Halofuginone [BSA]

Study objectives: Narcolepsy type 1 (NT1) is a chronic sleep disorder characterized by loss of hypocretin-producing neurons. Increased NT1 incidence was observed in Sweden following mass-vaccination with Pandemrix (R). Genetic association to HLA DQB1*06:02 implies an autoimmune origin, but target autoantigen remains unknown. Candidate autoantigens for NT1 have previously been identified in solid-phase immunoassays, while autoantibodies against conformation-dependent epitopes are better detected in radiobinding assays. The aims are to determine autoantibody levels against nine candidate autoantigens representing (1) proteins of the hypocretin transmitter system; Preprohypocretin (ppHypocretin), Hypocretin peptides 1 and 2 (HCRT1 and HCRT2) and Hypocretin receptor 2 (HCRTR2); (2) proteins previously associated with NT1; Tribbles homologue 2 (TRIB2), Pro-opiomelanocortin/alpha-melanocyte-stimulating-hormone (POMC/alpha-MSH) and Prostaglandin D2 Receptor DP1 (DP1); (3) proteins suggested as autoantigens for multiple sclerosis (another HLA DQB1*06:02-associated neurological disease); ATP-dependent Inwardly Rectifying Potassium Channel Kir4.1 (KIR4.1) and Calcium-activated chloride channel Anoctamin 2 (ANO2). Methods: Serum from post-Pandemrix (R) NT1 patients (n = 31) and their healthy first-degree relatives (n = 66) were tested for autoantibody levels in radiobinding assays separating autoantibody bound from free labelled antigen with Protein A-Sepharose. I-125-labelled HCRT1 and HCRT2 were commercially available while S-35-methionine-labelled ppHypocretin, HCRTR2, TRIB2, alpha-MSH/POMC, DP1, KIR4.1 or ANO2 was prepared by in vitro transcription translation of respective cDNA. In-house standards were used to express data in arbitrary Units/ml (U/ml). Results: All radiolabelled autoantigens were detected in a concentration-dependent manner by respective standard sera. Levels of autoantibodies in the NT1 patients did not differ from healthy first-degree relatives in any of the nine candidate autoantigens. Conclusions: None of the nine labelled proteins proposed to be autoantigens were detected in the radiobinding assays for conformation-dependent autoantibodies. The results emphasise the need of further studies to identify autoantigen(s) and clarify the mechanisms in Pandemrix (R)-induced NT1.

Hypocretin system is composed of hypocretins (hcrts) and their receptors (hcrtrs), which hasmultiple vital functions. Hypocretins work via hypocretin receptors and it is reported that functional differentiation occurred in hcrtrs. It is necessary to figure out the evolution process of hypocretin receptors. In our study, we adopt a comprehensive approach and various bioinformatics tools to analyse the evolution process of HCRTR gene family. It turns out that the second round of whole genome duplication in early vertebrate ancestry and the independent round in fish ancestry may contribute to the diversity of HCRTR gene family. HCRTR1 of fishes and mammals are not the same receptor, which means that there are three members in the family. HCRTR2 is proved to be the most ancient one in HCRTR gene family. After duplication events, the structure of HCRTR1 diverged from HCRTR2 owing to relaxed selective pressure. Negative selection is the predominant evolutionary force acting on the HCRTR gene family but HCRTR1 of mammals is found to be subjected to positive selection. Our study gains insight into the molecular evolution process of HCRTR gene family, which contributes to the further study of the system.