Multiple antibody detection in 'seronegative' myasthenia gravis patients.

BACKGROUND AND PURPOSE: Myasthenia gravis (MG) is an autoimmune disease caused by antibody mediated impairment in the neuromuscular junction. Seronegative MG (SNMG) without antibodies against acetylcholine receptor (AChR) and muscle-specific kinase (MuSK) by routine assays accounts for about 20% of all MG patients. METHODS: Plasma from 81 Chinese MG patients previously found to be seronegative was tested by routine assays for AChR and MuSK antibodies. These samples were screened by (i) a novel, highly sensitive radioimmunoassay for AChR antibodies; (ii) cell-based assays for clustered AChR, MuSK and lipoprotein receptor-related protein 4 (LRP4) antibodies; (iii) a radioimmunoassay for titin antibodies. RESULTS: Antibodies to AChR, MuSK, LRP4 and titin were found in 25% (20/81), 4% (3/81), 7% (6/81) and 6% (5/78) of SNMG patients, respectively. In total, 37% of SNMG patients were found to be positive for at least one of the tested antibodies. AChR antibody positive patients had more severe disease (P = 0.008) and a trend towards fewer remissions/minimal manifestations than AChR antibody negative patients. The four patients with coexistence of antibodies had more severe disease, whilst the seronegative patients had milder MG (P = 0.015). CONCLUSIONS: Detection of multiple muscle antibodies by more sensitive assays provides additional information in diagnosing and subgrouping of MG and may guide MG treatment.

Titin antibodies in "seronegative" myasthenia gravis--A new role for an old antigen.

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.

MuSK autoantibodies in myasthenia gravis detected by cell based assay--A multinational study.

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.

Specific adsorbents for myasthenia gravis autoantibodies using mutants of the muscle nicotinic acetylcholine receptor extracellular domains.

Myasthenia gravis (MG) is usually caused by antibodies against the muscle acetylcholine receptor (AChR). Plasmapheresis and immunoadsorption are often used to treat non-responsive patients. Antigen-specific immunoadsorption would remove only the pathogenic autoantibodies reducing side-effects. We expressed AChR extracellular domain mutants for use as specific adsorbents, and characterized them. Antigenicity and capacity for autoantibody binding were improved compared to the wild-type proteins. Adsorption appeared to be fast, as high plasma flow-rates could be applied. The bound autoantibodies were eluted repeatedly, allowing column reuse, without compromise in efficiency. Overall, the adsorbents were specific, efficient and suitable for use in therapy.

A comprehensive analysis of the epidemiology and clinical characteristics of anti-LRP4 in myasthenia gravis.

Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation (∼2-50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7-32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.

A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model.

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) gamma-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms.

Epidemiological and immunological profile of muscle-specific kinase myasthenia gravis in Greece.

OBJECTIVES: The purposes of this study were to determine the epidemiological characteristics of muscle-specific kinase-myasthenia gravis (MuSK-MG) in Greece and the IgG subclass of the anti-MuSK antibodies. METHODS: This population-based study was performed on MuSK-MG patients in Greece between 1 January 1986 and 30 June 2006. Epidemiological and clinical data for 33 patients were collected. In addition, the distribution of anti-MuSK IgG autoantibody subclasses in the sera of 14 patients was determined by immunoprecipitation. RESULTS: The average annual incidence was 0.32 patients/million population/year. On 1st July 2006, there were 33 prevalent cases, giving a point prevalence rate of 2.92/million (women 4.56 and men 1.25). In females, onset of MuSK-MG occurred after the age of 30, whilst, in males, the disease appears in any decade. The female:male incidence ratio was 3.33:1, whilst the prevalence ratio was 3.65:1. Most patients presented with involvement of the facial and bulbar muscles. Amongst about 800 MG patients seropositive for antibodies against either the AChR or MuSK, one patient was found to be seropositive for anti-MuSK antibodies and ambiguous for anti-acetylcholine receptor (anti-AChR) antibodies. The vast majority of anti-MuSK antibodies were IgG4, whilst total IgG4 levels in these patients were similar to those in two healthy controls. CONCLUSIONS: The incidence and prevalence of MuSK-MG in Greece are amongst the highest reported previously for other countries. MuSK-MG in Greece affects both sexes, but mainly females. The main epidemiological indices were calculated. The vast majority of anti-MuSK antibodies were IgG4.

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.

Conjugation of acetylcholine receptor-protecting Fab fragments with polyethylene glycol results in a prolonged half-life in the circulation and reduced immunogenicity.

Antibodies to the acetylcholine receptor (AChR) cause AChR loss, resulting in the disease, myasthenia gravis (MG). The majority of the pathogenic antibodies seem to be directed against the main immunogenic region (MIR) of the AChR. In contrast to the intact antibodies, Fab fragments of anti-AChR antibodies are not themselves pathogenic and such fragments of anti-MIR monoclonal antibodies (mAbs) protect the AChR in vitro and in vivo against the pathogenic antibodies. However, Fab fragments have a very short in vivo half-life and are immunogenic, obstacles which must be overcome before their clinical use can be envisaged. We investigated the effect of conjugating Fab fragments to polyethylene glycol (PEG), a method known to increase the in vivo half-life and reduce the immunogenicity of proteins. When the Fab' fragments of two rat anti-MIR mAbs (nos. 35 and 195) were conjugated to methoxy-PEG-maleimide, the conjugates retained about 10% of their AChR binding activity and efficiently protected the AChR against the binding and modulating activity of myasthenic antibodies. Their in vivo half-life in rats was approximately 15 times longer than that of the unconjugated Fab' fragment and they were much less immunogenic in mice. This work represents an important step towards the clinical use of AChR-protective anti-MIR Fabs, but further improvements are needed before their clinical use is attempted.

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.

Crystal structure of Fab198, an efficient protector of the acetylcholine receptor against myasthenogenic antibodies.

The crystal structure of the Fab fragment of the rat monoclonal antibody 198, with protective activity for the main immunogenic region of the human muscle acetylcholine receptor against the destructive action of myasthenic antibodies, has been determined and refined to 2.8 A resolution by X-ray crystallographic methods. The mouse anti-lysozyme Fab D1.3 was used as a search model in molecular replacement with the AMORE software. The complementarity determining regions (CDR)-L2, CDR-H1 and CDR-H2 belong to canonical groups. Loops CDR-L3, CDR-H2 and CDR-H3, which seem to make a major contribution to binding, were analyzed and residues of potential importance for antigen-binding are examined. The antigen-binding site was found to be a long crescent-shaped crevice. The structure should serve as a model in the rational design of very high affinity humanized mutants of Fab198, appropriate for therapeutic approaches in the model autoimmune disease myasthenia gravis.

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.

Severe congenital myasthenic syndrome due to homozygosity of the 1293insG epsilon-acetylcholine receptor subunit mutation.

Recently, a congenital myasthenic syndrome (CMS) with end-plate acetylcholine receptor (AChR) deficiency due to missense mutations in the genes for the AChR subunit was described. The first observed patient with this CMS was heteroallelic for the two epsilon-AChR subunit mutations epsilon1101insT and epsilon1293insG. This patient had only a moderate phenotype with mild muscle weakness and abnormal fatigue. We have now found homozygosity for the epsilon1293insG mutation in a severely affected CMS patient, who lost the ability to walk in midchildhood and shows profound weakness and muscle wasting. Our observation allows a genotype-phenotype correlation illustrating how differences in the AChR mutation haplotype can profoundly influence disease severity.

The conformation of the main immunogenic region on the alpha-subunit of muscle acetylcholine receptor is affected by neighboring receptor subunits.

Myasthenia gravis (MG) is caused by autoantibodies to the acetylcholine receptor (AChR). Experiments with fetal (alpha(2)betagammadelta) and adult (alpha(2)betaepsilondelta) AChR and with recombinant subunit dimers showed that some monoclonal antibodies (mAbs) against the main immunogenic region (MIR), located on the alpha-subunit of the AChR, bind better to fetal AChR and to alphagamma subunit dimer than to adult AChR and alphaepsilon dimer and equally to both alphabeta and alphadelta. However, other anti-MIR mAbs prefer adult AChR and alphaepsilon dimer, bind well to alphabeta but weakly to alphadelta. These results suggest that the MIR conformation is affected by the neighboring gamma/epsilon- and delta-subunits and may contribute to understanding the antibody specificities in MG.

Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the receptor in tethered membranes was investigated with Fourier transform infrared spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed against the ligand-binding site which yielded dissociation constants in agreement with radioligand binding assays. The presented method for the functional immobilisation of the nicotinic acetylcholine receptor in tethered membranes might be generally applicable to other membrane proteins.

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.

Relative spatial position of a snake neurotoxin and the reduced disulfide bond alpha (Cys192-Cys193) at the alpha gamma interface of the nicotinic acetylcholine receptor.

We determined the distances separating five functionally important residues (Gln(10), Lys(27), Trp(29), Arg(33), and Lys(47)) of a three-fingered snake neurotoxin from the reduced disulfide bond alpha(Cys(192)-Cys(193)) located at the alphagamma interface of the Torpedo nicotinic acetylcholine receptor. Each toxin position was substituted individually for a cysteine, which was then linked to a maleimido moiety through three different spacers, varying in length from 10 to 22 A. We estimated the coupling efficiency between the 15 toxin derivatives and the reduced cystine alpha(192-193) by gel densitometry of Coomassie Blue-stained gels. A nearly quantitative coupling was observed between alphaCys(192) and/or alphaCys(193) and all probes introduced at the tip of the first (position 10) and second (position 33) loops of Naja nigricollis alpha-neurotoxin. These data sufficed to locate the reactive thiolate in a "croissant-shaped" volume comprised between the first two loops of the toxin. The volume was further restrained by taking into account the absence or partial coupling of the other derivatives. Altogether, the data suggest that alphaCys(192) and/or alphaCys(193), at the alphagamma interface of a muscular-type acetylcholine receptor, is (are) located in a volume located between 11.5 and 15.5 A from the alpha-carbons at positions 10 and 33 of the toxin, under the tip of the toxin first loop and close to the second one.

Nicotinic receptor subtypes in human brain ageing, Alzheimer and Lewy body diseases.

Human brain ageing is associated with reductions in a variety of nicotinic receptors subtypes, whereas changes in age-related disorders including Alzheimer's disease or Parkinson's disease are more selective. In Alzheimer's disease, in the cortex there is a selective loss of the alpha4 (but not alpha3 or 7) subunit immunoreactivity and of nicotine or epibatidine binding but not alpha-bungarotoxin binding. Epibatidine binding is inversely correlated with clinical dementia ratings and with the level of Abeta1-42, but not related to plaque or tangle densities. In contrast, alpha-bungarotoxin binding is positively correlated with plaque densities in the entorhinal cortex. In human temporal cortex loss of acetylcholinesterase catalytic activity is positively correlated with decreased epibatidine binding and in a transgenic mouse model over expressing acetylcholinesterase, epibatidine binding is elevated. In Parkinson's disease, loss of striatal nicotine binding appears to occur early but is not associated with a loss of alpha4 subunit immunoreactivity. Tobacco use in normal elderly individuals is associated with increased alpha4 immunoreactivity in the cortex and lower densities of amyloid-beta plaques, and with greater numbers of dopaminergic neurons in the substantia nigra pars compacta. These findings indicate an early involvement of the alpha4 subunit in beta-amyloidosis but not in nigro-striatal dopaminergic degeneration.