Myasthenia gravis patients with ryanodine receptor antibodies have distinctive clinical features

Myasthenia gravis (MG) is an autoimmune disease caused in 85% of the patients by acetylcholine receptor (AChR) antibodies. Non-AChR muscle antibodies, against titin and ryanodine receptor (RyR) are mainly found in sera of patients with thymoma or late-onset MG. The occurrence of RyR antibodies increases the risk for severe MG and should lead to active immunomodulating treatment already at MG onset. The aim in this study was to describe the association between symptoms at MG onset and antibody profile in 152 patients. Patients with RyR antibodies had the highest rate of bulbar, respiratory and neck involvement at MG onset. They also had the highest frequency of non-limb MG symptoms. Neck weakness occurred in 40%. Respiratory difficulties at MG onset occurred in patients with titin antibodies, with and without RyR antibodies. Patients with RyR antibodies have a distinctive non-limb MG symptom profile, with bulbar, ocular, neck, and respiratory symptoms. These features, identified as early as at the first examination by a neurologist, characterize the RyR antibody positive subgroup at MG onset.     

雷诺丁受体抗体阳性的重症肌无力临床特点

雷诺丁受体（ryanodine receptor, RyR）是存在于内质网/肌质网中的一种重要钙离子通道，在骨骼肌兴奋收缩偶联机制中起重要作用。RyR抗体阳性的重症肌无力（myasthenia gravis, MG）患者常合并胸腺瘤，对常规治疗不敏感，会导致延误临床早期识别及治疗。血清RyR抗体水平与患者临床症状的严重程度显著相关。该文就4例RyR抗体阳性MG患者的临床特点及治疗过程进行讨论并文献复习，旨在提高对RyR抗体阳性MG的认识及诊疗水平。     

Ryanodine receptor antibodies in myasthenia gravis: epitope mapping and effect on calcium release in vitro

Patients with myasthenia gravis can have antibodies against skeletal muscle ryanodine receptor (Ry1), the sarcoplasmic reticulum calcium-release channel, which plays a crucial role in excitation-contraction coupling. We have screened a panel of overlapping Ry1 fusion proteins with Ry1 antibody-containing myasthenia gravis sera to identify the main immunogenic region. The pc2 Ry1 fusion protein representing a Ry1 region close to the N-terminus (residues 799-1172) was identified as the main immunogenic region for the antibodies. The binding kinetics of the Ry1 antibodies to the pc2 Ry1 fusion protein were tested using an optical biosensor. Ry1 antibodies in the IgG fraction from sera of patients with myasthenia gravis bound with high affinity and with a stoichiometry of 1:1. The functional effect of these Ry1 antibodies was tested in an in vitro Ca2+-release assay. The Ry1 antibodies induced a twofold increase of the half-maximal concentration for 4-Cl-m-cresol-induced Ca2+ release from terminal cisternae vesicles but had no effect on V(max). The effect on 4-Cl-m-cresol-induced Ca2+ release was specific, as preincubation of the active IgG fraction with the pc2 Ry1 fusion protein abolished the inhibition. These data suggest that the Ry1 sequence defined by residues 799-1172 is involved in the regulation of Ry1 function, and that this regulation could be functionally affected in vivo in patients with myasthenia gravis.     

Disease severity and outcome in thymoma myasthenia gravis: a long-term observation study

Thymomas occurring in myasthenia gravis (MG) are usually of the cortical subtype and are usually treated by thymectomy. However, the factors that influence MG outcome in thymoma MG patients are not known. In a long-term study, MG severity and treatment was observed in 24 thymoma and 24 non-thymoma MG patients for up to 30 years, and the occurrence of muscle autoantibodies was assayed. The rate of complete stable remission was low and did not differ between the two groups. There was no significant difference in MG severity between thymoma and non-thymoma MG patients at any time during the study. Titin and ryanodine receptor (RyR) antibody occurrence was significantly higher in thymoma MG patients. Four thymoma (all titin and RyR antibody positive) and two non-thymoma (both titin and one RyR antibody positive) MG patients died from MG-related respiratory insufficiency. Seventy percent of thymoma and 75% of non-thymoma MG patients were treated with immunosuppressive drugs. The number of patients who received plasmapheresis did not differ in those who were treated because of acute MG deterioration, irrespective of planned surgery.This study shows equal MG severity and outcome in thymoma and non-thymoma MG, but the presence of RyR antibodies in thymoma MG and titin/RyR in non-thymoma MG indicates a less favorable prognosis.     

Regional [H]acetylcholine and [H]nicotine binding in developing mouse brain

The postnatal development of nicotine-like binding sites in the cortex, hippocampus, midbrain and cerebellum of 3-, 7-, 12-, 17- and 30-day-old mice was studied. Two different nicotinic cholinergic ligands, namely [³H]acetylcholine ([³H]ACh) and [³H]nicotine ([³H]NIC) were used to detect the nicotine-like binding sites in in vitro binding assays. The postnatal development of the binding sites of [³H]NIC increased gradually with age in all brain regions studied. The [³H]ACh binding, on the other hand, showed a marked peak on day 12 in the cerebellum and midbrain but did not change notably with age in the hippocampus and cortex, except for a slight temporary increase in the cortex on day 7. The time-course for the appearance of nicotinic binding sites as observed with [³H]ACh was found to be rather similar to that earlier described for [³H]alpha-bungarotoxin binding sites, whereas that for [³H]NIC differed from that described for other nicotinic ligands.     

High affinity [3H]imipramine and [3H]paroxetine binding sites in suicide brains

Summary Specific binding of [3H]imipramine and [3H]paroxetine was simultaneously examined in human brains (frontal cortex, temporal cortex, cingulate cortex, hypothalamus, hippocampus and amygdala) from 11 controls and 11 depressed suicide victims. A single saturable high affinity site was obtained for both radioligands. Age was not related to significant changes in [3H]imipramine and [3H]paroxetine binding parameters, which indicates the stability of the brain serotonergic system with increasing age.A major finding of the present study concerns the existence of a significant decrease in the maximum number (Bmax) of [3H]imipramine binding sites in hippocampus from depressed suicides as compared with the control group, without changes in the binding affinity (Kd). In contrast, when [3H]paroxetine was used as radioligand, no changes in either Bmax or Kd were detected in any of the brain regions studied. These findings suggest that [3H]imipramine may be a better marker than [3H]paroxetine when alterations in the presynaptic serotonergic uptake site are to be detected.     

Enzyme-linked immunosorbent assay for detection of antibodies to extractable muscle antigens in myasthenia gravis

A purified citric acid soluble extract from human skeletal muscle (AEMA) and a phosphate-buffered saline extract from rabbit muscle acetone powder (EMA) were used to coat polystyrene beads in an enzyme-linked immunosorbent assay (ELISA). From 54 patients with myasthenia gravis, positive results were observed in 14. Five of 6 patients with thymoma had high levels of antibodies. With the diagnostic difficulties in detecting small and medium-sized thymoma, a sensitive assay for detection of antibodies to muscle antigen may be an important supplementary tool to detect tumors at early stages.     

Endogenous material in brain inhibiting [3H]nicotine and [3H]acetylcholine binding

The supernatant obtained from mouse brain homogenates contains material that inhibits the saturable binding of [³H]nicotine in mouse cerebral cortex. This inhibitory material was further purified by heat denaturation, ultrafiltration through an Amicon PM-10 membrane filter, and gel chromatography on Sephadex G-10. The material inhibited the binding of [³H]acetylcholine with the same potency as it did that of [³H]nicotine. It also had some affinity for the sites that specifically bind [³H]D-Ala, D-Leu enkephalin, but had much lower affinity for the binding sites for [³H]quinuclidinyl benzilate (QNB), [³H]spiroperidol, [³H]naloxone, or [³H]imipramine. Acid hydrolysis destroyed the activity. These preliminiary results suggest the presence in brain of “nicotinelike” substances, one of which may be the endogenous ligand for the sites that specifically bind [³H]nicotine.     

Evidence for loss of brain [H]spiroperidol and [H]ADTN binding sites in rabbit brain with aging

[³H]Spiroperidol and [³H]2-amino-6,7-dihydroxyl-1,2,3,4,-tetrahydronaphthalene hydrochloride (ADTN) binding were measured in various central nervous system regions of 5 month and 5.5 year old rabbits. In striatum, young animals had a 38% higher number of [³H]spiroperidol binding sites and a 140% higher number of [³H]ADTN binding sites than did the older animals. In frontal cortex and anterior limbic cortex there were respectively 42% and 26% more [³H]spiroperidol binding sites in the young animals. There was no change in the binding site number or affinity for [³H]spiroperidol in retina with aging. Pharmacological characterization demonstrated that [³H]spiroperidol binds to a dopamine receptor in striatum and to a serotonin receptor in cortex.     

Do acetylcholine receptor and striated muscle antibodies predict the presence of thymoma in patients with myasthenia gravis?

Introduction: Acetylcholine receptor (AChR) and striated muscle antibodies (StrAbs) are found frequently in myasthenia gravis (MG) patients with thymoma. In this study we aimed to determine the positive predictive value (PPV) and negative predictive value (NPV) of these antibodies for thymoma in patients with MG. Methods: Antibody findings, thymic histology, and onset age were reviewed for 1141 patients with MG. PPV and NPV of these antibodies for thymoma were determined. Results: The PPV of AChR binding antibodies plus StrAbs was highest (50.0%) with onset before the age of 40 years. The PPV of all antibodies was low (<9%) after age 40. Higher StrAb levels did not increase the PPV. The NPV of AChR binding antibodies was high (99.7%) for all ages. Conclusions: Patients without AChR binding antibody are not likely to have a thymoma. StrAbs and AChR binding antibodies are not diagnostic for thymoma, but in early‐onset MG their presence should raise the clinical suspicion for thymoma. Muscle Nerve 49 : 30–34, 2014     

Myasthenia gravis associated with Charcot-Marie-Tooth neuropathy: report of a case

We report the case of a 24 year old woman who developed myasthenia gravis in the course of a mild form of Charcot-Marie-Tooth neuropathy. We describe the clinical manifestations together with the neurophysiological, pathological, serological findings and response to therapy and discuss the unusual association in the light of the relevant literature.

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Sommario Si riferisce di un caso di Miastenia Gravis in una donna di 24 anni affetta da una forma non invalidante di malattia di Charcot-Marie-Tooth. Vengono riportate le manifestazioni cliniche, i risultati elettrofisiologici, bioptici e sierologici, nonché l'approccio terapeutico utilizzato. Questa inusuale associazione viene discussa alla luce dei dati riportati in letteratura.

     

Characterization and distribution of [3H]ohmefentanyl binding sites in the human brain

Binding properties and localization of [3H]ohmefentanyl, a new ligand for mu opioid receptors, were investigated on normal human brain sections. Binding assays performed at the level of the basal ganglia revealed: (1) a steady-state binding reached after 60 min incubation at room temperature, (2) the presence, in saturation experiments, of an apparent single class of binding sites with a Kd = 1.68 +/- 0.45 nM and a Bmax = 162 +/- 9 fmol/mg protein, (3) an order of potency to inhibit [3H]ohmefentanyl binding as follows: ohmefentanyl greater than [D-Ala2, MePhe4, Gly-ol5] enkephalin (DAGO) greater than ethylketocyclazocine (EKC) much greater than Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu) (BUBU) and U-50,488H. Quantitative autoradiography showed an heterogeneous distribution of [3H]ohmefentanyl binding sites with the highest densities in amygdala, medical geniculate body, thalamus, and caudate nucleus. Binding characteristics and anatomical distribution also show that [3H]ohmefentanyl may bind to a small proportion of additional sites called "DAGO-inaccessible [3H]ohmefentanyl specific binding sites." [3H]Ohmefentanyl binding to these sites can be partly inhibited by sigma ligands such as 1,3-di-o-tolylguanidine (DTG) and haloperidol. However, unlabeled DAGO inhibited more than 80% of [3H]ohmefentanyl specific binding in most of the human brain regions studied, suggesting that the major population of sites labeled by [3H]ohmefentanyl represented mu opioid receptors.     

Autoradiographic distribution of binding sites for the non-NMDA receptor antagonist [H]CNQX in human motor cortex, brainstem and spinal cord

The distribution of non-NMDA receptors in the normal human motor cortex, brainstem and spinal cord has been investigated using [³H]CNQX. In the motor and premotor cortex, specific [³H]CNQX binding was present in all cortical laminae with the highest density of binding sites in laminae I, II and the upper part of III. In the normal brainstem, non-NMDA receptors labelled by [³H]CNQX had a heterogenous distribution. Brainstem motor nuclei subserving eye movements, which tend to be spared in motor neuron disease (MND), had a higher density of [³H]CNQX binding sites compared to other cranial nerve motor nuclei (VII, X, XII) which tend to be affected. Specific [³H]CNQX binding was present throughout the spinal grey matter, the greatest density of binding being found in the substantia gelatinosa. Excitotoxicity at non-NMDA receptors has been implicated in chronic neurodegenerative diseases such as motor neuron disease. This study suggests that the density of non-NMDA receptors, labelled by [³H]CNQX, does not account for selective vulnerability of motor neurons in this disorder.     

3H]1-[2-(2-thienyl)cyclohexyl]piperidine labels two high-affinity binding sites in human cortex: further evidence for phencyclidine binding sites associated with the biogenic amine reuptake complex

Previous work demonstrated two high-affinity PCP binding sites in guinea pig brain labeled by [3H]TCP (1-(1-[2-thienyl]cyclohexyl)piperidine): site 1 (N-methyl-D-aspartate [NMDA]-associated) and site 2 (dopamine-reuptake complex associated). The present study examined brain membranes prepared from various species, including human, for the presence of site 2, defined as binding in the presence of (+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d]cyclohepten-5,10-imine maleate ((+)-MK801) minus binding in the presence of 10 microM TCP (nonspecific binding). Studies were conducted in absence of sodium which was found to be inhibitory to [3H]TCP binding. The results demonstrated detectable levels of site 2 in brain membranes of guinea pig, rabbit, pig, mouse, sheep, and human but not in the rat or chicken. Using human cortical membranes, site 2 was the predominant binding site. Detailed studies conducted with human cortical tissue showed that high-affinity dopamine (1-[2- [bis(4-fluorophenyl)-methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909)], [1,2]benzo(b)thiophenylcyclo-hexylpiperidine (BTCP), and serotonin (fluoxetine) uptake inhibitors produced a wash-resistant inhibition of [3H]TCP binding to site 2, but not site 1. Preincubation of guinea pig brain membranes with BTCP was shown to produce an increase in the dissociation rate of [3H]TCP from PCP site 2. Structure activity studies with various uptake inhibitors showed that GBR12909, benztropine, fluoxetine, and BTCP have higher affinity for site 2 than for site 1. (+)-MK801, ketamine, and tiletamine were very selective for site 1, whereas dexoxadrol and TCP were moderately selective for site 1. These results suggest that human cortex possesses high-affinity PCP binding sites associated with biogenic reuptake binding sites, and that guinea pig brain, but not rat brain, may be an appropriate animal model for studying PCP site 2 in human brain.     

Heterogeneity of antibodies directed against the α-bungarotoxin binding site on human acetylcholine receptor and severity of myasthenia gravis

A rapid and sensitive radioimmunological method is described, using decamethonium (DC), which revealed antibodies which blocked α-bungarotoxin (α-Bgt) binding to human acetylcholine receptor (AChR) in 98% of myasthenia gravis (MG) patients' sera tested. These sera had anti-AChR antibody titres by the conventional assay. The titre of blocking antibodies (1 to 110 nM) could be measured and was found to produce from 1 to 54% inhibition of α-Bgt binding. No relationship was found between these titres and anti-AChR antibody titres. MG sera were divided into 2 major groups on the basis of their blocking effects, with and without DC, but there was no correlation between these and the clinical status, as defined by Osserman's classification. However, no sera from asymptomatic or ocular MG patients had the dual capacities of blocking α-Bgt binding, directly and in the presence of DC.     

[H]Kainic acid binding and choline acetyltransferase activity in alzheimer''s dementia

The binding of [³H]kainic acid to caudate nucleus membranes prepared from brains of examples of Alzheimer's dimentia and controls has been determined. No changes were detected in either the affinity of the number of kainate binding sites in the Alzheimer samples compared to control, although there was a large decrease in choline acetyltransderase activity.     

Computer-assisted image analysis to quantify regional and specific receptor ligand binding: Upregulation of [H]QNB and [H]WB4101 binding in denervated hippocampus

Quantitative regional analysis of receptor autoradiographs using the Nikon Magiscan image analysis system permits resolution of regional variations in specific binding in non-homogeneous CNS structures, such as the hippocampus. Cholinergic denervation, produced by fimbrial transections, elicits a 24% increase in atropine-displaceable [³H]QNB binding in whole coronal sections of the hippocampal formation, which is greatest in the dorsal subiculum, CA3 and dentate gyrus. This lesion also elicits a 69% increase in lower affinity [³H]WB4101 binding which is displaceabte by phentolamine, but not by prazosin. This represents a sum of increases and decreases in binding in several subregions. Taken together, these findings serve to emphasize the need for normalized regional evaluation of subtracted images which have been calibrated, and linearized or transformed, to reveal binding specific to a single site.     

Correlation of C3 level with severity of generalized myasthenia gravis

Acute exacerbation of generalized myasthenia gravis (GMG) can cause swallowing impairment, respiratory failure, or death. It is important to identify immunological factors that might be regarded reliably as an index of the patient's clinical condition, response to treatment, and measure of certain immune aberrations of MG. In this study we investigated correlations between complement component C3, acetylcholine receptor antibody (AChRab) titer, and clinical severity of GMG. AChRab titer and C3 concentration were determined by radioimmunoassay and nephelometry, respectively. The clinical severity of GMG was assessed by the quantitative MG score (QMGS) according to Besinger and colleagues. Our findings indicate that the C3 level correlates with clinical severity of AChRab‐positive GMG. Muscle Nerve, 2009