RESUMO
We have developed a B cell immortalization method for low B cell numbers per well using simultaneous B cell stimulation by CpG2006 and B cell infection by Epstein-Barr virus (EBV), followed by an additional CpG2006 and interleukin-2 (IL-2) stimulus. Using this method, immunoglobulin G (IgG)-producing immortalized B cell lines were generated from peripheral blood IgG(+)CD22(+) B cells with an efficiency of up to 83%. Antibody can already be obtained from the culture supernatant after 3-4 weeks. Moreover, clonality analysis demonstrated monoclonality in 87% of the resulting immortalized B cell lines. Given the high immortalization efficiency and monoclonality rate, evidence is provided that no further subcloning is necessary. An important application of this B cell immortalization method is the characterization of (autoreactive) antibodies from patients with autoimmune disease. This could eventually lead to the identification of new autoantigens, disease markers or targets for therapy.
Assuntos
Anticorpos Monoclonais/biossíntese , Linfócitos B/metabolismo , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Imunoglobulina G/biossíntese , Interleucina-2/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos B/virologia , Técnicas de Cultura de Células/métodos , Transformação Celular Viral/efeitos dos fármacos , Transformação Celular Viral/imunologia , Células Clonais , DNA/análise , Herpesvirus Humano 4/patogenicidade , Humanos , Hibridomas , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/biossínteseRESUMO
Objectives - The use of telencephalin as a possible marker for altered cortical function as demonstrated by functional MRI was investigated in a pilot study with 16 patients with localization-related epilepsy and secondarily generalized seizures. Materials and methods - Functional MRI of verbal working memory performance (Sternberg paradigm) and self-regulatory control processes (Stroop paradigm) was used to examine cortical activation in 16 patients with localization-related epilepsy and secondarily generalized seizures. Additionally, blood serum concentrations of soluble telencephalin (marker for neuronal damage) were determined. Results - In three patients (one temporal and two frontal focus), telencephalin was detected. All three patients had lower functional MRI activation in the frontotemporal region (P = 0.04), but not in other regions (P > 0.35) compared with patients without detectable telencephalin. Additionally, an association of levetiracetam and frontotemporal activation was observed. Conclusions - These preliminary data in a heterogeneous group suggest an association between decreased frontotemporal activation on fMRI and both detectable telencephalin serum levels and levetiracetam use. Future longitudinal studies with larger patient groups are required to confirm these observations. It is hypothesized that altered local function of the frontotemporal cortex in localization-related epilepsy might be better predicted by the biochemical marker telencephalin than epilepsy characteristics such as seizure focus.
Assuntos
Encéfalo/patologia , Moléculas de Adesão Celular/sangue , Epilepsias Parciais/sangue , Epilepsias Parciais/patologia , Imageamento por Ressonância Magnética , Proteínas do Tecido Nervoso/sangue , Adulto , Anticonvulsivantes/uso terapêutico , Biomarcadores/sangue , Encéfalo/efeitos dos fármacos , Epilepsias Parciais/tratamento farmacológico , Feminino , Humanos , Levetiracetam , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Projetos Piloto , Piracetam/análogos & derivados , Piracetam/uso terapêuticoRESUMO
Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction of skeletal muscles. Triple-seronegative MG (tSN-MG, without detectable AChR, MuSK and LRP4 antibodies), which accounts for ~10% of MG patients, presents a serious gap in MG diagnosis and complicates differential diagnosis of similar disorders. Several AChR antibody positive patients (AChR-MG) also have antibodies against titin, usually detected by ELISA. We have developed a very sensitive radioimmunoprecipitation assay (RIPA) for titin antibodies, by which many previously negative samples were found positive, including several from tSN-MG patients. The validity of the RIPA results was confirmed by western blots. Using this RIPA we screened 667 MG sera from 13 countries; as expected, AChR-MG patients had the highest frequency of titin antibodies (40.9%), while MuSK-MG and LRP4-MG patients were positive in 14.6% and 16.4% respectively. Most importantly, 13.4% (50/372) of the tSN-MG patients were also titin antibody positive. None of the 121 healthy controls or the 90 myopathy patients, and only 3.6% (7/193) of other neurological disease patients were positive. We thus propose that the present titin antibody RIPA is a useful tool for serological MG diagnosis of tSN patients.
Assuntos
Autoanticorpos/sangue , Conectina/imunologia , Miastenia Gravis/sangue , Miastenia Gravis/diagnóstico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Cooperação Internacional , Proteínas Relacionadas a Receptor de LDL/imunologia , Masculino , Miastenia Gravis/epidemiologia , Ensaio de Radioimunoprecipitação , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologiaRESUMO
Gangliosides inhibit basal, thyrotropin-induced and fluoride-induced adenylate cyclase activity of human thyroid membranes in physiological conditions. In contrast neutral glycolipids, phospholipids and neuraminic acid containing oligosaccharides show no effect. The efficacy of inhibition is more dependent upon the position of the sialic acid residues than upon their absolute number. In general gangliosides with disialyl groups are more inhibitory than those with single sialyl moieties. The inhibitory effects of the individual gangliosides on the two modes of stimulation are parallel. This parallelism suggests that the inhibitory effect is located at the postreceptor level and that the gangliosides interact directly with the adenylate cyclase system. A possible role of thyroid membrane gangliosides as suppressive cofactors of adenylate cyclase is discussed in relation to recent findings of stimulating anti-ganglioside antibodies in Graves' disease.
Assuntos
Inibidores de Adenilil Ciclases , Gangliosídeos/farmacologia , Glândula Tireoide/enzimologia , Humanos , Membranas/enzimologia , Fluoreto de Sódio/farmacologia , Glândula Tireoide/efeitos dos fármacos , Tireotropina/farmacologiaRESUMO
Seronegative myasthenia gravis (MG) presents a serious gap in MG diagnosis and understanding. We applied a cell based assay (CBA) for the detection of muscle specific kinase (MuSK) antibodies undetectable by radioimmunoassay. We tested 633 triple-seronegative MG patients' sera from 13 countries, detecting 13% as positive. MuSK antibodies were found, at significantly lower frequencies, in 1.9% of healthy controls and 5.1% of other neuroimmune disease patients, including multiple sclerosis and neuromyelitis optica. The clinical data of the newly diagnosed MuSK-MG patients are presented. 27% of ocular seronegative patients were MuSK antibody positive. Moreover, 23% had thymic hyperplasia suggesting that thymic abnormalities are more common than believed.
Assuntos
Autoanticorpos/sangue , Miastenia Gravis/sangue , Miastenia Gravis/diagnóstico , Receptores Proteína Tirosina Quinases/imunologia , Adulto , Idoso , Feminino , Citometria de Fluxo , Humanos , Cooperação Internacional , Proteínas Relacionadas a Receptor de LDL/imunologia , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/patologia , Neuromielite Óptica/diagnóstico , Radioimunoensaio , Receptores Colinérgicos/imunologia , Timo/patologia , Hiperplasia do Timo/diagnósticoRESUMO
Possible regulation of the synthesis of murine thyroglobulin (mTg) and a possible role for the major histocompatibility complex in this process were evaluated in 18 inbred strains of mice. A double antibody RIA was first developed to measure mTg in serum. The isolated mTG was labeled with 131I since 125I rapidly degraded the mTg molecule. The sensitivity of this mTg assay allowed detection of 15-20 ng/ml. Specificity was demonstrated by the minimal cross-reactivity with rat Tg (0.008%) and the total lack of cross-reactivity with human Tg. There was no correlation between H-2 haplotype and serum mTg levels. In five strains of mice with the H-2k haplotype, mTg levels varied from 30 +/- 2 to 48 +/- 11 ng/ml (mean +/- SD); however, only aged AKR/J mice (H-2k) exceeded this range (196 +/- 27 ng/ml). Strains with other haplotypes (a, b, d, g, q, v) demonstrated a similar range of mTg levels, but none had this age-related increase in mTg levels. The high levels of mTg were not caused by a decrease in the half-life of this protein and probably not caused by virus-induced alterations in the thyroid economy. Thyroids from the AKR/J mice, however, had larger follicles and flatter epithelia compared to thyroids from other strains. These studies suggest that AKR/J mice may represent a useful animal model for the study of goitrogenesis.
Assuntos
Tireoglobulina/sangue , Glândula Tireoide/crescimento & desenvolvimento , Envelhecimento , Animais , Imunodifusão , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos , Microscopia Eletrônica , Radioimunoensaio/métodos , Especificidade da Espécie , Tireoglobulina/análise , Glândula Tireoide/citologia , Glândula Tireoide/ultraestruturaRESUMO
A panel of monoclonal antibodies (mAbs) against human thyroglobulin (hTg) was obtained by somatic fusion of the nonsecreting myeloma cell line P3X66 Ag8/0 and spleen cells of Balb/c mice immunized with purified hTg. Antibody secreting clones were selected by solid phase enzyme immunoassay and analyzed for cross-reaction with Tg from several animal species. Twelve out of 15 mAbs cross-reacted with both rat and mouse Tg and 11 Mabs cross-reacted with bovine Tg. The cross-reaction with mouse Tg paralleled that of rat Tg, whereas discrete differences between the cross-reactivity patterns with bovine Tg were observed. Two clones secreted mAbs specific for hTg. We further characterized the mAbs and found that three mAbs recognized T4-containing determinants and one mAb reacted with both T4 and T3-containing determinants on the Tg molecule. The binding of the mAbs to hormonogenic determinants depended upon the thyroid hormone content of the molecule and the integrity of the three dimensional structure of Tg. One other mAb reacted with four peptides of CNBr-cleaved hTg, indicating the recognition of a repetitive determinant in the hTg molecule distinct from the hormonogenic regions.
Assuntos
Anticorpos Monoclonais , Tireoglobulina/imunologia , Animais , Linhagem Celular , Epitopos/análise , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Plasmocitoma , Tireoglobulina/análise , Glândula Tireoide/análise , Glândula Tireoide/citologiaRESUMO
Myasthenia gravis (MG) is an organ-specific autoimmune disease caused by an antibody-mediated assault on the muscle nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Binding of antibodies to the AChR leads to loss of functional AChRs and impairs the neuromuscular signal transmission, resulting in muscular weakness. Although a great deal of information on the immunopathological mechanisms involved in AChR destruction exists due to well-characterized animal models, it is not known which etiological factors determine the susceptibility for the disease. This review gives an overview of the literature on the AChR, MG and experimental models for this autoimmune disease.
Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Miastenia Gravis/imunologia , Receptores Colinérgicos/imunologia , Adulto , Animais , Especificidade de Anticorpos , Doenças Autoimunes/epidemiologia , Doenças Autoimunes/etiologia , Linfócitos B/imunologia , Modelos Animais de Doenças , Peixe Elétrico/imunologia , Epitopos/imunologia , Feminino , Humanos , Imunidade Celular , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/epidemiologia , Miastenia Gravis/etiologia , Prevalência , Coelhos , Ratos , Receptores Colinérgicos/química , Receptores Colinérgicos/fisiologia , Linfócitos T/imunologiaRESUMO
The effect of target organ manipulation by means of denervation and treatment with anabolic steroids on the severity of disease in EAMG was assessed in inbred rats. Unilateral limb denervation, a procedure known to increase the AChR content of muscle, 'protected' the denervated leg against antibody-mediated AChr loss in acute EAMG induced by passive transfer of mAb 35 directed against the main immunogenic region. Also in chronic EAMG, brought about by immunizing rats with AChR in complete Freund's adjuvant, the AChR loss of the denervated leg was about one fourth (13.5 vs. 53%) of the control leg. In both acute and chronic EAMG the amount of AChR complexed with antibody was lower in the denervated leg. This lower AChR occupancy with antibody in the denervated leg occurred also in conditions of marked antibody excess and was therefore due to enhanced AChR synthesis. Next the effect of treatment with a weakly virilizing anabolic steroid nandrolone in chronic and acute EAMG was examined in order to examine whether a hypothesized enhanced synthesis of AChR would protect animals from disease. In the absence of an immunosuppressive effect, in terms of concentration of antibodies to AChR, nandrolone treatment protected the rats from severe disease in the chronic EAMG model as shown by the fact that of the 9 rats 6 showed mild (1+) disease and 3 no disease at all; conversely 6 out of 9 control rats showed severe (3+) disease. Rats treated with nandrolone showed a 48 +/- 1.7% loss of AChR compared to a loss of 58 +/- 3.6% in the control rats, suggesting enhanced AChR synthesis. When nandrolone-pretreated rats were given acute EAMG by passive transfer of mAb 35 a paradoxical effect was seen. In contrast to the controls all of the rats pretreated with nandrolone showed severe signs of EAMG; this was associated with a higher loss of AChR and increased consumption of complement C4 as suggested by decreased concentrations of C4 in the serum. Results show increased AChR synthesis to protect against chronic EAMG both in terms of clinical disease (nandrolone) as well as AChR loss (nandrolone, denervation). In addition it was shown that nandrolone increases serum C4 consumption which in the complement-dependent acute EAMG model causes enhancement of the severity of clinical disease and increased AChR loss.
Assuntos
Miastenia Gravis/metabolismo , Receptores Colinérgicos/biossíntese , Doença Aguda , Animais , Anticorpos Monoclonais/administração & dosagem , Doença Crônica , Complemento C3/metabolismo , Complemento C4/metabolismo , Modelos Animais de Doenças , Feminino , Imunização Passiva , Denervação Muscular , Miastenia Gravis/etiologia , Miastenia Gravis/imunologia , Nandrolona/farmacologia , Ratos , Ratos Endogâmicos Lew , Receptores Colinérgicos/efeitos dos fármacosRESUMO
Myasthenia gravis (MG) is an organ-specific autoimmune disease in which autoantibodies against nicotinic acetylcholine receptors (AChR) at the postsynaptic membrane cause loss of functional AChR and disturbed neuromuscular transmission. The immunopathogenic mechanisms responsible for loss of functional AChR include antigenic modulation by anti-AChR antibodies, complement-mediated focal lysis of the postsynaptic membrane, and direct interference with binding of acetylcholine to the AChR or with ion channel function. The loss of AChR and subsequent defective neuromuscular transmission is accompanied by increased expression of the different AChR subunit genes, suggesting a role for the target organ itself in determining susceptibility and severity of disease. Experimental autoimmune myasthenia gravis (EAMG) is an animal model for the disease MG, and is very suitable to study the immunopathogenic mechanisms leading to AChR loss and the response of the AChR to this attack. In this article the current concepts of the structure and function of the AChR and the immunopathological mechanisms in MG and EAMG are reviewed.
Assuntos
Doenças Autoimunes/imunologia , Miastenia Gravis/imunologia , Animais , Autoanticorpos/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Miastenia Gravis/fisiopatologia , Coelhos , Receptores Colinérgicos/imunologiaRESUMO
OBJECTIVE: To investigate the role of blocking antibodies in neonatal Guillain-Barré syndrome (GBS) occurring 12 days postpartum in a child born to a mother with ongoing GBS. METHODS: We studied plasma filtrate, purified IgG, and monovalent Fab fragments from the affected mother and serum from the neonate as well as serum samples after recovery from disease 3 months later. Experiments were performed on the hemidiaphragms of adult mice and neonatal and juvenile rats. Quantal endplate currents were recorded with the perfused macro-patch clamp electrode. RESULTS: A dual effect was seen. Serum from mother and infant depressed quantal content by approximately 90% and reduced the amplitude of postsynaptic currents by 30 to 40%. The antibody nature of the blockade could be confirmed by showing that monovalent Fab fragments were similarly effective as purified immunoglobulin (Ig) G. No IgG antibodies to gangliosides, fetal or adult nicotinic acetylcholine receptor, or voltage-gated calcium channels could be detected, but IgM antibodies to the ganglioside GM1 were present. After recovery from GBS no blocking activity was seen in the sera of mother and infant. To elucidate why neonatal disease onset was delayed we examined the possible influence of early developmental changes in functional properties of the neuromuscular junction and applied the mother's active serum to postnatal rats. Although blockade was present in 23-day-old rats, it was absent in 5-day-old rats. CONCLUSION: Transplacentally transferred blocking antibodies may be specifically directed at epitopes of the mature but not the fetal neuromuscular junction.
Assuntos
Anticorpos Bloqueadores/imunologia , Síndrome de Guillain-Barré/imunologia , Síndrome de Guillain-Barré/fisiopatologia , Adulto , Animais , Feminino , Humanos , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Endogâmicos LewRESUMO
Aged rats resistant to acetylcholine receptor loss in passive transfer experimental autoimmune myasthenia gravis (EAMG) do not reveal infiltrating macrophages at the neuromuscular junction (NMJ) as observed in susceptible rats. It was investigated whether this age-related resistance is due to impaired macrophage function in these aged rats. Reconstitution of aged rats with bone marrow from young donors did not lead to macrophage infiltration, nor did it abolish resistance to EAMG. Subsequently, it was investigated whether macrophages are a primary cause of acetylcholine receptor (AChR) loss in EAMG or are attracted to the NMJ secondary to tissue damage. In lethally irradiated young susceptible rats infiltrating macrophages were absent from the NMJ. However, similar AChR losses were observed in irradiated and non-irradiated rats. These results suggest that macrophages do not contribute to acetylcholine receptor loss in the effector phase of passive transfer EAMG and that age related resistance to passive transfer EAMG is not primarily determined by the absence of infiltrating macrophages.
Assuntos
Envelhecimento/fisiologia , Macrófagos/fisiologia , Miastenia Gravis/imunologia , Miastenia Gravis/patologia , Junção Neuromuscular/patologia , Receptores Colinérgicos/metabolismo , Animais , Transplante de Medula Óssea , Movimento Celular , Suscetibilidade a Doenças , Feminino , Macrófagos/efeitos da radiação , Miastenia Gravis/metabolismo , Ratos , Ratos Endogâmicos BN , Irradiação Corporal TotalRESUMO
In experimental autoimmune myasthenia gravis anti-rat nicotinic acetylcholine receptor (AChR) antibody titers correlated significantly with the AChR-antibody complexes found in muscle. It was shown that at least a large part of the AChR-antibody complexes are formed in vitro, which can be prevented by washing of the muscle homogenate. Using a modified assay, no differences in AChR-antibody complexes could be detected between rats with and without symptoms of experimental autoimmune myasthenia gravis. Also no difference in AChR loss nor in inhibition of alpha-bungarotoxin binding to AChR was found between these groups of rats. However, a significant difference in the reduction of AChR function was found, using an assay measuring agonist-induced 22Na+ flux into the TE671 cell line.
Assuntos
Complexo Antígeno-Anticorpo/análise , Doenças Autoimunes/imunologia , Músculos/imunologia , Miastenia Gravis/imunologia , Receptores Colinérgicos/imunologia , Animais , Feminino , Ratos , Ratos Endogâmicos Lew , Receptores Colinérgicos/análiseRESUMO
Injection of anti-AChR antibodies in passive transfer experimental autoimmune myasthenia gravis (EAMG) results in increased degradation of acetylcholine receptor (AChR) and increased synthesis of AChR alpha-subunit mRNA. Passive transfer of anti-Main Immunogenic Region (MIR) mAb 35 in aged rats does not induce clinical signs of disease nor AChR loss. The expression of the AChR subunit genes was analyzed in susceptible and resistant rats. In aged EAMG resistant rats, no increase in the amount of AChR alpha-subunit mRNA was measured. In vivo AChR degradation experiments did not show any increase in AChR degradation rates in aged resistant rats, in contrast to young susceptible rats. Taken together, these data demonstrate that resistance of the AChR protein to antibody-mediated degradation is the primary mechanism that accounts for the resistance to passive transfer EAMG in aged rats.
Assuntos
Autoimunidade/imunologia , Miastenia Gravis/imunologia , Receptores Colinérgicos/genética , Receptores Colinérgicos/imunologia , Envelhecimento/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Biópsia , Modelos Animais de Doenças , Feminino , Expressão Gênica/imunologia , Denervação Muscular , Músculo Esquelético/imunologia , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Junção Neuromuscular/química , Junção Neuromuscular/imunologia , Junção Neuromuscular/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos BN , Receptores Colinérgicos/metabolismo , Nervo Isquiático/cirurgia , Sinapses/química , Sinapses/imunologia , Sinapses/metabolismo , Regulação para Cima/genéticaRESUMO
The immunoglobulin heavy chain (VH) gene family usage in experimental autoimmune myasthenia gravis (EAMG) model was investigated by RNA slot blot hybridization using VH gene family specific probes. Anti-acetylcholine receptor (AChR) monoclonal antibodies (mAbs) isolated from susceptible C57BL/6 and resistant BALB/c mice were found to be encoded by VH genes from at least six different families. The Vgam3.8 family was overrepresented in alpha-bungarotoxin blocking mAbs. Expression of cross-reactive idiotypes by anti-AChR mAbs was irrespective of the VH gene family usage. Strain dependent differences in susceptibility for EAMG were not reflected in an aberrant VH gene family usage of anti-AChR mAbs.
Assuntos
Doenças Autoimunes/imunologia , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Miastenia Gravis/imunologia , Receptores Colinérgicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Autoanticorpos/genética , Autoanticorpos/imunologia , Doenças Autoimunes/genética , Idiótipos de Imunoglobulinas/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miastenia Gravis/genética , Especificidade da Espécie , TorpedoRESUMO
Antibodies against the HuD antigen expressed in small-cell lung cancer (SCLC) cross-react with proteins expressed in neurons of the central and peripheral nervous system and are associated with paraneoplastic encephalomyelitis and sensory neuropathy (PEM/SN). We isolated anti-HuD Fab fragments from an antibody phage display library that was constructed from mRNA of a metastatic lymph node from a patient with SCLC and Pem/SN. Fab GLN495 recognized HuD and other related proteins (HuC and Hel-N1, or Hu antigens) in immunoblots of these recombinant proteins and in immunohistochemical and Western blot analysis of SCLC and neurons. Fab GLN495 inhibited up to 75% of the anti-Hu antibodies of the patient from which it was derived, suggesting that recognizes a dominant epitope in the polyclonal anti-Hu antibody response. Fab GLN495 also competed with anti-Hu sera from most but not all patients with PEM/SN, indicating that the same epitope is recognized by a large subgroup of patients. Human monoclonal anti-HuD antibodies may be useful in diagnosis of HuD expressing tumors and in clarifying the autoimmune etiology of PEM/SN. This study, the first to demonstrate that tumor specific recombinant antibodies can be isolated from metastatic lymph node tissue, shows that this approach may be generally applicable to isolate human antibodies against tumor specific antigens.
Assuntos
Carcinoma de Células Pequenas/imunologia , Encefalomielite/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Neoplasias Pulmonares/imunologia , Síndromes Paraneoplásicas/imunologia , Proteínas de Ligação a RNA/imunologia , Sequência de Aminoácidos , Anticorpos/imunologia , Bacteriófagos/imunologia , Carcinoma de Células Pequenas/complicações , Proteínas ELAV , Proteína Semelhante a ELAV 4 , Encefalomielite/complicações , Epitopos/imunologia , Deleção de Genes , Humanos , Neoplasias Pulmonares/complicações , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/imunologia , Neurônios/imunologia , Síndromes Paraneoplásicas/complicações , Proteínas de Ligação a RNA/genética , Proteínas RecombinantesRESUMO
Myasthenia gravis (MG) susceptibility is partially determined by allelic heterogeneity of immune-modulatory genes. IgG receptors (FcgammaR) link the humoral and cellular branches of the immune system, and regulate immune responses and inflammation. Three FcgammaR subclasses (FcgammaRIIa, FcgammaRIIIa, and FcgammaRIIIb) exhibit functional polymorphisms, which affect efficiency of FcgammaR-mediated functions. FcgammaRIIa genotypes, but not FcgammaRIIIa and FcgammaRIIIb genotypes, were differentially distributed among 107 MG patients as compared to 239 healthy controls (Pz.Lt;0.01), with a relative increase of the FcgammaRIIa-R/R131 genotype (Odds ratio 2.4, 95% confidence interval 1.4-3.9). These data suggest that the FcgammaRIIa-R/R131 genotype is a marker for susceptibility to MG.
Assuntos
Antígenos CD/genética , Predisposição Genética para Doença , Miastenia Gravis/genética , Miastenia Gravis/imunologia , Receptores de IgG/genética , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Alelos , Criança , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/epidemiologia , Países Baixos/epidemiologia , Polimorfismo Genético , Timoma/genéticaRESUMO
Myasthenia gravis (MG) is a prototype antibody-mediated autoimmune disease in which antibodies against nicotinic acetylcholine receptors (AChR) induce loss of functional receptors at the neuromuscular junction. Germinal centers present in MG hyperplastic thymus contain activated B-cells spontaneously producing anti-human AChR (huAChR) Ab in vitro. In order to access the anti-huAChR repertoire phage display Fab libraries of thymic lymphocytes were constructed from two MG patients. A total of four Fabs highly specific for huAChR were isolated that bind to determinants in or near the main immunogenic region (MIR). These anti-huAChR Fabs showed evidence of significant somatic mutations supporting the notion that the anti-huAChR Ab response in MG patients is driven by antigen. A total of two Fabs were able to inhibit up to 90% of donor serum anti-huAChR antibodies. Competition with serum anti-huAChR Ab was also observed in unrelated MG patients and indicate that anti-huAChR Fabs bind to epitopes on huAChR recognized by the majority of MG patients. In vitro antigenic modulation studies demonstrated that anti-huAChR Fabs were able to induce AChR loss when cross-linked by an anti-Fab antibody but not as monovalent Fab. Moreover, anti-huAChR Fabs were able to protect against AChR loss by antigenic modulation induced by MG serum antibodies suggesting a potential therapeutic role for these recombinant Fabs in patients with a myasthenic crisis.
Assuntos
Fragmentos Fab das Imunoglobulinas/imunologia , Miastenia Gravis/imunologia , Receptores Nicotínicos/imunologia , Especificidade de Anticorpos/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Bacteriófagos , Vetores Genéticos , Humanos , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Biblioteca de Peptídeos , Timo/citologia , Timo/imunologiaRESUMO
The in vivo effects of neonatal administration of varying doses of anti-idiotype antibodies on serum anti-acetylcholine receptor (AChR) antibody titers, idiotype expression, and disease severity was studied in experimental autoimmune myasthenia gravis. Polyclonal affinity purified anti-idiotype antibodies and monoclonal anti-idiotype antibodies directed at anti-AChR monoclonal antibody 65 were administered in dosages varying from the nanogram to the microgram range. Mab 65 is directed against the main immunogenic region of mammalian AChR. In 1 out of 4 experiments administration of a nanogram dosage of anti-idiotype antibodies led to an enhanced anti-AChR antibody response after immunization with AChR. But no enhancing effect on idiotype expression could be demonstrated during this experiment. Adoptive transfer of spleen cells from rats pretreated with a nanogram dosage of anti-idiotype antibodies resulted in an significantly increased antibody response against rat AChR after immunization. From these experiments we conclude that in vivo administration of polyclonal or monoclonal anti-idiotypes does not reproduceably modify the serum antibody level against the acetylcholine receptor, nor influences the idiotype profile of the immune response. Secondly, the idiotype mediated manipulation of the immune response against large antigens, like the acetylcholine receptor, is clearly more complicated than that against small haptens. Adoptive transfer models, might be helpful in analysing the possibilities of anti-idiotype treatment in myasthenia gravis in more detail.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Idiótipos de Imunoglobulinas/análise , Miastenia Gravis/imunologia , Receptores Colinérgicos/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Monoclonais , Modelos Animais de Doenças , Feminino , Peixes , Ratos , Ratos Endogâmicos Lew , TorpedoRESUMO
Experimental autoimmune myasthenia gravis (EAMG) can be induced in a large number of animal species by active immunization (AI) AChR, by passive transfer (PT) of anti-AChR antibodies, by autologous bone marrow transplantation and cyclosporin (BMT-Cy), or spontaneously. Depending on the model used, different immunological mechanisms are operational. In the AI model, the T cell is pivotal in directing the anti-AChR antibody production towards pathogenic, that is, cross-linking and complement-fixing antibodies. Injection of anti-AChR antibodies alone suffices to induce EAMG, excluding the role of specific cell-mediated immune responses in the effector phase of the disease. Aged animals are resistant to the induction of AI and PT EAMG. This resistance is localized at the postsynaptic membrane containing more AChR-anchoring proteins, including S-laminin and rapsyn in aged animals. In BMT-CyA EAMG, a dysregulation of the immune system in the absence of immunization is capable of inducing myasthenia. The role of these animal models in relation to pathogenesis and immunotherapy is discussed.