Simple semi-permanent blockade against rigid varus foot in a case with spasticity: possible practical benefits in ambulatory adults.

A 55-year-old ambulatory woman with hemiplegia and varus foot deformity had several problems in her daily life, including load pain and stance instability in the affected foot, easy fatigue of the non-paralysed leg, low back pain, neck stiffness and rapid shoe-rubber wear on the deformed side. We began repeated focal blockades using botulinum toxin to the tibialis posterior muscle to control varus spasticity. Distant influences presenting in the whole body were relieved soon after the first blockade, and shoe wear also stopped. Although, neither the deformed appearance nor foot contact pattern on walking changed in the initial period after beginning the blockade, the foot contact pattern revealed gradual improvement over several years. Generally, surgical correction is indicated for the treatment of deformed feet. The present case suggests that, in case of varus-deformed foot with some spastic elements, trial of focal blockade for varus spasticity may be worthwhile.

Decreased bone mineral density in experimental myasthenia gravis in C57BL/6 mice.

Experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis (MG), can be induced in C57BL/6 (B6, H-2 b) mice by 2-3 injections with Torpedo californica AChR (tAChR) in complete Freund's adjuvant. Some EAMG mice exhibit weight loss with muscle weakness. The loss in body weight, which is closely associated with bone structure, is particularly evident in EAMG mice with severe muscle weakness. However, the relationship between muscle weakness and bone loss in EAMG has not been studied before. Recent investigations on bone have shed light on association of bone health and immunological states. It is possible that muscle weakness in EAMG developed by anti-tAChR immune responses might accompany bone loss. We determined whether reduced muscle strength associates with decreased bone mineral density (BMD) in EAMG mice. EAMG was induced by two injections at 4-week interval of tAChR and adjuvants in two different age groups. The first tAChR injection was either at age 8 weeks or at 15 weeks. We measured BMD at three skeletal sites, including femur, tibia, and lumbar vertebrae, using dual energy X-ray absorptiometry. Among these bone areas, femur of EAMG mice in both age groups showed a significant decrease in BMD compared to control adjuvant-injected and to non-immunized mice. Reduction in BMD in induced EAMG at a later-age appears to parallel the severity of the disease. The results indicate that anti-tAChR autoimmune response alone can reduce bone density in EAMG mice. BMD reduction was also observed in adjuvant-injected mice in comparison to normal un-injected mice, suggesting that BMD decrease can occur even when muscle activity is normal. Decreased BMD observed in both tAChR-injected and adjuvant-injected mice groups were discussed in relation to innate immunity and bone-related immunology involving activated T cells and tumour necrosis factor-related cytokines that trigger osteoclastogenesis and bone loss.

Injection of inactive Bordetella pertussis and complete Freund's adjuvant with Torpedo californica AChR increases the occurrence of experimental autoimmune myasthenia gravis in C57BL/6 mice.

An animal model of myasthenia gravis (MG), termed experimental autoimmune MG (EAMG), is an important tool for investigations of disease mechanisms and/or methods of treatment for this disease. EAMG can be induced in C57BL/6 (B6, H-2b) mice by 2-3 times injections at 4 weeks intervals with Torpedo californica (t) acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the protocol especially with a two-injection schedule occasionally produces a poor incidence of EAMG. We have investigated the efficacy of the additional adjuvant, inactive organisms of Bordetella pertussis (iBP), on the induction with a two-injection schedule. In a group immunized with tAChR in CFA + iBP, 76% of mice developed EAMG (average grade in exercise test, 1.02). Whereas, 46% of mice were found EAMG-positive (average grade, 0.73) in a group injected with tAChR/CFA alone. Thus, the combined use of CFA and iBP significantly increased both the occurrence and severity of clinical MG in the immunized mice. This was accompanied by higher antibody (Ab) and T-cell responses to tAChR. The effect on disease occurrence of the iBP use in a three-injection protocol was also described.

Sympathetic skin response and heart rate variability as diagnostic tools for the differential diagnosis of Lewy body dementia and Alzheimer's disease: a diagnostic test study.

OBJECTIVE: The purpose of this study is to investigate the usefulness of sympathetic skin response (SSR) and heart rate variability (HRV) for the differential diagnosis of patients with dementia with Lewy bodies (DLB). DESIGN: A diagnostic test study. SETTING: Single centre in Japan. PARTICIPANTS: We examined 20 patients with probable Alzheimer's disease (AD) diagnosed with NINCDS-ADRDA criteria and 20 with probable DLB diagnosed with the criteria of the third international DLB workshop. METHODS: For the SSR measurement, surface electrodes were used: the active recording electrode was placed on the palm of the hand and the reference electrode was placed on the dorsum of the same hand. SSR was induced by a median nerve electrical stimulation at an amplitude of 20 mA. For the HRV measurement, the A-A intervals were measured twice for 2 min with an interval of 5 min in a sitting position after a rest of 5 min. From the low-frequency power (LF; 0.02-0.15 Hz) and high-frequency power (HF; 0.15-0.50 Hz), the ratio of LF to HF power (LF/HF) was calculated using the maximal entropy method. RESULTS: SSR and HRV could detect the abnormality of autonomic function in patients with DLB at sensitivities of 85% and 90%, respectively. On the other hand, SSR and HRV detected an abnormality of autonomic function in patients with AD at sensitivities of 15% and 25% (p<0.05). The combination of the SSR and the HRV (double-positive) indicated abnormal autonomic function was recorded in only 1 of 20 patients (5%) with AD. In contrast, this combination indicated autonomic abnormality in 15 of 20 patients with DLB by our criteria (75%). CONCLUSIONS: SSR and HRV can be applied to differentiate DLB from AD.

Autoantibody-induced internalization of nicotinic acetylcholine receptor α3 subunit exogenously expressed in human embryonic kidney cells.

Autoantibody against nicotinic acetylcholine receptor (nAChR) α3 subunit has been implicated in the pathogenesis of paraneoplastic neurological syndrome. To examine the effect of anti-α3 subunit autoantibody on cell-surface nAChRs, we established human embryonic kidney 293 cells stably co-expressing α3 and ß4 subunits. Upon incubation with seropositive patient's serum, this cell line showed co-accumulation of patient's IgG and α3 subunits in the cytoplasm. These data support the hypothesis that anti-α3 subunit autoantibody induces internalization of cell-surface nAChRs and thereby impairs synaptic transmission.

Single turnovers of fluorescent ATP bound to bipolar myosin filament during actin filaments sliding.

The nucleotide turnover rates of bipolar myosin thick filament along which actin filament slides were measured by the displacement of prebound fluorescent ATP analog 2'(3')-O-[N-[2-[(Cy3)]amindo]ethyl] carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP) upon flash photolysis of caged ATP. The fluorescence of the thick filament where actin filament slides decayed with two exponential processes. The slower rate constant was the same as that without actin filament. Along bipolar myosin thick filament, actin filaments slide at a fast speed towards the central bare zone (forward), but more slowly away from the bare zone (backward). The displacement rate constant of fluorescent ATP from the myosin filament where actin filament moved forward was 5.0 s(-1), whereas the rate constant where the actin filament slid backward was 1.7 s(-1). These findings suggest that the slow ADP release rate is responsible for the slow backward sliding movement.

Up-regulation of MHC class I and class II in the skeletal muscles of myasthenia gravis.

The up-regulation of MHC in muscles is thought to be associated with inflammatory myopathies. The expression of MHC class I and class II was examined in muscles of myasthenia gravis (MG). MG muscle specimens from all 5 patients with thymoma and 2 of 5 without thymoma showed MHC class I up-regulation and 3 of 5 with thymoma showed MHC class II. The up-regulation of MHC in MG muscles is thus considered to be a common phenomenon. MG muscles expressed not only MHC class I but also class II, and these muscles could thus act as immunoregulating cells in MG.

A case of myasthenia gravis with cardiac fibrosis and easily provoked sustained ventricular tachycardia.

A 65-year-old male, who had been diagnosed to have myasthenia gravis (MG) 25 years previously, was admitted to our hospital with faintness. Cardiac ultrasonography showed decreased left ventricular function. Magnetic resonance imaging depicted delayed contrast enhancement in localized regions. No significant coronary artery stenosis was found, and due to the reproducible susceptibility for sustained ventricular tachycardia, he underwent cardioverter-defibrillator implantation. Although relatively uncommon, cardiac manifestations should not be overlooked in MG patients, as they may be associated with ventricular arrhythmias and cardiac dysfunction.

Autoimmune targets of heart and skeletal muscles in myasthenia gravis.

OBJECTIVE: To investigate the clinical, histological, and immunological features of patients with myasthenia gravis (MG) who also developed myocarditis and/or myositis. DESIGN: Observational and retrospective case series. SETTING: Keio University, Hanamaki General Hospital, Kanazawa University, Nagasaki University, and Juntendo University. PATIENTS: A cohort of 8 patients with MG with clinically defined inflammatory myopathies. INTERVENTIONS: Clinical and histological features were described. Serological analyses included MG-related antistriational autoantibodies (those to titin, ryanodine receptor, muscular voltage-gated potassium channel Kv1.4) and myositis-specific autoantibodies. RESULTS: Of 924 patients with MG, 8 (0.9%) had inflammatory myopathies. The mean (SD) onset age of MG was 55.3 (10.3) years. All patients showed severe symptoms with bulbar involvement; 5 patients had myasthenic crisis and 4 had invasive thymoma. Myocarditis was found in 3 patients and myositis in 6. Myocarditis, developing 13 to 211 months after the MG onset, was characterized by heart failure and arrhythmias. Myositis, developing before or at the same time as MG, affected limb and paraspinal muscles. Histological findings of skeletal muscles showed CD8(+) lymphocyte infiltration. Seven patients had 1 of these antistriational autoantibodies but not myositis-specific autoantibodies. Immunomodulatory therapy was required for all patients and was effective for both MG and inflammatory myopathies, although 1 patient died. CONCLUSIONS: Heart and skeletal muscles are autoimmune targets in some patients with MG. This autoimmunity has a broad clinical spectrum with antistriational autoantibodies.

Autoantibody to dihydropyridine receptor in myasthenia gravis.

To investigate autoantibodies related to excitation-contraction (E-C) coupling in patients with myasthenia gravis (MG), we developed a novel method to detect autoantibodies against dihydropyridine receptor (DHPR). Using this method, we detected DHPR antibody in 37% (11 out of 30) of MG patients with thymoma. Antibodies were not detected in normal nor disease controls. The titer of DHPR antibodies showed no significant correlation with autoantibodies to acetylcholine nor ryanodine receptors. The DHPR antibody is another marker for thymoma in MG, and it might have some role in clinical symptoms related to E-C coupling.

Is excitation-contraction coupling impaired in myasthenia gravis?

OBJECTIVE: To investigate whether excitation-contraction (E-C) coupling of muscle is impaired in patients with myasthenia gravis (MG). METHODS: In 51 patients with generalized MG and 35 normal subjects, compound muscle action potentials (CMAPs) of the abductor pollicis brevis, and movement-related potentials using an accelerometer placed at the thumb tip were simultaneously recorded after median nerve stimulation at the wrist. The E-C coupling time (ECCT) was estimated by a latency difference between CMAP and movement-related potential. Antibodies against acetylcholine receptor (AChR), ryanodine receptor (RyR), and muscle specific receptor tyrosine kinase (MuSK) were measured by immunoassays. RESULTS: The mean ECCT was significantly longer in patients with MG (mean+/-SEM; 2.79+/-0.1 ms; p=0.002) than in normal controls (2.52+/-0.1 ms). Among MG patients, the mean ECCT was longer for patients with thymoma than for those without it (P=0.04), and was shorter for patients treated with FK506 (an immunosuppressant and also an enhancer of RyR related Ca(2+) release) than for those not receiving this treatment (p=0.04). ECCT had no significant correlation with anti-AChR, anti-RyR, or anti-MuSK antibodies. CONCLUSIONS: In MG, E-C coupling appears to be impaired, particularly in patients with thymoma, and FK506 possibly facilitates E-C coupling. SIGNIFICANCE: The functional implication of impaired E-C coupling is not established, but it may contribute to muscle weakness in patients with MG.

Use of alum and inactive Bordetella pertussis for generation of antibodies against synthetic peptides in mice.

We have investigated the efficacy of the combined use of Alum and inactive Bordetella pertussis (iBP) adjuvants for eliciting anti-peptide antibodies. ICR mice were immunized four times at 3-week intervals with each of 7 free (i.e., not conjugated to any carrier) synthetic peptides of 15-17 amino acid residues in Alum + iBP, in the commonly used adjuvant protocols (CFA; CFA (initial) followed by IFA), or in CFA + iBP. Serum samples after 3 and 4 injections were tested by RIA. Use of Alum + iBP greatly increased the production of antibodies for most of the peptides. The results have important implications for human vaccine formulation involving peptides.

Myasthenia gravis induced in mice by immunization with the recombinant extracellular domain of rat muscle-specific kinase (MuSK).

UNLABELLED: Myasthenia gravis (MG) is mostly caused by anti-acetylcholine receptor (AChR) auto-antibodies (Abs). Such Abs are undetectable in 10-15% of MG patients, but many have anti-muscle-specific kinase (MuSK) Abs. We injected recombinant rat-MuSK extracellular domain in H-2(a), H-2(b), H-2(bm12) and H-2(d) mice. Certain strains exhibited exercise-induced fatigue, tremors, weight loss, and some died after 2-3 injections. Compound muscle action potentials showed decrement with low-frequency repetitive nerve stimulation. Miniature endplate potentials decreased, suggesting lower numbers of endplates functional AChRs. Myasthenic sera inhibited agrin-induced AChR aggregation in C2C12 myotubes. CONCLUSION: Anti-MuSK Abs induce MG, which might also result from blocking the agrin-signaling pathway.

Voglibose inhibits postprandial hypotension in neurologic disorders and elderly people.

The authors examined blood pressure, glucose, insulin, and neurotensin before and after intake of 75 g glucose with or without voglibose in 28 neurologic patients and 20 healthy controls. Voglibose significantly prevented hypotension and neurotensin increment after glucose intake and had no influence on glucose or insulin increment. These results suggest that voglibose benefits postprandial hypotension.

Elevation of IL-12 p40 and its antibody in myasthenia gravis with thymoma.

We examined the serum levels of cytokines, interferon (IFN)-alpha, IFN-gamma, interleukin (IL)-4, IL-12 p40, and IL-12 p70; those that affect the T helper 1 and 2 balance in patients with myasthenia gravis (MG). Among the cytokines tested, only IL-12 p40, together with the serum titer of anti-IL-12 p40 antibody, was significantly elevated in MG with thymoma. Their elevation was independent of the histopathology of thymoma. Thymectomy decreased the levels of IL-12 p40 accompanied by the anti-acetylcholine receptor antibody, but not anti-IL-12 p40 antibodies. These data strongly suggest the association of IL-12 p40 and its autoantibody with the immunopathology of MG with thymoma.

Inhibition by human sera of botulinum neurotoxin-A binding to synaptosomes: a new assay for blocking and non-blocking antibodies.

The mouse protection assay (MPA), which is an in vivo assay, is currently the most widely used method for monitoring blocking antibodies (Abs) in botulinum neurotoxin (BoNT)-treated patients. In recent studies we found that a number of the regions on the heavy (H) subunit of BoNT/A that bind blocking mouse Abs coincided, or overlapped, with the regions that bind to mouse synaptosomes (snps). This suggested that blocking anti-BoNT/A Abs would be expected to inhibit BoNT/A binding to snps. In the present work, we analyzed sera from 58 cervical dystonia (CD) patients who had been treated with BOTOX (a preparation of BoNT/A serotype) for blocking Abs by MPA and by their abilities to inhibit in vitro the binding of 125I-labeled active BoNT/A or inactive toxin (toxoid) to mouse brain snps. With active 125I-labeled BoNT/A-snps binding, the MPA-positive sera (n = 30) displayed inhibition levels that were distinctly higher (mean = 21.1 +/- 5.8) than those obtained with MPA-negative sera (n = 28) (mean = -1.3 +/- 3.9; p < 0.0001) or control sera (n = 19) (mean = -3.4 +/- 2.8; p < 0.0001). Similarly, inhibition levels by MPA-positive sera of 125I-labeled toxoid snp-binding (mean = 48.6 +/- 8.7) were distinctly higher than inhibition by MPA-negative sera (mean=10.0+/-7.6; p < 0.0001) or control sera (mean = 1.8 +/- 6.9; p < 0.0001). Thus, using labeled active toxin or toxoid, the inhibition assay correlated very well with the MPA. The inhibitory activity of the non-protective sera generally correlated with the duration of survival after toxin challenge (correlation coefficients of inhibition: active toxin = 0.445; p = 0.0167; inactive toxoid = 0.774; p < 0.0001). It is concluded that the snp-inhibition assay reported here is reliable, reproducible and correlates very well with the MPA. It requires much less serum (0.75% of the amount needed for the MPA) and is considerably less costly than the MPA. With either 125I-labeled active toxin or toxoid, it is possible to distinguish CD sera that have blocking Abs from those that lack such Abs. Since the results with the toxoid were as discriminating as those of the active toxin, it would not even be necessary to use active toxin in these assays.

Vaccination with a MHC class II peptide in Alum and inactive pertussis strongly ameliorates clinical MG in C57BL/6 mice.

We have investigated the efficacy of immunization against peptides from predisposing MHC class II molecules in human-compatible adjuvants for ameliorating experimental autoimmune myasthenia gravis (EAMG). C57BL/6 mice were immunized three times with the peptide I-Abetab62-76 in Alum+killed pertussis organisms (PT) prior to two injections with tAChR. The treatment greatly reduced the occurrence and severity of clinical MG relative to controls that received saline/Alum+PT or none. It also reduced antibody and T-cell responses against tAChR. The results have important implications for the possible immunotherapy of MG by targeting disease-associated MHC.

Mapping of the synaptosome-binding regions on the heavy chain of botulinum neurotoxin A by synthetic overlapping peptides encompassing the entire chain.

The purpose of this work was to map, on the heavy (H) chain of botulinum neurotoxin A (BoNT/A), the regions that bind to mouse brain synaptosomes (snps). We prepared 60 synthetic overlapping peptides that had uniform size and overlaps and encompassed the entire H chain (residues 499 to 1296) of BoNT/A. The ability of each peptide to inhibit the binding of 125I-labeled BoNT/A to mouse brain snps was studied. The binding of 125I-labeled BoNT/A to mouse brain snps was completely inhibited by free unlabeled BoNT/A, but not by unrelated proteins, indicating that the binding of BoNT/A to mouse brain snps was a specific event. Inhibition studies with the individual peptides showed that, on the H(N) domain, inhibitory activities greater than 10% were exhibited, in decreasing order, by peptides 799-817, 659-677, 729-747, 533-551, 701-719, and 757-775. Lower inhibitory activities (between 5.6% and 8.7%) were exhibited by five other peptides, 463-481, 505-523, 519-537, 603-621 and 645-663. The remaining 18 H(N) peptides had little or no inhibitory activity. In the H(C) domain, peptides 1065-1083, 1163-1181 and 1275-1296 had the highest inhibitory activities (between 25% and 29%), followed (10-12% inhibitory activity) by peptides 1107-1125, 1191-1209 and 1233-1251. Two other peptides, 1079-1097 and 1177-1195, had very low (5.8% and 4.9%) inhibitory activities. The remaining 23 H(C) peptides had no inhibitory activity. Inhibition with mixtures of equimolar quantities of the most active 6 peptides of HN, 5 of H(C) or all 11 of H(N) and H(C) revealed that the peptides contain independent non-competing binding regions. Comparison of the locations of the snp-binding regions on the H-subunit with the regions that bind blocking mouse anti-BoNT/A Abs helped explain the protecting ability of these Abs. In the three-dimensional structure of BoNT/A, the snp-binding regions that completely coincide or significantly overlap with the antigenic regions occupy surface locations and most of them reside in the last half of the H(C) domain. But some of the regions reside in the HN domain and might play a role in the translocation event.