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Commercially available antibodies that bind to the human muscle acetylcholine receptor (ACHR) have been validated previously for flow cytometric use ( Keefe et al., 2009 ; Leite et al., 2008 ; Lozier et al., 2015 ). Despite a multitude of commercially available antibodies to other nicotinic ACHRs, validation in a wide variety of immunoassay formats is lacking; when studied, a large proportion of these antibodies have been deemed not fit for most research purposes ( Garg and Loring, 2017 ). We have recently described a flow cytometric immunomodulation assay for the diagnosis of Autoimmune Autonomic Ganglionopathy (AAG) ( Urriola et al., 2021 ) that utilises the monoclonal antibody mab35( Urriola et al., 2021 ) which is specific for ganglionic ACHR (gnACHR) that contain α3 subunits ( Vernino et al., 1998 ). Other fluorescent ligands for α3-gnACHR have not been validated for flow cytometric use. We investigated 7 commercially sourced antibodies and 3 synthetic fluorescent novel conotoxins purported to specifically bind to the extracellular domains of the gnACHR, and compared the results to staining by mab35, using flow cytometry with the neuroblastoma cell line IMR-32. We also evaluated the degree of non-specific binding by depleting the cell membrane of the relevant acetylcholine receptor with a pre-incubation step involving the serum from a patient with Autoimmune Autonomic Ganglionopathy containing pathogenic antibodies to the ganglionic acetylcholine receptor. None of the assessed conotoxins, and only one antibody (mab35) was found to perform adequately in flow cytometric staining of the native ganglionic acetylcholine receptor.

The autoimmune disorder of Lambert-Eaton myasthenic syndrome (LEMS) associates with small cell lung carcinoma (SCLC) in 50-60% of cases. It has been postulated that patients who harbour paraneoplastic neurological syndromes such as LEMS have an improved tumour prognosis compared to other patients with the tumour but without neurological deficit. In this intermediate report of an ongoing prospective study, 100 consecutive patients with biopsy-proven SCLC underwent full neurological examination and serum was taken for autoantibody analysis. Antibodies to voltage-gated calcium channels were detected in 10 patients, however only 4 had clinical and electrophysiological features of LEMS, 1 had limbic encephalitis, whilst the remaining 5 had no neurological signs. A further 6 patients had onconeural antibodies; only one had a paraneoplastic syndrome, sensory neuropathy. The median survival of the four antibody positive LEMS patients (19.6 months) was considerably greater than that for the antibody negative (8.9 months) or antibody positive patients as a whole (10.5 months). Although preliminary, these results suggest that functionally effective antibodies present in the sera of patients with LEMS may confer a survival advantage.

Opsoclonus-myoclonus syndrome (OMS) in children is a rare disorder including a severe eye movement disturbance, myoclonia, ataxia and often developmental retardation. Both OMS forms, idiopathic or neuroblastoma-associated (paraneoplastic), have been suspected to be autoimmune. Recently, autoantibodies have been found in OMS sera. We here show that autoantibodies in OMS, both intracellular and surface binding, belong mainly to the IgG3 subclass, although the total serum IgG3 level is normal. These results support the autoimmune hypothesis and point to a protein autoantigen as antigenic target.

Ion channels are crucial elements in neuronal signaling and synaptic transmission, and defects in their function are known to underlie rare genetic disorders, including some forms of epilepsy. A second class of channelopathies, characterized by autoantibodies against ligand- and voltage-gated ion channels, cause a variety of defects in peripheral neuromuscular and ganglionic transmission. There is also emerging evidence for autoantibody-mediated mechanisms in subgroups of patients with central nervous system disorders, particularly those involving defects in cognition or sleep and often associated with epilepsy. In all autoimmune channelopathies, the relationship between autoantibody specificity and clinical phenotype is complex. But with this new information, autoimmune channelopathies are detected and treated with increasing success, and future research promises new insights into the mechanisms of dysfunction at neuronal synapses and the determinants of clinical phenotype.

We describe a new antibody, called anti-glial nuclear antibody (AGNA), in patients with paraneoplastic neurological syndromes (PNS) and small-cell lung carcinoma (SCLC). AGNA was initially identified in 24 sera of our archives by immunohistochemistry on rat cerebellum. AGNA positive sera showed a characteristic nuclear staining of the Bergmann glia in the Purkinje cell layer. Immunoblots and probing a cerebellar expression library with AGNA sera did not identify the antigen. Twenty of the 24 patients with AGNA had PNS and all but two had lung cancer. AGNA was identified in 13/113 (11.5%) patients with SCLC compared with 0/122 with other types of cancer (p

To assess the survival impact of the presence of P/Q-type calcium channel antibodies in patients with small cell lung carcinoma (SCLC), we examined the frequency of the antibodies and Lambert-Eaton myasthenic syndrome (LEMS) in 148 consecutive patients with SCLC, and in 30 patients with paraneoplastic cerebellar degeneration and SCLC, and studied their relation with survival. In both series, only patients with LEMS had a remarkably long survival, whereas presence of the antibodies without LEMS did not result in a better prognosis.

Background CNS disorders can be caused by IgG antibodies to neuronal surface proteins, and these antibodies have the potential to cross the placenta. Since some of them target proteins involved in neurodevelopment, such as contactin-associated protein-2 (CASPR2), we hypothesised that they could alter developing neuronal circuits in utero and lead to neurodevelopmental disorders in the children. Methods A dual approach to the study was undertaken. First, we studied pregnancy serum samples from two population-based Danish cohorts: a cohort of women with postpartum psychosis and a cohort of mothers of children with mental retardation and other disorders of psychological development (MR–DPD), each with individually matched controls. We screened them for the presence of antibodies to CASPR2. Then, we assessed the effects of in-utero exposure to the antibodies in a mouse maternal-to-fetal model. Findings The combined results from these two cohorts showed that maternal antibodies to CASPR2 were associated with an increased risk of MR–DPD in the children: eight (4·4%) of 182 mothers of MR–DPD children had CASPR2 antibodies compared with only three (0·9%) of 347 mothers of children without this diagnosis (p=0·01). This association was explored by injection of mouse dams with IgG purified from CASPR2-antibody-positive patients, or from healthy controls. The mouse offspring had long-term behavioural sequelae, manifested as deficits in social interaction and social activities, and increased repetitive, non-social behaviours. When they were culled at 12 months, their brains showed abnormal migration of cortical projection neurons, increased microglial activation, and reduction in the number of glutamatergic synapses, confirming that there had been long-term changes in neurodevelopment. Interpretation CASPR2 was identified as a specific target for maternal antibodies that can alter brain development in utero, resulting in long-term behavioural deficits and permanent abnormalities at the cellular and synaptic level. These results should encourage further epidemiological and experimental studies to confirm and explore further how CASPR2 and other maternal antibodies can lead to neurodevelopmental disorders in children. Funding EC was funded by the Programme for Advanced Medical Education at the Calouste Gulbenkian Foundation. This work was also supported by the Stanley Medical Research Institute. Resources were provided by the Nuffield Department of Clinical Neurosciences.

Antibodies to neuronal antigens are found in a range of different neurological disorders, both central and peripheral, and often indicate the presence of particular underlying tumours. This conference and workshop was an opportunity to discuss the clinical associations of these antibodies, the relevance of new antibody specificities, and to establish protocols for their detection.

The Papua New Guinean small-eyed snake (Micropechis ikaheka) is recognised as a cause of life-threatening envenoming in certain parts of New Guinea. The clinical features suggest the presence of toxins acting at the neuromuscular junction and on muscle. We have used the mouse phrenic nerve hemidiaphragm preparation, a phospholipase A2 assay, and 125I-neurotoxin-binding radioimmunoassays to look for toxic activities in the crude venom and in preliminary high-performance liquid chromatography (HPLC) fractions. Micropechis ikaheka venom at 1 and 3 micrograms/ml completely abolished nerve-evoked muscle twitch within 70 min at 37 degrees C. There was also a sustained contracture of the muscle and some reduction in twitch tension evoked by direct stimulation; these were explained by the presence of phospholipase A2 activity. The venom inhibited the binding of 125I-alpha-bungaro-toxin to detergent-extracted human muscle acetylcholine receptor (AChR), and inhibited acetylcholine receptor function in a muscle cell line. It also inhibited binding of 125I-omega-conotoxin GVIA to detergent-extracted human frontal cortex voltage-gated calcium channels, but this appeared to be dependent on the phospholipase A2 activity. Identification of the main neurotoxic fractions following HPLC are shown.

The immunochemical and functional properties of IgG fractions from patients with Lambert-Eaton myasthenic syndrome (LEMS) were examined in chick and rat synaptosomes. LEMS IgG immunoprecipitated 125I-omega conotoxin GVIA (125I-omega CgTx) labeled N-type calcium channels solubilized from both tissues, and reacted with a 65 kDa protein band in immunoblots of rat synaptosomes. Depolarization-induced 45Ca2+ influx into chick synaptosomes was partially inhibited by omega CgTx, whereas influx into rat synaptosomes was insensitive to omega CgTx. No effect of LEMS sera or IgG on 45Ca2+ uptake was apparent in either preparation.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of neuromuscular transmission. Immunoglobulin G (IgG) autoantibodies act presynaptically to cause functional loss of Ca2+ channels by down-regulation. This in turn causes reduced release of acetylcholine in response to nerve stimulation (i.e. reduced quantal content). We show here that in vitro application of LEMS IgG for 24 h was as effective in causing reduced quantal content in mice as injecting animals with the antibody for 2 days. Furthermore, incubation with divalent (F(ab')2) fragments also significantly reduced quantal content. However, monovalent (Fab) fragments were without significant effect. No antibody or antibody fragment preparation affected spontaneous release. Whole LEMS IgG and F(ab')2 (but not Fab) fragments also inhibited K(+)-evoked 45Ca2+ flux in a small cell carcinoma cell line. These findings present functional evidence that LEMS IgG-induced down-regulation of nerve terminal Ca2+ channels arises due to antibody cross-linking of these channels.

Background: The N-methyl-D-aspartate receptor (NMDAR) is a type of ionotropic receptor belonging to the glutamate receptor superfamily found abundantly in the cortex, basal ganglia, and sensory pathways of the nervous system. The receptor is highly permeable to calcium ions and plays a key role in the plasticity of synapses, which is believed to underlie memory and learning. The NMDAR has been associated with a range of neurological disorders including ischemic brain damage and epilepsy, andmore speculatively with neurodegenerative disorders such as Alzheimer's and Huntington's chorea. Anti-NMDA receptor encephalitis has been recently identified as treatment responsive encephalitis associatedwith anti-NMDA receptor antibodies (Iizuka and Sakai, 2008). These antibodies bind to the NR1/NR2 heteromers of the NMDAR and antibody levels are known to correlate with the clinical severity of the disease. Clinical features seen in neuropsychiatric disorders such as Tourette's syndrome (TS) overlap with NMDAR receptor encephalitis, which might make it a part of the spectrum of anti-NMDAR disorders. Aims: Pathogenic antibodies against the NMDAR have been implicated in a range of disorders, however, the full clinical spectrum and treatment responses are still unclear. Therefore, in the present study we analysed serum samples from children diagnosed with TS for the presence of anti-NMDAR antibodies. Materials and methods: Sera from 21 TS patients with a mean age of 11 years were analysed for the presence of antibodies against the NMDAR using HEK293 cells transfected with NR1:NR2B subunits and co-transfected with enhanced green fluorescent protein (EGFP). According to the number of cells stained and intensity of staining the samples were scored between 0 and 4 (0= negative, 4= strongly positive). Results: Out of the 21 samples tested in the assay 4 were found positive for antibodies against the NMDAR. All 4 serum samples came frommale patients diagnosed with TS with an age range of 8 to 14 years. Conclusions: In the present study the presence of anti-NMDAR antibodies in the sera of 4 TS patients has been demonstrated. TS is co-morbid with multiple conditions like obsessive compulsive disorder (OCD) and attention deficit hyperactivity disorder (ADHD) so an understanding into its pathogenic mechanism would have major implications in the field of neuropsychiatry.

Background Autoantibodies to neuronal surface proteins are found in adult seizure-related disorders such as anti-N-methyl-D-aspartate receptor antibody (NMDAR-Ab) encephalitis and limbic encephalitis. We have previously shown, in new-onset childhood epilepsy, that CNS autoantibodies are also found in a proportion of these patients. We aimed to determine whether these antibodies are pathogenic and epileptogenic in vivo. Methods IgG from three patients with NMDAR-Ab encephalitis and two healthy controls was purified for passive transfer. Wireless electroencephalogram (EEG) transmitters were implanted into 15 mice. Nine of these mice were injected intracerebroventricularly with IgG from NMDAR-Ab positive patients and six mice with IgG from controls. 5 days of continuous electrophysiology recordings were used to detect spontaneous and induced epileptiform activity. A library of signature events was created to analyse the EEG recordings of all mice. Seizure susceptibility was tested 48 h after injection with a subthreshold dose of the proconvulsant pentylenetetrazole. Seizures were scored according to a modified Racine scale: 1 non-epileptic behavioural change, 2 partial clonus, 3 generalised clonus, and 4 lethal seizure. Findings Mice injected with NMDAR-Ab positive IgG had increased seizure susceptibility, with all having stage 3 seizures compared with only half of the control-IgG injected group (p=0·04). The number of stage 3 seizures was also significantly higher (mean 7·7 [SD 8·4] vs 0·8 [1·0], p=0·003), as was the total seizure score calculated at the end of 60 min of observation (38 [29·5] vs 7·5 [4·1], p=0·003). Preliminary analysis of the post-mortem tissue showed human IgG in the hippocampi of NMDAR-Ab injected but not in control-IgG injected mice. Interpretation Successful passive transfer of NMDAR-Ab positive IgG shows the epileptogenic potential of these antibodies. EEG analysis and investigation of the underlying pathogenic mechanism in ex-vivo slices is in progress. Funding Wellcome Trust.

Sample Group O D @ 6 5 0 n m The Presence of Autoantibodies to Tumour-Associated Antigens can Predate Clinical Diagnosis of Lung Cancer Caroline Chapman(1), D Isenberg(2), A Murray(3), A Thorpe(1), J McElveen(3), J Allen(3), J Robertson(1) (1) Division of Surgery, School of Clinical Sciences, University of Nottingham, Nottingham UK. (2) Centre For Rheumatology, University of London, UK. (3) Oncimmune Ltd, Nottingham City Hospital, Nottingham, UK. email: caroline.chapman@nottingham.ac.uk

The Lambert-Eaton myasthenic syndrome (LEMS) is a disorder of neuromuscular transmission, often associated with small cell lung carcinoma (SCLC), and characterized by reduced quantal release of acetylcholine from the motor nerve terminals. Another neuromuscular transmission disorder, myasthenia gravis, has a well-understood autoimmunological cause. This review discusses the evidence for a similar autoimmunological effect in the development of LEMS. Injection of LEMS IgG into mice passively transfers the physiological and morphological abnormalities, which include paucity and disorganized arrangement of active zone particles believed to represent the voltage-gated calcium channels (VGCCs). Calcium influx via VGCCs into SCLC cells is reduced by LEMS IgG suggesting that in SCLC-associated LEMS, antibodies may be triggered by VGCCs expressed on these cells; this immunological cross-reactivity may lead to the neurological abnormality. Similar VGCCs on neuronally derived cells may trigger the disorder in those without a tumour. The disorder provides another example of the complicated relationships between the nervous and immune systems and tumorigenic processes.

The myasthenic (Eaton-Lambert) syndrome, associated with carcinoma of the bronchus in one patient and with immunological disorders in two others, improved after plasma exchange--observations supported by electromyographic evidence in two cases. Prednisolone and azathioprine treatment led to almost complete remission in one of the non-neoplastic cases and to improvement in the other. The IgG fraction of plasma from all three patients, injected daily (10 mg) into mice for 37-77 days, significantly reduced the initial compound muscle action potential and the quantal content of the end-plate potential measured in the diaphragm, when compared with control human IgG. These results indicate that an IgG autoantibody, binding to nerve terminal determinants, may be responsible for the disorder of neuromuscular transmission in the myasthenic syndrome, and that immunosuppressive drugs may be useful in treating the nonneoplastic form of the disease.