Experimental autoimmune myasthenia gravis: can pretreatment with 125I-labeled receptor prevent functional damage at the neuromuscular junction?

Rats were immunized with purified receptor from electric fish to induce experimental autoimmune myasthenia gravis (EAMG). It is implied by the clonal selection theory that antigens react only with receptors on specific immunocompetent cell subpopulations. In an attempt to damage these specific cells with the aid of highly radioactive antigen, one group of rats was pretreated with an additional injection of radiolabeled receptor of high specific activity 3 days before the basic immunization. The success of the immunization was monitored by measuring changes in the following three parameters: antibody titers against nicotinic acetylcholine receptor; number of alpha-bungarotoxin-binding sites at endplates; and number of acetylcholine-operated ionic endplate channels, using quantitative electrophysiologic methods. Conventionally immunized animals showed the classical signs of EAMG: elevated antibody titers against nicotinic acetylcholine receptor and a reduction of the number of alpha-bungarotoxin-binding sites, as well as reduction of the number of acetylcholine-operated ionic channels. The same symptoms were found in animals pretreated with unlabeled receptor and in animals pretreated with radioactive albumin. Animals pretreated with radioactively labeled receptor showed far less reduction of functional nicotinic acetylcholine receptor and only slightly raised antibody titers. This study suggests that preimmunization with radioactive antigen selectively eliminates immunocompetent cells, thus precluding the production of antibodies by a subsequent immunization procedure. The same protective effect cannot be obtained by either preimmunization with unlabeled antigen or by radioactively labeled unspecific antigen.     

Immunological studies of acetylcholine receptors

Immunochemical techniques for the study of acetylcholine receptors are described. Immunization of rabbits, rats, guinea pigs, and goats with acetylcholine receptor protein purified from Electrophorus electric organ tissue results in muscular weakness and death due to impaired neuromuscular transmission. Serum from immunized animals contains high concentrations of antibodies directed at receptors from the electric organ and low concentrations of antibodies directed at receptors from skeletal muscle. The detailed similarities between the disease of receptor-immunized animals, “experimental autoimmune myasthenia gravis” (EAMG), and myasthenia gravis are compared. Reactions of antisera from animal with EAMG with receptor from Electrophorus and Torpedo are studied. Antireceptor antibodies in these antisera are directed predominantly at determinants other than the acetylcholine-binding site.     

Is experimental autoimmune myasthenia gravis induced only by acetylcholine receptors?

EAMG has been induced in a wide variety of animals by using AcChR purified from electric organ and muscle sources. Electrophoresis of SDS polyacrylamide gels heavily loaded with purified AcChR often reveals the presence of minor contaminants. To test whether these contaminants or any other components present in Torpedo californica AcChR preparations could induce EAMG, solubilized Torpedo membrane fragments were depleted of AcChR by passage over an alpha-BuTx-conjugated resin and then injected into Lewis rats in an attempt to induce EAMG. The results demonstrated that some of the minor contaminants present in purified AcChR preparations were antigenic, but EAMG could not be induced with preparations enriched in these contaminants or containing other Torpedo non-AcChR components and lacking AcChR. The conclusion drawn from this study was that the acetylcholine receptor was the only component present in Triton X-100-solubilized Torpedo californica membrane fragments that could induce EAMG.     

Purification and characterization of a nicotinic acetylcholine receptor from chick brain

Immunohistochemical studies have previously shown that both the chick brain and chick ciliary ganglion neurons contain a component which shares antigenic determinants with the main immunogenic region of the nicotinic acetylcholine receptor from electric organ and skeletal muscle. Here we describe the purification and initial characterization of this putative neuronal acetylcholine receptor. The component was purified by monoclonal antibody affinity chromatography. The solubilized component sediments on sucrose gradients as a species slightly larger than Torpedo acetylcholine receptor monomers. It was affinity labeled with bromo[3H]acetylcholine. Labeling was prevented by carbachol, but not by alpha-bungarotoxin. Two subunits could be detected in the affinity-purified component, apparent molecular weights 48 000 and 59 000. The 48 000 molecular weight subunit was bound both by a monoclonal antibody directed against the main immunogenic region of electric organ and skeletal muscle acetylcholine receptor and by antisera raised against the alpha subunit of Torpedo receptor. Evidence suggests that there are two alpha subunits in the brain component. Antisera from rats immunized with the purified brain component exhibited little or no cross-reactivity with Torpedo electric organ or chick muscle acetylcholine receptor. One antiserum did, however, specifically bind to all four subunits of Torpedo receptor. Experiments to be described elsewhere (J. Stollberg et al., unpublished results) show that antisera to the purified brain component specifically inhibit the electrophysiological function of acetylcholine receptors in chick ciliary ganglion neurons without inhibiting the function of acetylcholine receptors in chick muscle cells. All of these properties suggest that this component is a neuronal nicotinic acetylcholine receptor with limited structural homology to muscle nicotinic acetylcholine receptor.     

Characterization of serum and mucosal antibody responses in white sturgeon (Acipenser transmontanus Richardson) following immunization with WSIV and a protein hapten antigen

Serum and cutaneous mucus antibodies were monitored in white sturgeon for 15 weeks following intraperitoneal immunization. Ten fish were immunized (50 microg) with white sturgeon iridovirus (WSIV) or white sturgeon gonad (WSGO) tissue culture cells emulsified with or without FCA. An additional group was immunized with FITC:KLH+FCA. Fish were booster immunized at 6 weeks. Fish immunized with FITC:KLH+FCA produced significant serum antibodies to FITC by 6 weeks and this response peaked at 12 weeks (average titer 31,000). Mucosal antibodies to FITC were first detected at 12 weeks and significantly elevated by 15 weeks (average titer 18). Anti-WSIV antibody titers were detected in the serum by 9 weeks in fish immunized with WSIV and WSIV+FCA, but only a small number responded to immunization. At 15 weeks, four fish immunized with WSIV produced serum antibodies (average titer 838) and one fish immunized with WSIV+FCA had a serum titer of 1600. Mucosal anti-WSIV antibody titers of 8 and 16 were observed in two fish from the WSIV group at 12 weeks while four different fish from this group responded at 15 weeks (average titer 4). Western Blot using a monoclonal antibody confirmed immunoglobulin in mucus, and specificity to WSIV was further demonstrated by immunocytochemistry using serum from fish immunized with WSIV. Specific antibody was not detected in mucus of fish immunized with WSIV+FCA, WSGO, or WSGO+FCA. Collectively, these experiments demonstrate that white sturgeon can generate a specific antibody response following immunization, and is the first report showing mucosal immunoglobulin is present in this species.     

Monoclonal anti-acetylcholine receptor antibodies with differing capacities to induce experimental autoimmune myasthenia gravis

To study the characteristics of the individual autoantibodies that are important in the development of an autoimmune disease, we produced 26 anti-acetylcholine receptor (anti-AChR) monoclonal antibodies (mcAb) and studied the experimental autoimmune myasthenia gravis (EAMG) induced by a number of them. The mcAb reactive with mammalian acetylcholine receptor (M-AChR) exhibited a wide range of dissociation rates from in situ M-AChR of motor endplates. All anti-M-AChR mcAb were capable of producing at least some degree of histopathologic change at the endplate indicative of EAMG, but their potencies varied markedly. One mcAb induced, even at large doses, only minor macrophage invasion without clinical or electromyographic effect. Others induced severe EAMG, and even death, at 1/200th the dose. Low potency was associated with high rate of mcAb dissociation from antigen. High potency was associated with intermediate avidity, not high avidity. These observations suggest that in EAMG, and perhaps in myasthenia gravis, the characteristics of the individual antibodies making up the autoimmune response can determine the severity of the autoimmune disease.     

Specific killing of lymphocytes that cause experimental autoimmune myasthenia gravis by ricin toxin-acetylcholine receptor conjugates

Experimental autoimmune myasthenia gravis (EAMG) is an autoimmune disease in which antibodies to acetylcholine receptor (AChR) cause loss of AChR from muscle, thereby impairing neuromuscular transmission. Here we report the use of a hybrid molecule that contains ricin toxin, irreversibly coupled to AChR to specifically suppress the immune response to AChR in vitro. Lymph node cell cultures from rats with EAMG pretreated with ricin toxin-AChR conjugates exhibited suppressed T helper cell proliferation and B cell antibody synthesis in response to the subsequent addition of AChR. Nonspecific toxicity of the conjugates was measured by suppression of the T cell proliferative response to the mitogen concanavalin A and the antigen keyhole limpet hemocyanin (KLH), and B cell antibody production to KLH. We have evaluated different pretreatment conditions and ricin toxin covalently coupled to AChR in different molar ratios to optimize specific immunosuppression. By varying the number of ricin molecules covalently bound to AChR in the immunotoxin, we were able to minimize the nonspecific toxicity while still maintaining specific killing of AChR-reactive lymphocytes. Furthermore, B cells were more susceptible to specific killing than were the T cells. The specific immunosuppression was potentiated by performing the pretreatment with immunotoxin in the presence of chloroquine. Chloroquine raises lysosomal pH and probably delays the degradation of immunotoxin in the cell. It should be noted that ricin toxin was covalently coupled to AChR by using a novel, non-reducible reaction. These in vitro results suggest that it may be feasible to use immunotoxin molecules to specifically suppress this autoimmune response in vivo.     

Binding of antibodies to acetylcholine receptors in Electrophorus and Torpedo electroplax membranes

Antisera against purified acetylcholine receptors from the electric tissues of Torpedo californica and of Electrophorus electricus were raised in rabbits. The antisera contain antibodies which bind to both autologous and heterologous receptors in solution as shown by an immunoprecipitation assay. Antibodies in both types of antisera bind specifically to the postjunctional membrane on the innervated surface of the intact electroplax from Electrophorus electric tissue as demonstrated by an indirect immunohistochemical procedure using horseradish peroxidase conjugated to anti-rabbit IgG. Only anti-Electrophorus receptor antisera, however, cause inhibition of the receptor-mediated depolarization of the intact Electrophorus electroplax. The lack of inhibition by anti-Torpedo receptor antibodies, which do bind, suggests that the receptor does not undergo extensive movement during activity. The binding of anti-Torpedo antibodies to receptor-rich vesicles prepared by subcellular fractionation of Torpedo electric tissue was demonstrated by both direct and indirect immunohistochemical methods using ferritin conjugates. These vesicles can be conveniently collected and prepared for electron microscopy on Millipore filters, a procedure requiring only 25 micrograms of membrane protein per filter. In addition, it was possible to visualize the binding of anti-Torpedo receptor antibodies directly, without ferritin. These anti-Torpedo receptor antibodies, however, do not inhibit the binding of acetylcholine or of alpha-neurotoxin to receptor in Torpedo microsacs but do inhibit binding of alpha-neurotoxin to Torpedo receptor in Triton X-100 solution. It is likely that the principal antigenic determinants on receptor are at sites other than the acetylcholine-binding sites and that inhibition of receptor function, when it occurs, may be due to a stabilization by antibody binding of an inactive conformational state.     

Immune response gene control of lymphocyte proliferation induced by acetylcholine receptor-specific helper factor derived from lymphocytes of myasthenic mice

The role of lymphokines secreted by acetylcholine receptor (AChR)-reactive lymphocytes in the regulation of an autoimmune response to AChR has not been studied in the human or murine model of myasthenia gravis. We investigated whether AChR-immune lymphocytes derived from mice with experimental autoimmune myasthenia gravis (EAMG) can produce an AChR-specific, genetically controlled soluble factor with biologic activity. AChR-reactive lymphocytes of mice with EAMG secreted an AChR-specific helper factor in vitro, which induced proliferation of AChR-immune but not Mycobacterium tuberculosis-immune lymphocytes. Recombinant, I-A mutant, and monoclonal anti-I-A antibody analyses suggest that AChR-specific helper factor-induced lymphocyte proliferation is controlled by an immune response gene at the I-A subregion of the murine major histocompatibility complex, and is mediated by the I-A molecule.     

Anti-CTLA-4 antibody treatment triggers determinant spreading and enhances murine myasthenia gravis

CTLA-4 appears to be a negative regulator of T cell activation and is implicated in T cell-mediated autoimmune diseases. Experimental autoimmune myasthenia gravis (EAMG), induced by immunization of C57BL/6 mice with acetylcholine receptor (AChR) in adjuvant, is an autoantibody-mediated disease model for human myasthenia gravis (MG). The production of anti-AChR Abs in MG and EAMG is T cell dependent. In the present study, we demonstrate that anti-CTLA-4 Ab treatment enhances T cell responses to AChR, increases anti-AChR Ab production, and provokes a rapid onset and severe EAMG. To address possible mechanisms underlying the enhanced autoreactive T cell responses after anti-CTLA-4 Ab treatment, mice were immunized with the immunodominant peptide alpha(146-162) representing an extracellular sequence of the ACHR: Anti-CTLA-4 Ab, but not control Ab, treatment subsequent to peptide immunization results in clinical EAMG with diversification of the autoantibody repertoire as well as enhanced T cell proliferation against not only the immunizing alpha(146-162) peptide, but also against other subdominant epitopes. Thus, treatment with anti-CTLA-4 Ab appears to induce determinant spreading, diversify the autoantibody repertoire, and enhance B cell-mediated autoimmune disease in this murine model of MG.     

Effect of conjugation methodology on the immunogenicity and protective efficacy of meningococcal group C polysaccharide-P64k protein conjugates

Neisseria meningitidis serogroup C polysaccharide (CCPS) was conjugated to the carrier protein P64k using two different conjugation procedures, condensation mediated by carbodiimide with adipic acid dihydrazide as spacer and the reductive amination method. BALB/c mice were immunized with the resultant polysaccharide-protein conjugates and the immune response was evaluated. All conjugates assayed generated at least 10-fold higher antibody titers than the free polysaccharide. The reductive amination method rendered the best conjugate (CCPS-P64kR) that was able to elicit antibody titers statistically higher than the titer elicited by the plain CCPS (P<0.001). The sera of the group immunized with CCPS-P64kR showed a three-fold higher bactericidal response than the sera of the group immunized with the plain CCPS and they were able to protect against challenge with meningococci in the infant rat protection model. In addition, three different conjugates were obtained from polysaccharides with molecular relative sizes of 2000-4000 Da, 4000-10,000 Da or 10,000-50,000 Da, but no differences were detected in the immune response obtained against the three conjugates. Our experiments demonstrate that it is possible to generate a protective, T-cell-dependent response against CCPS using the P64k protein as carrier.     

Cellular immune response to acetylcholine receptors in murine experimental autoimmune myasthenia gravis: inhibition with monoclonal anti-I-A antibodies

Gene(s) at the I-A subregion of the murine major histocompatibility complex influence susceptibility to experimental autoimmune myasthenia gravis. C57Bl/6 mice immunized with acetylcholine receptors (AChR) in complete Freund's adjuvant demonstrated cellular and humoral immune responses to AChR. They developed muscle weakness characteristic of myasthenia gravis and demonstrated a reduction in the muscle AChR content. The kinetics of AChR-specific lymphocyte proliferation generally correlate with anti-AChR antibody response. AChR-specific lymphocyte proliferation was also observed in C57Bl/6 splenocytes after secondary immunization with AChR. The in vitro cellular reactivity to AChR in experimental autoimmune myasthenia gravis (EAMG) mice (C57Bl/6) was suppressed by monoclonal anti-I-Ab antibodies directed against private (Ia20) or public (Ia8) specificities, suggesting a critical role for these Ia determinants in the cellular immune response to AChR in murine EAMG.     

Analysis of antibody response to synthetic peptides derived from the fusion protein of measles virus

A computer program combining of hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus (MV) fusion protein was used to select four possible epitopes. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using the homobifunctional cross-linker bis-sulfosuccinimidyl suberate. Immune stimulating complexes were prepared with the peptides conjugated to the purified protein derivative carrier using a dialysis method. All antisera raised in rabbits against the peptide conjugates had a high titer to the homologous peptides and reacted well with denatured MV as tested by plate ELISA. None of the sera had neutralizing antibody. Human sera positive for MV antibody reacted strongly with the synthesized peptides indicating that the selected locations function as partial antigenic sites. Antisera against peptide conjugates reacted weakly in immunofluorescence and none of these antisera reacted with purified MV proteins in Western blot. The results obtained in this study indicated that although the computer program could not predict epitopes important for the neutralization of the MV, the predicted epitopes are useful for detecting antibodies against MV.     

Physiochemical and immunological properties of acetylcholine receptors from human muscle

Summary The acetylcholine receptor protein from human muscle was extracted with the non-ionic detergent Triton X-100 and purified by affinity chromatography on -Naja toxin sepharose 4B. Further purification on Dicap-MP sepharose 4B, a choline analog compound, led to ACHR preparations with specific activities of 2–7 nmol/mg protein. The isolated receptor, labeled with ¹²⁵I--bungarotoxin was characterized by different methods and compared to ACHRs from Torpedo californica electroplax and rat-denervated skeletal muscle. Gel filtration on Ultrogel AcA 34 resulted in a stokes radius of 70 Å for the receptor monomer and 99 Å for the dimeric form. Sucrose density gradient centrifugation showed sedimentation coefficients of 9.1 S and 13.5 S. From these data the molecular weight of the ACHR monomer was estimated as 254 000 D and 540 000 D for the receptor dimer. The isoelectric point of the ¹²⁵I--bgt-ACHR complex was determined by thin-layer isoelectric focussing to be pH 5.Purified ACHRs were used for immunization of rats and mice which developed an EAMG as verified by clinical observation and electrophysical measurements. Sera from the immunized animals as well as from myasthenia gravis patients were subsequently used to compare the cross-reactivity of ACHR preparations from different sources. While antibodies of rats immunized with Torpedo ACHRs cross-reacted with ACHR preparations from rat and human skeletal muscle, antibodies from mice immunized with rat ACHR only reacted with preparations from rats and mice. Antibodies from mice immunized with ACHR of human origin exhibited a broad cross-reactivity, as did antibodies from MG patients.Abbreviations AB antibody - ACHR nicotinic acetylcholine receptor - BSA bovine serum albumin - Dicap-MP methyl-[N-(6-aminocaproyl-6aminocaproyl)-3-amino]pyridinbromide - EAMG experimental autoimmune myasthenia gravis - EDTA ethylenediaminetetraaceticacid - MG myasthenia gravis - PMSF phenylmethylsulfonylfluoride Recipient of a postdoctoral grant from Deutsche Forschungsgemeinschaft; present address: Neurologische Klinik, Medizinische Einrichtungen der Universität Düsseldorf.     

Cathepsin S is required for murine autoimmune myasthenia gravis pathogenesis

Because presentation of acetylcholine receptor (AChR) peptides to T cells is critical to the development of myasthenia gravis, we examined the role of cathepsin S (Cat S) in experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. Compared with wild type, Cat S null mice were markedly resistant to the development of EAMG, and showed reduced T and B cell responses to AChR. Cat S null mice immunized with immunodominant AChR peptides showed weak responses, indicating failed peptide presentation accounted for autoimmune resistance. A Cat S inhibitor suppressed in vitro IFN-gamma production by lymph node cells from AChR-immunized, DR3-bearing transgenic mice. Because Cat S null mice are not severely immunocompromised, Cat S inhibitors could be tested for their therapeutic potential in EAMG.     

抗谷胱甘肽S转移酶多克隆抗体的制备及特异性鉴定

目的:制备谷胱甘肽S转移酶(GST)的兔多抗,并鉴定该抗体的特异性。方法:用纯化的GST标签蛋白(纯度>98%)免疫新西兰大白兔,获得GST的兔抗血清,并经HiTrap rProtein A柱纯化获得高效价高特异性的抗体;用间接ELISA法检测抗体效价,Western印迹检测抗体的特异性,并与商业化抗体进行对比。结果:通过免疫法得到了GST的兔多克隆抗体血清,抗体效价达1∶1×106,经rProtein A柱纯化后获得了高效价高特异性的抗体,其高效高特异性已达商业化抗体水平。结论:获得了GST的高效价高特异性的兔多克隆抗体。     

Hapten concentrations in the circulation of animals actively immunized with hapten-protein conjugates.

Animals immunized with hapten-protein conjugates subsequently circulate high concentrations of hapten bound by antibody. The levels of hapten detected are capable of significantly reducing antibody titer in the sera immunized animals. In the case of steroid-protein conjugates, the main source of increased plasma steroid concentration is the immunizing conjugate, although a contribution from increased host secretion may also occur. The results for rabbits immunized with digoxin-BSA indicate that the appearance of circulating digoxin followed the appearance of circulating antibody to digoxin. Appearance of digoxin in circulation appears to coincide with the operation of the immune response and may be related to macrophage activity. Similar conclusions are drawn from results obtained for circulating morphine in the serum of a sheep immunized with morphine-BSA. Injected hapten-protein antigens are probably processed by macrophage to produce low molecular weight haptenic fragments which are maintained in circulation for prolonged periods in the form of antibody-hapten complexes.     

Preparation of immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horseradish peroxidase with human antibodies to HIV-1]

By using three different linkage methods with carbodiimide, glutaraldehyde and periodite, immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horse radish peroxidase with human antibodies to HIV-1 were prepared. The human antibodies were purified by the affinity procedure on Protein-A-Sepharose 6B. The conjugates were tested in a solid phase immunoenzymatic system for the HIV-1 antigen. It was shown that the conjugates prepared by the carbodiimide linkage method had the highest titer, the beta-lactamase conjugate being superior by its titer to the respective peroxidase conjugate. In the lyophilized state the conjugates prepared with the carbodiimide linkage method were stable.     

B7-1 costimulatory molecule is critical for the development of experimental autoimmune myasthenia gravis

Following immunization with acetylcholine receptor (AChR), MHC class II-restricted, AChR-specific CD4 cell activation is critical for the development of experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 mice. To study the contributions of B7-1 and B7-2 costimulatory molecules in EAMG, B7-1, B7-2, and B7-1/B7-2 gene knockout (KO) mice were immunized with Torpedo AChR in CFA. Compared with wild-type C57BL6 mice, B7-1 and B7-1/2 KO mice were resistant to EAMG development. B7-1 KO mice had reduced anti-AChR Ab compared with C57BL/6 mice. However, neither B7-1 nor B7-2 gene disruption impaired AChR-induced or dominant alpha(146-162) peptide-induced in vitro lymphoproliferative responses. Blocking of the B7-1 or B7-2 molecule by specific mAbs in vivo led to a reduction in the AChR-specific lymphocyte response, and the reduction was more pronounced in mice treated with anti-B7-2 Ab. The findings implicate B7-1 molecules as having a critical role in the induction of EAMG, and the resistance of B7-1 KO mice is associated with suppressed humoral, rather than suppressed AChR-specific, T cell responses. The data also point to B7-2 molecules as being the dominant costimulatory molecules required for AChR-induced lymphocyte proliferation.