Autoantibodies against M1 muscarinic acetylcholine receptor in myasthenic disorders.

The Lambert-Eaton myasthenic syndrome (LEMS), often associated with small-cell lung carcinoma (SCLC), is a disorder of acetylcholine (ACh) release from motor nerve terminals. In most patients, it is caused by autoantibodies against the P/Q-type voltage-gated calcium channels (VGCC) that trigger ACh release. However, these antibodies are not detected in approximately 15% of clinically and electrophysiologically typical cases. The M1-type pre-synaptic muscarinic ACh receptor (M1 mAChR) modulates cholinergic neuromuscular transmission by linking to P/Q-type VGCC, and may partially compensate for the reduced calcium entry. Immunoblotting against solubilized human M1 mAChR, we detected autoantibodies in: (a) 14 of 20 (70%) anti-VGCC-positive LEMS patients; (b) all five anti-VGCC-negative LEMS patients, one of whose serum had previously passively transferred LEMS-type electrophysiological defects to mice; (c) all five LEMS patients with autonomic symptoms; (d) seven of 25 (28%) myasthenia gravis (MG) patients in whom increased ACh release partially compensates for post-synaptic defects; (e) none of 10 SCLC patients without LEMS. Although not proving primary pathogenicity of anti-M1 mAChR antibodies, the present results highlight their potential to affect synaptic compensatory mechanisms, more in LEMS than MG.

Seronegative Lambert-Eaton myasthenic syndrome: study of 110 Japanese patients.

The authors characterized the clinical and immunologic features of 110 patients with Lambert-Eaton myasthenic syndrome (LEMS). Anti-P/Q-type voltage-gated calcium channels (VGCC) antibodies were detected in 85% of the patients (seropositive) but not in the rest (seronegative). Except for the indication that small cell lung carcinoma is less common in seronegative patients, no significant differences were found in the clinical characteristics of patients who had or did not have anti-P/Q-type VGCC antibodies. The results of passive transfer experiments suggest that seronegative LEMS is also an autoantibody-mediated disorder.

Congenital myasthenic syndrome caused by low-expressor fast-channel AChR delta subunit mutation.

OBJECTIVE: To determine the molecular basis of a disabling congenital myasthenic syndrome (CMS) observed in two related and one unrelated Arab kinship. BACKGROUND: CMS can arise from defects in presynaptic, synaptic basal lamina-associated, or postsynaptic proteins. Most CMS are postsynaptic, and most reside in the AChR epsilon subunit; only two mutations have been reported in the AChR delta subunit to date. METHODS: Cytochemistry, electron microscopy, alpha-bungarotoxin binding studies, microelectrode and patch-clamp recordings, mutation analysis, mutagenesis, and expression studies in human embryonic kidney cells were employed. RESULTS: Endplate studies showed AChR deficiency, fast decaying, low-amplitude endplate currents, and abnormally brief channel opening events. Mutation analysis revealed a novel homozygous missense mutation (deltaP250Q) of the penultimate proline in the first transmembrane domain (TMD1) of the AChR delta subunit. Expression studies indicate that deltaP250Q (1) hinders delta/alpha subunit association during early AChR assembly; (2) hinders opening of the doubly occupied closed receptor (A(2)R); and (3) speeds the dissociation of acetylcholine from A(2)R. Mutagenesis studies indicate that deltaP250L also has fast-channel effects, whereas epsilon P245L and epsilon P245Q, identical mutations of the corresponding proline in the epsilon subunit, have mild slow-channel effects. CONCLUSIONS: deltaP250Q represents the third mutation observed in the AChR delta subunit. The severe phenotype caused by deltaP250Q is attributed to endplate AChR deficiency, fast decay of the synaptic response, and lack of compensatory factors. That the penultimate prolines in TMD1 of the delta and epsilon subunits exert a reciprocal regulatory effect on the length of the channel opening bursts reveals an unexpected functional asymmetry between the two subunits.

Plasmapheresis affects T helper type-1/T helper type-2 balance of circulating peripheral lymphocytes.

Plasmapheresis not only removes humoral factors, but may also modulate cellular immunity. We investigated whether plasmapheresis influenced T helper type-1/T helper type-2 (Th1/Th2) cytokine-producing-cell balance in 3 patients with neuroimmunological disease. The production of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and IL-4 in the culture supernatant of peripheral blood mononuclear cells stimulated by anti-CD3 and anti-CD28 was assayed. In 2 of 3 patients, plasmapheresis (immunoadsorption or plasma exchange) reduced Th1/Th2 cytokine ratio. The results may suggest that plasmapheresis induces a shift of Th1/Th2 balance in peripheral blood.

Down-regulation of E-cadherin by antisense oligonucleotide enhances basement membrane invasion of pancreatic carcinoma cells.

Pancreatic carcinoma shows a marked invasiveness around tissues lymph node and/or hematogenous metastases resulting in poor prognoses of the patients. We examined on whether E-cadherin is associated with these malignant behaviors of pancreatic carcinoma cells using a human pancreatic adenocarcinoma cell line, JHP-1. Immunohistochemically, E-cadherin expression of JHP-1 cells was remarkably inhibited by treatment with E-cadherin antisense oligonucleotide. By invasion-MTT assay, JHP-1 cells treated with E-cadherin antisense oligonucleotide showed a significant increase of invasiveness compared to those treated with the control oligonucleotide (P < 0.001), whereas the proliferation of JHP-1 cells was not affected by the presence of either E-cadherin antisense or control oligonucleotide. Thus, down-regulation of E-cadherin of pancreatic carcinoma cells induced the invasiveness into the basement membrane. These results suggest that the reduction in E-cadherin expression plays a key role not only in detachment of cell-cell adhesion but also in invasion and metastasis of pancreatic carcinoma cells.

Short-term effects of prednisolone on neuromuscular transmission in the isolated mdx mouse diaphragm.

To determine the mechanism of the beneficial effects of prednisolone on Duchenne muscular dystrophy (DMD), we examined the short-term effects of prednisolone on neuromuscular transmission by using conventional microelectrode methods in the mdx mice. High (56 micromol/liter) and low (2.8 micromol/liter) concentrations of prednisolone were applied to a bath containing phrenic nerve-diaphragm preparations from mdx mice, and several parameters related to neuromuscular transmission were recorded. The high dose of prednisolone significantly decreased parameter n on quantal release by nerve impulse and decay time-constant of end-plate potentials, which showed adverse effect on neuromuscular transmission. The low dose of prednisolone did not significantly increase quantal content, but could assist the compensatory reaction to maintain the safety margin of neuromuscular transmission in the mdx mice. Our results suggest that the latter effect represents one of the possible mechanisms of the therapeutic effects of prednisolone on DMD.

Myasthenic crisis with delayed recovery after plasmapheresis.

We report on 2 elderly patients with myasthenia gravis in whom recovery from crisis was prolonged despite intensive plasmapheresis (PP). In both patients, the anti-acetylcholine (anti-AChR) titer failed to fall sufficiently after completing PP. These patients might have had antibodies that produced a more pronounced effect on the degradation of AChR, or the synthesis of AChR might have been reduced by aging. The anti-AChR titer did not correlate with a reduction of IgG after PP in 1 patient. Successful treatment was achieved by keeping the anti-AChR titer at a low level via the concomitant use of prednisolone with PP.

Myopathy, myasthenic syndrome, and epidermolysis bullosa simplex due to plectin deficiency.

Plectin, an intermediate filament linking protein, is normally associated with the sarcolemma, nuclear membrane, and intermyofibrillar network in muscle, and with hemisdesmosomes in skin. A 20-year-old female with epidermolysis bullosa simplex since birth had progressive ocular, facial, limb, and trunkal weakness and fatigability since age 9, fivefold CK elevation, a 25% decrement with myopathic motor unit potentials and increased electrical irritability on electromyography, and no anti-acetylcholine receptor (AChR) antibodies. Plectin expression was absent in muscle and severe plectin deficiency was noted in skin. Morphologic studies revealed necrotic and regenerating fibers and a wide spectrum of ultrastructural abnormalities: large accumulations of heterochromatic and lobulated nuclei, rare apoptotic nuclei, numerous cytoplasmic and few intranuclear nemaline rods, disarrayed myofibrils, thick-filament loss, vacuolar change, and pathologic alterations in membranous organelles. Many endplates (EPs) had an abnormal configuration with chains of small regions over the fiber surface and a few displayed focal degeneration of the junctional folds. The EP AChR content was normal. In vitro electrophysiologic studies showed normal quantal release by nerve impulse, small miniature EP potentials, and fetal as well as adult AChR channels at the EP. Our findings support the notion that plectin is essential for the structural integrity of muscle and skin, and for normal neuromuscular transmission.

Quinidine normalizes the open duration of slow-channel mutants of the acetylcholine receptor.

Quinidine is a long-lived open-channel blocker of the wild-type endplate acetylcholine receptor (AChR). To test the hypothesis that quinidine can normalize the prolonged channel opening events of slow-channel mutants of human AChR, we expressed wild-type AChR and five well characterized slow-channel mutants of AChR in HEK 293 cells and monitored the effects of quinidine on acetylcholine-induced channel currents. Quinidine shortens the longest component of channel opening burst (tau3b) of both wild-type and mutant AChRs in a concentration-dependent manner, and 5 microM quinidine reduces tau3b of the mutant AChRs to that of wild-type AChRs in the absence of quinidine. Because this concentration of quinidine is attainable in clinical practice, the findings predict a therapeutic effect for quinidine in the slow-channel congenital myasthenic syndrome.

Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor epsilon subunit.

We describe the genetic and kinetic defects in a congenital myasthenic syndrome caused by heteroallelic mutations of the acetylcholine receptor (AChR) epsilon subunit gene. The mutations are an in-frame duplication of six residues in the long cytoplasmic loop (epsilon1254ins18) and a cysteine-loop null mutation (epsilonC128S). The epsilon1254 ins18 mutation causes mode switching in the kinetics of receptor activation in which three modes activate slowly and inactivate rapidly. The epsilon1245ins18-AChR at the endplate shows abnormally brief activation episodes during steady state agonist application and appears electrically silent during the synaptic response to acetylcholine. The phenotypic consequences are endplate AChR deficiency, simplification of the postsynaptic region, and compensatory expression of fetal AChR that restores electrical activity at the endplate and rescues the phenotype.

Slow-channel myasthenic syndrome caused by enhanced activation, desensitization, and agonist binding affinity attributable to mutation in the M2 domain of the acetylcholine receptor alpha subunit.

We describe a novel genetic and kinetic defect in a slow-channel congenital myasthenic syndrome. The severely disabled propositus has advanced endplate myopathy, prolonged and biexponentially decaying endplate currents, and prolonged acetylcholine receptor (AChR) channel openings. Genetic analysis reveals the heterozygous mutation alphaV249F in the propositus and mosaicism for alphaV249F in the asymptomatic father. Unlike mutations described previously in the M2 transmembrane domain, alphaV249F is located N-terminal to the conserved leucines and is not predicted to face the channel lumen. Expression of the alphaV249F AChR in HEK fibroblasts demonstrates increased channel openings in the absence of ACh, prolonged openings in its presence, enhanced steady-state desensitization, and nanomolar rather than micromolar affinity of one of the two binding sites in the resting activatable state. Thus, neuromuscular transmission is compromised because cationic overloading leads to degenerating junctional folds and loss of AChR, because an increased fraction of AChR is desensitized in the resting state, and because physiological rates of stimulation elicit additional desensitization and depolarization block of transmission.

Transient elevation of nerve growth factor content in the rat hippocampus and frontal cortex by chronic ethanol treatment.

The nerve growth factor (NGF) content in the hippocampus and frontal cortex of chronic ethanol-treated rats was measured and compared with that of control rats, using a two-site enzyme immunoassay (EIA) system. The different time periods of chronic ethanol treatment caused transient elevation of the NGF content in both the hippocampus and frontal cortex. The NGF content in the hippocampus was significantly elevated in rats undergoing ethanol treatment of 2 weeks and 1 month. Nerve growth factor content of the 1 month treatment was higher than that of the 2 week treatment. However, a 3 month administration of ethanol reduced the NGF content to the control level. The NGF content in the frontal cortex increased significantly in the 2 week administration, but decreased to the control level in the 1 month administration. The increase of NGF may be caused by the proliferation of glial cells or the enhancement of neuronal production of NGF.

'Split tolerance' induction by intrathymic injection of acetylcholine receptor in a rat model of autoimmune myasthenia gravis; implications for the design of specific immunotherapies.

Experimental autoimmune myasthenia gravis (EAMG) in the Lewis rat, induced by a single injection of acetylcholine receptor (AChR) protein, is a model used to study human myasthenia gravis (MG). The production of anti-AChR antibodies in the animal model and human MG is T cell-dependent, and AChR-specific T cells have been considered as a potential target for specific immunotherapy. Intrathymic injection of antigens induces antigen-specific tolerance in several T cell-mediated autoimmune models. We examined the effect of intrathymic injection of AChR on T cell responses and the production of antibodies to AChR in EAMG rats. Primed lymph node cells from rats receiving intrathymic injection of AChR exhibited reduced proliferation to AChR with marked suppression of interferon-gamma (IFN-gamma) secretion in the antigen-stimulated culture, compared with those of rats injected with PBS. However, neither anti-Narke AChR nor anti-rat AChR antibody production was suppressed or enhanced in intrathymically AChR-injected animals compared with that of animals injected intrathymically with PBS or perithymically with AChR. This 'split tolerance' may be attributable to the suppression of type-1 T helper cells (Th1). Our results suggest that the suppression of Th1 function alone may not be sufficient for the prevention of antibody-mediated autoimmune diseases.

[A case of cerebral venous sinus thrombosis occurring in ulcerative colitis].

A 33-year-old man with a 13-year history of ulcerative colitis developed generalized clonic convulsions after transient right hemiparesis. A computed tomographic scan revealed a hemorrhagic lesion in the left frontal lobe and contrast tomography demonstrated delta-sign in the occipital area. A sagittal gadolinium-contrast magnetic resonance imaging scan demonstrated a low signal intensity area in the superior sagittal sinus. The venous phase of his carotid angiogram showed a lack of filling of the superior sagittal sinus. Coagulation studies revealed abnormal findings, elevated fibrinogen and increased aggregation of platelets. Cerebral venous or sagittal thrombosis occurring in ulcerative colitis is very rare in Japan. But thromboembolism is known as one of the extraintestinal complications of ulcerative colitis, and a hypercoagulable state may occur even in the inactive state. Ulcerative colitis should be considered as one of the etiologies of thromboembolism in addition to the results of coagulation studies for the prevention of cerebrovascular diseases.