Experimental autoimmune myasthenia: A model of myasthenia gravis in rats and guinea pigs.

Immunization of animals with acetylcholine receptor (AChR) protein from the electric organs of Electrophorus electricus and Torpedo californica induces an autoimmune response to the AChR of mammalian skeletal muscle. Rats and guinea pigs develop experimental autoimmune myasthenia gravis (EAMG) after a single inoculation with small quantities of AChR and adjuvant. The indicence and severity of disease appears to depend on the dose of AChR and stability of the emulsion. EAMG is strikingly similar to myasthenia gravis (MG) of man in its clinical picture and its electrophysiological abnormalities. The presence of antibodies to syngeneic rat muscle AChR in the serum of rats with EAMG documents the existence of autoimmunity in the experimental disease. A common immunopathogenesis is suggested for both EAMG and mg.

An antiserum specific for cholinergic synaptic vesicles from electric organ.

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle-specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.

Antibodies to synaptic vesicles purified from Narcine electric organ bind a subclass of mammalian nerve terminals.

Antibodies were raised in rabbits to synaptic vesicles purified to homogeneity from the electric organ of Narcine brasiliensis, a marine electric ray. These antibodies were shown by indirect immunofluorescence techniques to bind a wide variety of nerve terminals in the mammalian nervous system, both peripheral and central. The shared antigenic determinants are found in cholinergic terminals, including the neuromuscular junction, sympathetic ganglionic and parasympathetic postganglionic terminals, and in those synaptic areas of the hippocampus and cerebellum that stain with acetylcholinesterase. They are also found in some noncholinergic regions, including adrenergic sympathetic postganglionic terminals, the peptidergic terminals in the posterior pituitary, and adrenal chromaffin cells. They are, however, not found in many noncholinergic synapse-rich regions. Such regions include the molecular layer of the cerebellum and those laminae of the dentate gyrus that receive hippocampal associational and commissural input. We conclude that one or more of the relatively small number of antigenic determinants in pure electric fish synaptic vesicles have been conserved during evolution, and are found in some but not all nerve terminals of the mammalian nervous system. The pattern of antibody binding in the central nervous system suggests unexpected biochemical similarities between nerve terminals heretofore regarded as unrelated.

Monoclonal antibody Tor 23 binds a subset of neural cells in the human cortex and displays an altered binding distribution in Alzheimer's disease.

Recent evidence suggests that alterations in molecules of the external neuronal surface may be pivotal factors in Alzheimer's disease (AD) either as primary or secondary lesions. We are studying neuronal surface components with a library of monoclonal antibodies (MAbs) made to highly purified, exclusively cholinergic nerve terminals of the Torpedo ray. The most extensively characterized of the Tor MAbs. Tor 23, binds the external membrane of some human neuronal cells in culture. Our present findings demonstrate that Tor 23, in situ, binds the apparent limiting membrane of rare neurons of the human isocortex. Tor 23 binds, in addition, internally within a subpopulation of subcortical white matter astrocytes, as identified by colocalization with glial fibrillary acidic protein. Neuronal surface binding of Tor 23 parallels our findings in other species: astrocyte staining was not observed in other species and may be unique to human. Immunoblot analysis of white matter reveals one polypeptide band with a relative mobility of 115,000 +/- 15,000 daltons. In the mid-frontal cortex from cases of AD. Tor 23 immunopositive neurons are greatly reduced in number and immunopositive astrocytes are completely absent. The reduction of the neuronal surface epitope defined by Tor 23 supports the recent hypothesis that surface molecules are altered in AD. The absence of Tor 23 positive astrocytes opens an area for specific investigation: namely, the role subcortical astrocytes may play in the pathogenesis of AD.

Monoclonal antibodies against the main immunogenic region of the acetylcholine receptor. Mapping on the intact molecule.

About two-thirds of the antibodies to the nicotinic acetylcholine receptor (AChR) in myasthenic patients, and in rats immunized with intact AChR, bind to the main immunogenic region (MIR) on the alpha-subunit. We tested all available anti-MIR monoclonal antibodies (mAbs) by competition experiments for binding on the intact AChR from Torpedo electric organ and human muscle. Practically complete competition between all possible paired combinations of anti-MIR mAbs was found. As a consequence, the MIR must be a very concrete and small region. Furthermore, the location of the MIR relative to some other less immunogenic regions was also determined.

Passive transfer of experimental autoimmune myasthenia gravis by monoclonal antibodies to the main immunogenic region of the acetylcholine receptor.

Experimental autoimmune myasthenia gravis (EAMG) was passively transferred to rats by injecting monoclonal antibodies (mAbs) directed at the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR). The MIR is located on the extracellular part of the AChR alpha-subunit. All four mAbs directed at the MIR which were tested were very efficient in inducing EAMG: within 2 days the rats became moribund or very weak and their muscle AChR content decreased to about 50% of normal. These mAbs are of two different IgG subclasses (IgG1 and IgG2a) and derived from rats immunized with AChR from either fish electric organs or mammalian muscles. One mAb directed at the extracellular side of the beta-subunit did not cause AChR loss or induce symptoms of EAMG. mAbs to the cytoplasmic side were, as expected, ineffective.

Selective interactions of electrolectins from eel electric organ and mouse thymus with mouse immature thymocytes.

The pure electrolectin, a beta-D-galactoside binding lectin from the electric organ of the electric eel Electrophorus electricus, was found to agglutinate selectively a subpopulation of mouse thymocytes. This cell population could be separated from non-agglutinated cells by 1 g sedimentation over fetal calf serum. The agglutinated cells could be identified as immature thymocytes on the basis of the density of theta-antigen they bear on their surfaces, their mitotic activity and the absence of response to the mitogenic action of phytohemagglutinin. The immature mouse thymocytes were found to bind the endogenous mouse thymic lectin (MTL) a protein that displays the same saccharide specificity as the eel electrolectin and with which it cross-reacts immunologically. MTL is secreted by mouse thymic reticulocytes in tissue culture and its specific activity is markedly increased after depleting the thymus of its thymocytes. This finding is an indication of the possible localization of MTL in the thymic epithelium. These results are discussed in the light of our recent findings that the eel electrolectin has prophylactic and therapeutic actions on the experimental auto-immune myasthenia gravis in rabbits.

Interaction of monoclonal antibodies to electroplaque acetylcholine receptors with the alpha-bungarotoxin binding site of goldfish brain.

Polyclonal and monoclonal antibodies raised against acetylcholine receptors from Torpedo californica and Electrophorus electricus electroplaque were tested for interaction with the [125I]alpha-bungarotoxin binding protein of goldfish brain. A subset of monoclonal antibodies which recognize the main immunogenic region of the alpha subunit of the Electrophorus acetylcholine receptor interacted at high affinity with the [125I]alpha-bungarotoxin binding protein. Using immunofluorescence, these antibodies were shown to label the same layers of the optic tectum as [125I]alpha-bungarotoxin.

Monoclonal antibodies associated with sodium channel block nerve impulse and stain nodes of Ranvier.

Monoclonal antibodies were generated against native eel electroplax sodium channels in their natural membrane. These antibodies block nerve conduction in rat central (optic) and peripheral (sciatic) nerve. The antibody binding to eel electroplax membrane fragments and to rat brain synaptosomes can be modulated by neurotoxins. Thus it implies that active sites of the sodium channels are immunogenic in their natural membrane. Unlike the antibodies described in the past, our antibodies recognize antigenic determinants which are associated with the physiological activity of the channel and have been conserved through evolution.

A novel cholinergic-specific antigen (Chol-2) in mammalian brain.

Three new antisera have been raised in sheep against cholinergic electromotor presynaptic plasma membranes prepared from the electric organs of the electric ray, Torpedo marmorata. They all recognized one or more cholinergic-specific antigens in the mammalian nervous system by the following criteria: they sensitized the cholinergic subpopulation of rat-brain synaptosomes--and only this subpopulation--to lysis by the complement system and, in an immunocytochemical study, selectively stained choline acetyltransferase-positive cholinergic neurons in the rat spinal cord. However, two of the three antisera failed to recognize Chol-1 alpha and -beta, two closely related minor gangliosides already identified as the cholinergic-specific antigens recognized by previous anti-Torpedo presynaptic plasma membrane antisera or indeed any other ganglioside and the third recognized only Chol-1 alpha. A further investigation of the antigen(s) recognized by the most antigenic of the new antisera indicated that it is proteinaceous in nature, but has epitopes in common with electric organ gangliosides.

Suppression of experimental autoimmune myasthenia gravis with new immunosuppressants: 15-deoxyspergualin and actinobolin.

In the search for a new drug to treat myasthenia gravis, we studied the efficacy of new immunosuppressants on experimental autoimmune myasthenia gravis (EAMG). 15-Deoxyspergualin (15-DSP), bactobolin and actinobolin were administered to some groups at the time of immunization and to other groups 10 days after. The most effective results were achieved with doses of 2.5 mg/kg daily of 15-DSP and 30 mg/kg daily of actinobolin administered from day 1. In both groups, the body weights of the rats increased as normally as those of controls and signs of myasthenia were mild. Immunoelectron microscopic examination of the neuromuscular junctions in rats treated with 2.5 mg/kg of 15-DSP appeared normal, even in the chronic phase (induced by a booster at week 4). Levels of anti-acetylcholine receptor antibodies were almost completely suppressed. Although the effects of these drugs were more remarkable when administered from day 1 than from day 10, the results suggest that they may prove useful in treating myasthenic patients.

Experimental autoimmune myasthenia gravis in the mouse.

Myasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or the electric ray. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography. In addition, EAMG is frequently characterized by the presence of serum antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody-mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content. Another hallmark of the disease is complement and IgG deposits located at the neuromuscular junction, which can be visualized by immunofluorescence techniques.

Interactions of antinicotinic acetylcholine receptor antibodies with rat brain and muscle antigenic determinants.

Studies were performed to determine whether antibodies prepared against nicotinic acetylcholine receptors (nAcChoR) from electric tissue are reactive toward nAcChoR-like antigenic determinants in rat brain. Reference experiments involved the use of Torpedo electroplax and rat innervated muscle as tissue controls and an anti-alpha-bungarotoxin antiserum as a probe for curaremimetic neurotoxin binding sites. As evinced by their ability to inhibit immunoprecipitation of Torpedo nAcChoR, brain or muscle membranes specifically interact with polyclonal antisera raised against Electrophorus electroplax nAcChoR. When the extent of polyclonal anti-nAcChoR antibody binding to muscle membranes is measured by protein A binding protocols, receptor-like antigenic determinants and toxin binding sites are found to be present in approximately equal quantities. In contrast, nAcChoR-like antigenic determinants on rat brain membranes are present at concentrations in excess of those of toxin binding sites. The results are consistent with the earlier observation that some antibodies prepared against nAcChoR from peripheral tissues recognize rat brain high-affinity alpha-bungarotoxin binding sites. The results also suggest the existence of nAcChoR-like entities in brain that do not bind toxin with a high affinity.

Presynaptic plasma membranes and synaptic vesicles of cholinergic nerve endings demonstrated by means of specific antisera.

Antisera were raised to cholinergic presynaptic plasma membranes and synaptic vesicles isolated from the electric organ of Torpedo marmorata and tested by immunochemical and immunohistochemical methods. The antisera responded to many antigens not specific to nerve endings, but it was possible to eliminate these antibodies by means of simple absorption procedures with fractions containing the unwanted antigens. After absorption, staining of thin sections of electric organ by immunofluorescence was limited to the region of nerve endings in the tissue. The remaining antibodies responded in the case of the plasma membrane antisera predominantly to a 33,000 molecular-weight polypeptide and a chloroform/methanol-soluble antigen. In cross reactivity studies it was found that this antiserum not only stains cholinergic nerve endings in Torpedo but also those in mammalian tissue. The antigen responsible for the cross reactivity is restricted to the chloroform/methanol-soluble material. The vesicle antiserum labels cholinergic nerve endings in mammalian tissue as well; the relevant antigen in this case is different from the one described above and is likely to be a glycosaminoglycan. The antisera provide valuable markers for cholinergic nerve terminals. In addition, the vesicle antiserum may now be used to study axonal transport and the life cycle of this organelle in the cholinergic neurone.