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Objective The objective of this study was to evaluate patients with ganglionic acetylcholine receptor antibody (gAChR-Ab) positive autoimmune autonomic ganglionopathy using a multimodal testing protocol to characterize their full clinical phenotype and explore biomarkers to quantify immunotherapy response. Methods We conducted a cohort study of 13 individuals (7 women, 21-69 years of age) with autonomic failure and gAChR-Ab >100 pM identified between 2005 and 2019. From 2018, all patients were longitudinally assessed with cardiovascular, pupillary, urinary, sudomotor, lacrimal and salivary testing, and Composite Autonomic Symptom Score (COMPASS-31) autonomic symptom questionnaires. The orthostatic intolerance ratio was calculated by dividing change in systolic blood pressure over time tolerated on head-up tilt. Eleven patients received immunotherapy. Results At first assessment, all 13 patients had cardiovascular and pupillary impairments, 7 of 8 had postganglionic sudomotor dysfunction, 9 of 11 had urinary retention and xeropthalmia, and 6 of 8 had xerostomia. After immunotherapy, there were significant improvements in orthostatic intolerance ratio (33.3 [17.8-61.3] to 5.2 [1.4-8.2], p = 0.007), heart rate response to deep breathing (1.5 [0.0-3.3] to 4.5 [3.0-6.3], p = 0.02), pupillary constriction to light (12.0 [5.5-18.0] to 19.0 [10.6-23.8]%, p = 0.02), saliva production (0.01 [0.01-0.05] to 0.08 [0.02-0.20] g/min, p = 0.03), and COMPASS-31 scores (52 to 17, p = 0.03). Orthostatic intolerance ratio correlated with autonomic symptoms at baseline (r = 0.841, p = 0.01) and following immunotherapy (r = 0.889, p = 0.02). Immunofluorescence analyses of skin samples from a patient 32 years after disease onset showed loss of nerve fibers supplying the dermal autonomic adnexa and epidermis, with clear improvements following immunotherapy. Interpretation Patients with autoimmune autonomic ganglionopathy demonstrated objective evidence of widespread sympathetic and parasympathetic autonomic failure, with significant improvements after immunotherapy. Quantitative autonomic biomarkers should be used to define initial deficits, guide therapeutic decisions, and document treatment response. ANN NEUROL 2021;89:753-768.

Background and aims: Acquired Neuromyotonia (NMT) is an autoimmune condition frequently associated with anti-contactin-associated-protein-like-2 (Caspr2) antibodies. NMT can occur as a paraneoplastic disorder in patients with thymoma, alone or in combination with Myasthenia Gravis (MG). Recently, antibodies against netrin-1-receptors (DCC and UNC5A) have been reported as predictors of thymoma in 6/9 patients with concomitant NMT and MG. We aimed to clinically characterize a large cohort of patients with thymoma-associated MG, and to explore serological correlation of NMT symptoms. Methods: 268 consecutive patients with thymoma-associated MG were retrospectively collected. NMT was defined as muscle twitching/cramps in at least 2 skeletal districts. Patients with NMT(23) were screened for anti-neuronal antibodies by immunohistochemistry on rat brain and cell based assay. Results: 23/268 patients developed NMT symptoms (muscle twitching, 3; cramps, 3, or both, 17). Overall, 33/268 patients with thymoma had a tumor recurrence, which was more frequent in those with (8/23) vs those without NMT (25/245, p=0.003). NMT onset preceded the tumor recurrence in 5/6 patients. In univariate analysis predictors of thymoma recurrence were younger age at thymectomy (odds ratio-OR:0.95, confidence interval-CI:0.93-0.97), Masaoka staging (OR:10.73, CI:2.38-48.36) and NMT (OR:4.69, CI:1.76-12.46). 6 patients with NMT had anti-neuronal antibodies (patient#1: Caspr2; patient#2: AMPAR; patient#3: DCC; patient#4: LGI1; patient#5: Caspr2+LGI1+DCC+UNC5A; patient#6: Caspr2+LGI1+DCC). Thymoma recurrence was found less frequently in negative (3/17) vs positive patients with NMT (4/6, #1, #2, #5 and #6; p=0.045). Conclusion: The occurrence of NMT symptoms in patients with thymoma-associated MG can predict tumor recurrence, and warrants a closer oncologic follow-up. Anti-neuronal surface autoantibodies may be useful to further stratify the recurrence risk. Disclosure: This work was funded by the ‘Ricerca finalizzata ministeriale 2015-2017’ provided by the Italian ministry of health

Summary Purpose Autoantibodies to specific neurologic proteins are associated with subacute onset encephalopathies, which often present with seizures that are poorly controlled by conventional antiepileptic drugs (AEDs). Previous cross-sectional studies have found specific neurologic antibodies in a small proportion of people with established epilepsy, but these investigations have seldom included patients with recent diagnosis. Methods We screened two large epilepsy cohorts to investigate the prevalence of multiple autoantibodies in adult patients with either established or newly diagnosed, untreated epilepsy. Key Findings Eleven percent of patients had antibodies to one or more antigen: voltage-gated potassium channel (VGKC) complex proteins (5%), glycine receptors (3%), and glutamic acid decarboxylase (GAD) and N-methyl-d-aspartate (NMDA) receptors (1.7% each). There was no difference in the prevalence of antibodies, individually or collectively, between patients with established and newly diagnosed epilepsy or with generalized or focal epilepsy. There was, however, a significantly higher prevalence of positive antibody titers in patients with focal epilepsy of unknown cause than in those with structural/metabolic focal epilepsy (14.8% vs. 6.3%; p

Autoantibodies to surface proteins that influence neuronal excitability are increasingly found in different forms of epilepsy or encephalitis in adults, and are also beginning to be identified in children. The conditions are often refractory to traditional antiepileptic drugs. Detection of these antibodies can help to identify forms of epilepsy that may respond to immunotherapies.

Background: GQ1b antibody (GQ1b-Ab) is detected in approximately two-thirds of sera of patients with Bickerstaff’s encephalitis (BE). Whilst some of the remaining patients have antibodies to other gangliosides, many patients with BE are reported to be seronegative. Methods and Results: Voltage-gated potassium channel antibody (VGKC-Ab) at high titer was detected during the diagnostic work-up of one patient with BE. Sera of an additional patient with BE and nine patients with Miller Fisher syndrome (MF) (all GQ1b-Ab positive) were investigated for VGKC-Ab and other anti-neuronal antibodies by radioimmunoprecipitation using 125I-dendrotoxin-VGKC and immunohistochemistry, respectively. Two patients with MF exhibited moderate titer VGKC-Abs. Regardless of positivity for VGKC or GQ1b antibodies, serum IgG of all patients with BE and MF reacted with the molecular layer and Purkinje cells of the cerebellum in a distinctive pattern. Conclusion: Voltage-gated potassium channel antibodies might be involved in some cases of BE or MF. The common staining pattern despite different antibody results suggests that there might be other, as yet unidentified, antibodies associated with BE and MF.

Background Treatment-resistant complex partial seizures (CPS) with orofacial involvement recently were reported in cats in association with hippocampal pathology. The features had some similarity to those described in humans with limbic encephalitis and voltage-gated potassium channel (VGKC) complex antibody. Hypothesis/Objectives The purpose of this pilot study was to evaluate cats with CPS and orofacial involvement for the presence of VGKC-complex antibody. Animals Client-owned cats with acute orofacial CPS and control cats were investigated. Methods Prospective study. Serum was collected from 14 cats in the acute stage of the disease and compared with 19 controls. VGKC-complex antibodies were determined by routine immunoprecipitation and by binding to leucine-rich glioma inactivated 1 (LGI1) and contactin-associated protein-like 2 (CASPR2), the 2 main targets of VGKC-complex antibodies in humans. Results Five of the 14 affected cats, but none of the 19 controls, had VGKC-complex antibody concentrations above the cut-off concentration (>100 pmol/L) based on control samples and similar to those found in humans. Antibodies in 4 cats were directed against LGI1, and none were directed against CASPR2. Follow-up sera were available for 5 cats in remission and all antibody concentrations were within the reference range. Conclusion and Clinical Importance Our study suggests that an autoimmune limbic encephalitis exists in cats and that VGKC-complex/LGI1 antibodies may play a role in this disorder, as they are thought to in humans.

Acetylcholine receptor (AChR) antibodies are present in around 85% of patients with myasthenia gravis (MG) as measured by the conventional radioimmunoprecipitation assay. Antibodies that block the fetal form of the AChR are occasionally present in mothers who develop MG after pregnancy, especially in those whose babies are born with arthrogryposis multiplex congenita. The antibodies cross the placenta and block neuromuscular transmission, leading to joint deformities and often stillbirth. In these mothers, antibodies made in the thymus are mainly specific for fetal AChR and show restricted germline origins, suggesting a highly mutated clonal response; subsequent spread to involve adult AChR could explain development of maternal MG in those cases who first present after pregnancy. In the 15% of "seronegative" MG patients without AChR antibodies (SNMG), there are serum factors that increase AChR phosphorylation and reduce AChR function, probably acting via a different membrane receptor. These factors are not IgG and could be IgM or even non-Ig serum proteins. In a proportion of SNMG patients, however, IgG antibodies to the muscle-specific kinase, MuSK, are present. These antibodies are not found in AChR antibody-positive MG and are predominantly IgG4. MuSK antibody positivity appears to be associated with more severe bulbar disease that can be difficult to treat effectively.

In Lambert-Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N-type (125I-omega conotoxin GVIA) and L-type ([3H]PN200-110) calcium channels. Sera with a high antibody titer (> 3 nM) against rat brain N-type channels contained autoantibodies that immunoprecipitated neuronal and muscle L-type channels. These IgG fractions stained a 55-kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the beta subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65-kDa protein that is unrelated to the beta subunit and displays properties similar to those of synaptotagmin.

In the Lambert-Eaton myasthenic syndrome (LEMS), immunoglobulin G (IgG) autoantibodies to presynaptic voltage-gated calcium channels (VGCCs) at the neuromuscular junction lead to a reduction in nerve-evoked release of neurotransmitter and muscle weakness. We have examined the action of LEMS IgGs on cloned human VGCCs stably expressed in transfected human embryonic kidney (HEK293) cell lines: 10-13 (alpha(1A-2), alpha(2b)delta, beta(4a)) and C2D7 (alpha(1B-1), alpha(2b)delta, beta(1b)). All LEMS IgGs studied showed surface binding to [(125)I]-omega-CTx-MVIIC-labeled VGCCs in the alpha(1A) cell line and two of six IgGs showed surface binding to [(125)I]-omega-CTx-GVIA-labeled VGCCs in the alpha(1B) cell line. We next studied the effect of LEMS IgGs (2 mg/ml) on whole-cell calcium currents in the alpha(1A) and alpha(1B) cell lines. Overnight treatment of alpha(1A) (10-13) cells with LEMS IgGs led to a significant reduction in peak current density without alteration of the current-voltage relationship or the voltage dependence of steady-state inactivation. In contrast, LEMS IgGs did not reduce peak current density in the alpha(1B) cell line. Overall these data demonstrate the specificity of LEMS IgGs for the alpha(1A) cell line and suggest that LEMS IgGs bind to and downregulate VGCCs in this cell line. Although several LEMS IgGs can be shown to bind to the alpha(1B) (C2D7) cell line, no functional effects were seen on this channel.

The neuromuscular junction is the target of a variety of autoimmune, neurotoxic and genetic disorders, most of which result in muscle weakness. Most of the diseases, and many neurotoxins, target the ion channels that are essential for neuromuscular transmission. Myasthenia gravis is an acquired autoimmune disease caused in the majority of patients by antibodies to the acetylcholine receptor, a ligand-gated ion channel. The antibodies lead to loss of acetylcholine receptor, reduced efficiency of neuromuscular transmission and muscle weakness and fatigue. Placental transfer of these antibodies in women with myasthenia can cause fetal or neonatal weakness and occasionally severe deformities. Lambert Eaton myasthenic syndrome and acquired neuromyotonia are caused by antibodies to voltage-gated calcium or potassium channels, respectively. In the rare acquired neuromyotonia, reduced repolarization of the nerve terminal leads to spontaneous and repetitive muscle activity. In each of these disorders, the antibodies are detected by immunoprecipitation of the relevant ion channel labelled with radioactive neurotoxins. Genetic disorders of neuromuscular transmission are due mainly to mutations in the genes for the acetylcholine receptor. These conditions show recessive or dominant inheritance and result in either loss of receptors or altered kinetics of acetylcholine receptor channel properties. Study of these conditions has greatly increased our understanding of synaptic function and of disease aetiology.

Antibodies to acetylcholine receptor (AChR) are found in 85% of patients with myasthenia gravis (seropositive MG [SPMG]) and are thought to be pathogenic; but in 15% of MG patients, the standard immunoprecipitation test for anti-AChR is negative (seronegative MG [SNMG]). Here, we used a novel approach, fluorescence-activated cell sorting analysis, to measure binding of SPMG and SNMG IgG antibodies to rhabdomyosarcoma cell lines that express human adult (TE671-e) or fetal (TE671-γ) AChR, and to human embryonic kidney (HEK) fibroblasts that express adult human AChR (HEK-AChR). We found that whereas most SPMG antibodies bind to all three cell lines, IgG from 8 of 15 SNMG sera/plasmas bind to the surface of both TE671 cell lines but not to HEK-AChR cells. These results indicate that SNMG antibodies bind to a muscle surface antigen that is not the AChR, which strongly supports previous studies that suggest that SNMG should be considered a distinct subtype of MG. Ann Neurol 2000;47:504–510.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder in which antibodies to presynaptic calcium channels at the neuromuscular junction lead to muscle weakness. Around 60% of patients with LEMS have an associated small cell lung carcinoma (SCLC), which is known to express voltage-gated calcium channels (VGCCs). Immunoglobulin (IgG) from patients with LEMS causes inhibition of calcium flux in an SCLC cell line.1 This inhibition has been shown to correlate with disease severity.1 Neuronal VGCCs can be classified into P-, Q-, N-, L-, and R-type according to their electrophysiological and pharmacological properties. Around 92% of LEMS patients have antibodies that immunoprecipitate P/Q-type (125I-CTx-MVIIC labeled) VGCCs and around 30% have antibodies to N-type (125I-CTx-GVIA labeled) VGCCs.2 In addition, antibodies from LEMS patients have been shown to abolish the component of neurotransmitter release subserved by both P-type and Q-type VGCCs in sympathetic and parasympathetic neurons of mice injected with LEMS IgG.3 Neuronal VGCCs consist of an 1 subunit with and 2 subunits. Several of the human genes encoding the different types of VGCC have now been cloned and sequenced, including the 1A, 1B, 1D, 1E, 1–4, and 2 subunits. Human embryonic

Background Autoantibodies directed against specific neuronal antigens are found in a significant number of patients with gastrointestinal neuromuscular diseases (GINMDs) secondary to neoplasia. This study examined the presence of antineuronal antibodies in idiopathic GINMD and GINMD secondary to South American Trypanosomiasis. The GI distribution of voltage-gated potassium channels (VGKCs) was also investigated. Methods Seventy-three patients were included in the study with diagnoses of primary achalasia, enteric dysmotility, chronic intestinal pseudo-obstruction, esophageal or colonic dysmotility secondary to Chagas’ disease. Sera were screened for specific antibodies to glutamic acid decarboxylase, voltage-gated calcium channels (VGCCs; P/Q subtype), nicotinic acetylcholine receptors (nAChRs; α3 subtype), and voltage-gated potassium channels (VGKCs, KV1 subtype) using validated immunoprecipitation assays. The distribution of six VGKC subunits (KV1.1–1.6), including those known to be antigenic targets of anti-VGKC antibodies was immunohistochemically investigated in all main human GI tract regions. Key Results Three patients (14%) with chagasic GI dysmotility were found to have positive anti-VGKC antibody titers. No antibodies were detected in patients with idiopathic GINMD. The VGKCs were found in enteric neurons at every level of the gut in unique yet overlapping distributions. The VGKC expression in GI smooth muscle was found to be limited to the esophagus. Conclusions & Inferences A small proportion of patients with GI dysfunction secondary to Chagas’ disease have antibodies against VGKCs. The presence of these channels in the human enteric nervous system may have pathological relevance to the growing number of GINMDs with which anti-VGKC antibodies have been associated.

In the freeze-fractured presynaptic membrane of the motor end-plate, the active zones consist of two parallel arrays and each array contains 10- to 12-nm particles arranged in two rows. In the Lambert-Eaton myasthenic syndrome (LEMS) and in mice treated with 10 mg/day of LEMS IgG, administered intraperitoneally for several weeks, there was a paucity and disorganization of the active zones, and clusters of 10- to 12-nm particles appeared. To further define the changes in the active zones, mice were studied that had been treated over 2 days with 104 to 180 mg of IgG. Treatment transferred the physiological defect of LEMS. Control mice received normal human IgG or no IgG. The spacing and density (number/unit area) of active-zone particles were evaluated in presynaptic membrane P-faces using computer-assisted stereometry. In the normal active zone, the distance between particles in a given row and between adjacent rows of an array was less than, but the distance between the two arrays was greater than, the distance between the two antigen-binding sites on human IgG. In mice treated with LEMS IgG, the initial alteration in the active zone was a Decemberrease in the distance between particles in a given row and between adjacent rows of an array; the distance between the two arrays remained unaltered. In more affected active zones, the parallel orientation of the rows was disturbed and the arrays became clusters. There was a significantly reduced density of active zones and of large-membrane particles associated with all active zones and clusters. The findings are consistent with the idea that LEMS IgG binds to the active-zone particles, cross-links these particles, and then modulates their density. This interpretation is in agreement with findings of the companion paper which follows in this issue of the Annals in which we immunolocalized LEMS IgG to the active zones at the motor end-plates.

1. The action of immunoglobulin G obtained from patients with Lambert-Eaton myasthenic syndrome (LEMS IgG) was investigated by injecting mice, followed by intracellular recordings from the mouse diaphragm. 2. End-plate potential quantal content was studied over a range of Ca2+ concentrations. Curves of log quantal content versus log Ca2+ concentration were shifted to the right by LEMS IgG. For low Ca2+ concentrations, release continued to follow Poisson statistics after LEMS IgG treatment. 3. Miniature end-plate potential (m.e.p.p.) frequency was measured in solutions containing high K+ concentrations. LEMS IgG significantly reduced m.e.p.p. frequency at each K+ concentration studied. 4. M.e.p.p. frequency was measured at fixed high-K+ concentration (15.9 mM) for a range of Ca2+ concentrations. The log-log plot of m.e.p.p. frequency versus Ca2+ concentration was shifted downwards throughout by LEMS IgG. 5. M.e.p.p. frequency was not affected by LEMS IgG in Ca2+-free solutions (K+ concentration 15.9 mM) or in solutions of low Ca2+ concentration (K+ concentration 5.9 mM). 6. At each Ca2+ concentration studied, m.e.p.p. amplitudes were not affected by LEMS IgG. 7. The data suggest that LEMS IgG acts on presynaptic voltage-dependent Ca2+ channels to cause their loss of function, probably by down-regulation.