Production of antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor coupled to protein carriers in different orientations.

The antibodies to nicotinic acetylcholine receptor alpha(181-192) synthetic peptides were elicited in rabbits and mice using the peptides conjugated to protein carriers in different orientations, either through C-terminal Cys (S-conjugates), or through amino groups (N-conjugates). S-conjugated peptides were less potent in eliciting peptide-specific antibodies compared to N-conjugates and this type of conjugation resulted in antibodies to the coupling reagent. However, the epitopes present in either S- or N-conjugated peptides appeared to be similar, indicating that amino acid residues, which form the epitope, were located in the middle part of the peptide and did not include both N- and C-terminal residues. Peptide conjugation to a protein carrier did not play a role in stabilizing the peptide conformation, but was necessary to concentrate the peptide epitopes on the carrier surface enabling bivalent antibody binding.

Structure of epitopes recognized by the antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptors: extrapolation to the structure of acetylcholine-binding domain.

Using the alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor (nAChR) and Ala-substituted peptide analogues, amino acid residues critical for specific monoclonal antibody (mAb) binding were identified. By means of 2D nuclear magnetic resonance (2D-NMR) analysis followed by molecular modeling, it was found that mAb binding resulted in stabilization of the free alpha3(181-192) peptide flexible conformation yielding an extended structure with residues 6-11 of the peptide being in direct contact with the Ab. Since the Ab binds the native AChR as well, it is suggested that the corresponding fragment of AChR alpha3 subunit is exposed to solution and also appears in extended conformation.

Epidemiology of seropositive myasthenia gravis in Greece.

OBJECTIVES: To study the epidemiological characteristics of myasthenia gravis in Greece. METHODS: A population based study was carried out of seropositive myasthenia gravis in Greece for the period from 1 January 1983 to 30 June 1997; 843 patients were studied. RESULTS: The average annual incidence for the period 1992-7, for which the database is complete, was 7.40/million population/year (women 7.14; men 7.66). On 1 July 1997, there were 740 prevalent cases. The point prevalence rate was 70.63/million (women 81.58; men 59.39). The average overall annual mortality rate in the patients was 0.67/million population (women 0.53; men 0.82), and the mortality rate attributed to myasthenia gravis was 0.43/million population (women 0.41; men 0.45). The average age at onset was 46.50 years (women 40.16; men 54.46), and the mean age of the prevalent patients was 52.58 (women 47.65; men 59.48). The women:men incidence ratio was 1:1.04, and the prevalence ratio was 1.41:1. It is predicted that the prevalence and women: men prevalence ratio would increase if the patient list included all patients with a date of onset before 1983. CONCLUSIONS: The largest epidemiological study ever performed on myasthenia gravis is presented. The most important epidemiological indexes are provided.

Anti-neuronal nicotinic receptor antibodies in MG patients with thymoma.

We tested 24 MG patients with thymoma (11 invasive) for the presence of anti-neuronal nicotinic receptor subtype antibodies and found that none had antibodies against the alpha 7-containing subtype, and only one (with invasive thymoma) had antibodies directed against the alpha 3-containing subtype. In this patient, the anti-alpha 3 antibodies recognized the extracellular part of the alpha 3 subunit. They developed later than the anti-alpha 1 muscle antibodies, with their titer increasing during the illness, while that of the anti-alpha1 muscle antibody decreased. Our data suggest that there is no correlation between the presence of anti-alpha 3 antibodies and thymoma.

Reconstitution of conformationally dependent epitopes on the N-terminal extracellular domain of the human muscle acetylcholine receptor alpha subunit expressed in Escherichia coli: implications for myasthenia gravis therapeutic approaches.

Myasthenia gravis (MG) is an autoimmune disease, caused by autoantibodies against the muscle acetylcholine receptor (AChR), an oligomeric transmembrane glycoprotein composed of alpha(2)beta gamma delta subunits. The alpha subunit carries in its N-terminal extracellular domain the main immunogenic region (MIR), a group of conformationally dependent epitopes that seems to be a major target for the anti-AChR antibodies in MG patients. Detailed epitope studies on pathogenic anti-AChR antibodies have been hindered because the binding of most of these antibodies is conformationally dependent, which precludes the use of denatured AChR fragments. The N-terminal extracellular fragment, residues 1-207, of the human AChR alpha subunit was expressed in Escherichia coli in a denatured form, solubilized in a guanidinium hydrochloride-containing buffer, purified, and renatured using a refolding approach which employs a detergent and a cyclodextrin as 'artificial chaperones'. Compared with the non-refolded protein, the refolded molecule exhibited a dramatic improvement in terms of the binding of all anti-MIR mAb tested. Anti-MIR mAb that normally bind weakly to the denatured alpha subunit bound approximately 30-100 times better to the refolded polypeptide and other anti-MIR mAb that bind exclusively to completely conformationally dependent epitopes also bound quite efficiently. These results, in addition to providing a means for the thorough investigation of the antigenic structure of the AChR, show that the conformationally dependent MIR epitopes do not require the participation of the oligosaccharide moiety of the alpha subunit nor the contribution of neighboring subunits for antibody binding. Such AChR fragments may be used in structural studies of the AChR autoantigen, and should prove valuable in the understanding and development of therapeutic approaches for MG.

Treatment of passively transferred experimental autoimmune myasthenia gravis using papain.

Antibody-mediated acetylcholine receptor (AChR) loss at the neuromuscular junction, the main cause of the symptoms of myasthenia gravis, is induced by bivalent or multivalent antibodies. Passive transfer of experimental autoimmune myasthenia gravis (EAMG) can be induced very efficiently in rats by administration of intact MoAbs directed against the main immunogenic region (MIR) of the AChR, but not by their monovalent Fab fragments. We tested whether papain, which has been used therapeutically in autoimmune and other diseases, is capable of preventing EAMG by in vivo cleavage of the circulating anti-AChR antibodies into Fab fragments. EAMG was induced in 4-week-old female Lewis rats by i.p. injection of anti-MIR mAb35. A total of 0.75 mg of papain was given as one or three injections 3-7 h after MoAb injection. The mAb35 + papain-treated animals developed mild weakness during the first 30 h and subsequently recovered, while all animals that received only mAb35 developed severe myasthenic symptoms and died within 24-30 h. Animals treated only with papain showed no apparent side effects for up to 2 months. Serum anti-AChR levels in mAb35 + papain-treated rats decreased within a few hours, whereas in non-papain-treated rats they remained high for at least 30 h. Muscle AChR in mAb35 + papain-treated animals was partially protected from antibody-mediated degradation. These results show that treatment of rats with papain can prevent passively transferred EAMG without any apparent harm to the animals, and suggest a potential therapeutic use for proteolytic enzymes in myasthenia gravis.

Alpha4 but not alpha3 and alpha7 nicotinic acetylcholine receptor subunits are lost from the temporal cortex in Alzheimer's disease.

Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the cerebral cortex in Alzheimer's disease (AD), but to date it has not been demonstrated which nicotinic receptor subunits contribute to this deficit. In the present study, autopsy tissue from the temporal cortex of 14 AD cases and 15 age-matched control subjects was compared using immunoblotting with antibodies against recombinant peptides specific for alpha3, alpha4, and alpha7 subunits, in conjunction with [3H]epibatidine binding. Antibodies to alpha3, alpha4, and alpha7 produced one major band on western blots at 59, 51, and 57 kDa, respectively. [3H]Epibatidine binding and alpha4-like immunoreactivity (using antibodies against the extracellular domain and cytoplasmic loop of the alpha4 subunit) were reduced in AD cases compared with control subjects (p < 0.02) and with a subgroup of control subjects (n = 9) who did not smoke prior to death (p < 0.05) for the former two parameters. [3H]Epibatidine binding and cytoplasmic alpha4-like immunoreactivity were significantly elevated in a subgroup of control subjects (n = 4) known to have smoked prior to death (p < 0.05). There were no significant changes in alpha3- or alpha7-like immunoreactivity associated with AD or tobacco use. The selective involvement of alpha4 has implications for understanding the role of nicotinic receptors in AD and potential therapeutic targets.

Triphosgene: an efficient carbonylating agent for liquid and solid-phase aza-peptide synthesis. Application to the synthesis of two aza-analogues of the AChR MIR decapeptide.

The N alpha/C alphaH exchange in aza-peptides has the advantage of preserving the side chain. Bis(trichloromethyl)carbonate or triphosgene is a solid, stable phosgene substitute which retains its high reactivity. Temperature and coupling times are greatly reduced with reference to other usually recommended carbonylating agents, while purity and yield are increased. It has been used, in both liquid- and solid-phase procedures, for the synthesis of various aza-analogues of dipeptides, tripeptides and decapeptides containing the alanine, aspartic acid and asparagine aza-residue.