The blots were scanned and densitometric analysis performed of the 50 kDa IgG heavy chain; IgG levels were calculated by comparison with a human IgG heavy chain standard curve. Antibody assays to detect specific neuronal surface antibodies Cell-based assays (eg.[20]) were used to measure antibodies to the VGKC-complex components, contactin2, CASPR2 (sera diluted 1:100) and LGI1, NMDAR, Rabbit Polyclonal to c-Met (phospho-Tyr1003) GABAA3 and dopamine (D2R) receptors (sera diluted 1:20). associated with encephalopathies that include disordered sleep, to rodent brain tissue including the lateral hypothalamus, and to live hippocampal neurons in culture. When sufficient sample was available, CSF levels of melanin-concentrating hormone (MCH) were measured. Sera from 44 H1N1-ASO3-vaccinated children without narcolepsy were also examined. None of these patients CSFs or sera was positive for NMDAR or CASPR2 antibodies or binding to neurons; 4/13 sera bound to orexin-neurons in rat brain tissue, but also to other neurons. MCH levels were a marginally raised (n = 8; p = 0.054) in orexin-deficient narcolepsy patients compared with orexin-normal children (n = 6). In the 44 H1N1-AS03-vaccinated healthy children, there was no rise in total IgG levels or in CASPR2 or NMDAR antibodies three weeks following vaccination. In conclusion, there were no narcolepsy-specific autoantibodies identified in type 1 narcolepsy sera or CSFs, and no evidence for a general increase in immune reactivity following H1N1-AS03 vaccination in the healthy children. Antibodies to other neuronal specific membrane targets, with their potential for directing use of immunotherapies, are still an important goal for future research. Introduction Narcolepsy is usually a lifelong and disabling condition, first described over 130 years ago [1]. It is characterised by dysregulation of the sleep-wake cycle with inappropriate penetration of rapid eye 7-Chlorokynurenic acid sodium salt movement (REM) sleep, and cataplexy, a sudden loss of motor tone brought on by emotion. Type 1 narcolepsy[2] is usually associated with a selective loss of neurons secreting neuropeptides orexin A and B, also called hypocretins 1 and 2 [3]. The disease is usually diagnosed from the history of severe sleepiness, in addition to the co-existence of cataplexy, and a positive multiple sleep latency test (MSLT), or very low or absent CSF orexin [4]. A tightly-linked HLA association is usually well established [5]. Twin discordance,[6] and the association of onset with streptococcal contamination [7] and pandemic flu immunisation [8] suggest an immunological trigger. In addition to the DQB1*06:02 association, [9] genome-wide association studies found polymorphisms in the T cell receptor alpha [10] as well as other immunity-linked genes. Antibodies to various CNS proteins, or candidate antigens, have been identified in narcolepsy using a variety of approaches, but none have yet led to development of disease-specific antibody assessments (reviewed in [11C13]), and immunotherapies have produced variable results (reviewed in [13]). Sleep disturbance is usually a common features of autoimmune forms of encephalitis associated with antibodies to the voltage-gated potassium channel (VGKC) complex proteins, contactin-associated protein 2 (CASPR2), or leucine- rich glioma-inactivated 1 (LGI1), or to N-methyl-D-aspartate receptor (NMDAR) which are all proteins uncovered on the surface of live neurons (reviewed in [14]). The recent recognition of children with narcolepsy and cataplexy, both presenting within months of Pandemrix vaccination (AS-03 adjuvanted) against the Pandemic 2009 H1N1 Swine Flu (H1N1-AS03), especially in northern European countries, [8, 15C18], provided an opportunity to look for potentially pathogenic antibodies in sera and CSFs close to onset. In addition, sera from healthy children before and after H1N1-AS03 vaccination [19] were examined for changes in total IgG and for CASPR2 and NMDAR antibodies. Methods Ethics statement The sera and CSFs from narcolepsy patients were obtained with ethical permission and written informed consent from subjects or their parents with approval of the coordinating 7-Chlorokynurenic acid sodium salt Ethical Boards of the University Hospital of Gothenburg and the Hospital District of Helsinki and Uusimaa. The study of H1N1 vaccination sera [19] was approved by the Oxfordshire Research Ethics Committee A (No 09/H0604/107), 7-Chlorokynurenic acid sodium salt and the UK Medicines and Healthcare Products Regulatory Agency (EUDRACT 2009-014719-11). All animal procedures were carried out in accordance with the UK Home Office guidelines under a project license granted by the Home Office to AV (Immunity in Neurological and Developmental Disorders, PPL No 30/1890). The adult rats were PBS-perfused under terminal barbiturate anaesthesia, and the newborn pups sacrificed by decapitation (Schedule 1). Compliance to 7-Chlorokynurenic acid sodium salt rules and regulations and adherence to the 3Rs principles was monitored by Biomedical Services, University of Oxford and Home Office inspectors. Post H1N1-AS03 vaccination narcolepsy samples The initial cohort was.