonal expansion of CD8(+) T cells was exclusively observed in Lewis but not in DA rats. This suggests that myelin-specific CD8(+) T cells are involved in the differentiation of encephalitogenic CD4(+) T cells in Lewis rats, whilst they do not impact CD4(+) T cell priming in DA rats. Hence, clonal expansion of CD8(+) T cells in secondary lymphoid organs appears to be linked to their ability to modulate CNS autoimmune responses.