Masaharu Takamori

Clinical study of FK506 in patients with myasthenia gravis

To investigate the usefulness of low‐dose FK506 for the treatment of myasthenia gravis (MG), we treated 19 patients with generalized MG in a 16‐week open clinical trial of FK506 (3–5 mg/day). At the end of the trial, total MG scores (range: 0–27 points) improved by 3 points or more in 7 of 19 patients (37%), and activities of daily living (ADL) scores (range: 0–6 points) also improved by 1 point or more in 8 of 19 patients (42%). Nine of 19 patients (47%) showed improvement in either MG or ADL scores. Significant reduction of anti‐acetylcholine receptor antibody titers and interleukin 2 production were observed at the end of this study. Minor but commonly observed side effects were an increase in neutrophil count and a decrease in lymphocyte count. No serious adverse events such as renal toxicity or diabetes mellitus were observed during the 16‐week treatment period. FK506 could safely serve as an adjunct to steroid therapy for MG at low dosage. Muscle Nerve 28: 570–574 2003

Patterns and severity of neuromuscular transmission failure in seronegative myasthenia gravis

Objectives: To compare the clinical and electrophysiological features of myasthenia gravis (MG) patients with (seropositive) or without (seronegative) antibodies to acetylcholine receptor. To investigate whether antibodies to muscle specific kinase (MuSK) and ryanodine receptor (RyR) are associated with particular features. Methods: Clinical profiles and single fibre electromyography (SFEMG) in the extensor digitorum communis (EDC) were reviewed in consecutive 57 seropositive and 13 seronegative patients. Antibodies to MuSK and RyR were measured by immunoassays. Results: Of the 13 seronegative patients, four (31%) were positive for MuSK antibodies and seven (54%) were positive for RyR antibodies, including all four MuSK positive patients. Clinical features were similar at presentation for seropositive and seronegative patients, but MuSK positive patients frequently developed myasthenic crises. Despite the similar clinical severities at the time of examination, the proportion with positive jitter (93% of seropositive patients, 50% of MuSK positive patients, and 44% of MuSK negative patients) and the extent of jitter (mean consecutive difference: 76 μs in seropositive patients, 36 μs in MuSK positive patients, and 30 μs in MuSK negative patients) were less in seronegative MG patients compared with seropositive MG patients. Conclusions: Seronegative MG is heterogeneous with respect to the presence of antibodies to MuSK. Impairment of neuromuscular synaptic transmission in EDC is less marked in seronegative than seropositive MG despite the similar clinical severity. This discrepancy may partly reflect the distribution of affected muscles in seronegative patients, but it is possible that other factors, such as impaired excitation-contraction coupling resulting from RyR antibodies, contribute to the clinical phenotype.

Anti-ryanodine receptor antibodies and FK506 in myasthenia gravis

Anti-ryanodine receptor (RyR) antibodies were measured in sera from 33 myasthenia gravis (MG) patients using three peptides from the human RyR1 sequence, two C-terminal peptides included in the functional calcium release channel, and an N-terminal peptide implicated in ion-conduction. Antibodies were more frequently positive against the two C-terminal peptides, particularly in thymoma-associated MG. In a preliminary open trial with FK506, immunosuppressant and enhancer of RyR-related sarcoplasmic calcium release, the authors observed the sustained benefits in anti-RyR-positive MG patients.

Beriberi neuropathy: Morphometric study of sural nerve

Seven biopsied sural nerves from patients with beriberi were morphometrically evaluated. In teased fiber analysis the mean frequency of myelinated fibers showing axonal degeneration and segmental demyelination was 37.5 and 5.3%, respectively. In two cases with frequency of segmental demyelination higher than 5%, segmental demyelination was shown by statistical criteria to have occurred on certain fibers in a clustered fashion. Therefore, the segmental demyelination in beriberi may be secondary to axonal degeneration. Electromyographic findings and slow improvement of muscle weakness were compatible with axonal degeneration of motor fibers. Determinations of fiber densities revealed preferential decrease of the density of large myelinated fibers with the preservation of the density of small myelinated and unmyelinated fibers. The preferential nerve fiber involvement in beriberi was not associated with pain in the lower limbs and this fact is contrary to the expectation of the proponents of the gate control theory.

Randomised, double-blind, placebo-controlled study of tacrolimus in myasthenia gravis

Objectives To evaluate the ability of tacrolimus to reduce the corticosteroid dose in patients with myasthenia gravis (MG) and the drugs safety in a double-blind, placebo-controlled, parallel group study. Methods Patients being treated with oral prednisolone at doses equivalent to 10–20 mg/day, and with stable symptoms, were randomised to tacrolimus or placebo in a 28-week double-blind study. The dose of corticosteroid was tapered with the procedures specified in the protocol. The primary efficacy endpoint was the mean daily prednisolone dose given in the last 12 weeks of the study. Results Eighty patients received the study drug (40 patients in each group) and were included in the full analysis set. In the full analysis set, there was no significant difference in the primary efficacy endpoint between the two groups (p=0.078). However, some secondary analyses suggested the steroid-sparing effect of tacrolimus. Tacrolimus was well tolerated, and no safety concerns were noted. Conclusions This study suggests that tacrolimus has a potential advantage as a steroid-sparing agent in the treatment of MG patients. Clinical trial registration number NCT00309088. Name of the trial registry: FK506 Phase 3 Study: A Study for Steroid Non-Resistant MG Patients.

Myasthenogenic significance of synthetic α-subunit peptide 183–200 of Torpedo californica and human acetylcholine receptor

Synthetic peptides of the Torpedo californica and human acetylcholine receptor alpha-subunit, Gly183-Asp200, were studied. Both peptides bound alpha-bungarotoxin to a significant extent, and inhibited toxin-binding with Torpedo or human acetylcholine receptor. The human peptide was, however, less potent than the Torpedo peptide. One can assume, therefore, that this sequence participates in cholinergic binding. The Torpedo alpha 183-200 segment was immunogenic in the induction of experimental autoimmune myasthenia gravis in rats, accompanied by elevation of anti-peptide and anti-rat acetylcholine receptor blocking antibody and reduced amplitudes of miniature end-plate potentials. Sera did not accelerate the degradation of rat acetylcholine receptor. Thus, one may interpret this to be the animal model induced by an immunopharmacological blockade of the acetylcholine-binding site. Furthermore, this Torpedo peptide was antigenic in the detection of antibody in human myasthenic sera. The human alpha 183-200 segment had neither of these 2 properties, although the corresponding anti-peptide antibody was elicited in immunized rats. It is feasible that the synthetic segment of this human sequence may not be maintained in such a conformation to be immunogenic.

Limb muscle endplates in ocular myasthenia gravis: Quantitative ultrastructural study

Motor endplate ultrastructure in the biceps brachii was quantitatively analyzed in five patients with ocular myasthenia gravis (56 endplates), six patients with generalized myasthenia gravis (83 endplates), and five controls (64 endplates). Ultrastructural changes of the motor endplates were observed in non weak limb muscles of patients with ocular myasthenia gravis as well as in generalized myasthenia; these changes were mostly restricted to the postsynaptic region. Decrease of the mean presynaptic and postsynaptic membrane length, and postsynaptic membrane density, were more remarkable in generalized myasthenia than in ocular myasthenia. Widening of the primary and secondary synaptic clefts was also observed more frequently in generalized myasthenia. There was no correlation between ultrastructural alterations and the duration of symptoms or treatment, severity of disease, or titers of antiacetylcholine receptor antibody.

Antibodies to synthetic peptides of the alA subunit of the voltage‐gated calcium channel in Lambert‐Eaton myasthenic syndrome

To search for antigenic sites in the molecular structure of alA subunit of the voltage-gated calcium channel (VGCC) (P/Q-type) in the Lambert-Eaton myasthenic syndrome (LEMS), we studied by immunoprecipitation assay serum samples from 30 LEMS patients (16 with small cell lung carcinoma (SCLC), 20 disease controls (10 with SCLC without LEMS and 10 with myasthenia gravis), and 15 healthy controls. Synthetic peptide antigens corresponded to the extracellular region (S5-S6 linker region) of each of the four domains forming the al subunit of P/Q-type VGCC. In addition, we studied serum samples for anti-P/Q-type VGCC antibodies by using w-conotoxin MVIIC-labeled extract of human cerebellum as an antigen. Among sera of 30 LEMS patients, nine samples (30%) (six with SCLC) were positive for antibodies to the domain IV S5-S6 linker peptide, and six samples (20%) (five with SCLC) were positive for antibodies to the domain II S5-S6 linker peptide. Only two of 15 antipeptide-positive sera were positive for both antibodies. Titers for antibodies to domain IV, as well as those for antibodies to domain II, correlated with those of anti-P/Q-type VGCC (human cerebellum extract) antibodies. The antipeptide antibody was present in only one of 20 disease controls, a patient with SCLC without LEMS. Our observations suggest two potential epitopes of LEMS antibodies.

Low-dose tacrolimus for intractable myasthenia gravis.

We treated two patients suffering from intractable myasthenia gravis (MG) with low-dose tacrolimus plus prednisolone. Both patients showed significant improvement of myasthenic signs, accompanied by suppressed serum anti-acetylcholine receptor antibody, waning in compound muscle action potential by repetitive nerve stimulation and IL-2 production by peripheral blood mononuclear cells. Low-dose tacrolimus plus prednisolone is a promising therapeutic regimen for intractable MG.

Lambert-Eaton myasthenic syndrome as an autoimmune calcium-channelopathy.

Lambert-Eaton myasthenic syndrome, often associated with small-cell lung carcinoma, is a disease of neuromuscular transmission in which antibodies directed against voltage-gated calcium channel (VGCC)(P/Q-type) in the motor nerve terminal play a crucial role in causing a deficient quantal release of acetylcholine. The motor nerve terminal and carcinoma cell may share a common antigen. The study using synthetic peptides and recombinant protein specified the extracellular S5-S6 linker regions in 3 of 4 domains as immunodominant sites in the molecular structure of P/Q-type VGCC alpha1 subunit. Also, the study by use of peptides and recombinant protein corresponding to synaptotagmin I suggested that in this functionally VGCC-associated presynaptic protein, the segment which exposes extracellularly during exocytosis can be immunogenic for the syndrome.