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The discovery of disease biomarkers, along with the use of \"liquid biopsies\" as a minimally invasive source of biomarkers, continues to be of great interest. In inflammatory diseases of the central nervous system (CNS), cerebrospinal fluid (CSF) is the most obvious biofluid source. Extracellular vesicles (EVs) are also present in CSF and are thought to be potential \"biomarker treasure chests\". However, isolating these CSF-derived EVs remains challenging. This small-scale pilot study developed and tested a protocol to enrich for CSF-EVs, both in relapsing remitting multiple sclerosis (RRMS) CSF and controls. These were subsequently compared, using an aptamer based proteomics array, SOMAscan™. EVs were enriched from RRMS patient (n = 4) and non-demyelinating control (idiopathic intracranial hypertension (IIH) (n = 3)) CSF using precipitation and mini size-exclusion chromatography (SEC). EV-enriched fractions were selected using pre-defined EV characteristics, including increased levels of tetraspanins. EVs and paired CSF were analysed by SOMAscan™, providing relative abundance data for 1128 proteins. CSF-EVs were characterised, revealing exosome-like features: rich in tetraspanins CD9 and CD81, size ~100 nm, and exosome-like morphology by TEM. Sufficient quantities of, SOMAscan™ compatible, EV material was obtained from 5 ml CSF for proteomics analysis. Overall, 348 and 580 proteins were identified in CSF-EVs and CSF, respectively, of which 50 were found to be significantly (t-test) and exclusively enriched in RRMS CSF-EVs. Selected proteins, Plasma kallikrein and Apolipoprotein-E4, were further validated by western blot and appeared increased in CSF-EVs compared to CSF. Functional enrichment analysis of the 50 enriched proteins revealed strong associations with biological processes relating to MS pathology and also extracellular regions, consistent with EV enrichment. This pilot study demonstrates practicality for EV enrichment in CSF derived from patients with MS and controls, allowing detailed analysis of protein profiles that may offer opportunities to identify novel biomarkers and therapeutic approaches in CNS inflammatory diseases.

Establishing biomarkers to predict multiple sclerosis diagnosis and prognosis has been challenging using a single biomarker approach. We hypothesised that a combination of biomarkers would increase the accuracy of prediction models to differentiate multiple sclerosis from other neurological disorders and enhance prognostication for people with multiple sclerosis. We measured 24 fluid biomarkers in the blood and cerebrospinal fluid of 77 people with multiple sclerosis and 80 people with other neurological disorders, using ELISA or Single Molecule Array assays. Primary outcomes were multiple sclerosis versus any other diagnosis, time to first relapse, and time to disability milestone (Expanded Disability Status Scale 6), adjusted for age and sex. Multivariate prediction models were calculated using the area under the curve value for diagnostic prediction, and concordance statistics (the percentage of each pair of events that are correctly ordered in time for each of the Cox regression models) for prognostic predictions. Predictions using combinations of biomarkers were considerably better than single biomarker predictions. The combination of cerebrospinal fluid [chitinase-3-like-1 + TNF-receptor-1 + CD27] and serum [osteopontin + MCP-1] had an area under the curve of 0.97 for diagnosis of multiple sclerosis, compared to the best discriminative single marker in blood (osteopontin: area under the curve 0.84) and in cerebrospinal fluid (chitinase-3-like-1 area under the curve 0.84). Prediction for time to next relapse was optimal with a combination of cerebrospinal fluid[vitamin D binding protein + Factor I + C1inhibitor] + serum[Factor B + Interleukin-4 + C1inhibitor] (concordance 0.80), and time to Expanded Disability Status Scale 6 with cerebrospinal fluid [C9 + Neurofilament-light] + serum[chitinase-3-like-1 + CCL27 + vitamin D binding protein + C1inhibitor] (concordance 0.98). A combination of fluid biomarkers has a higher accuracy to differentiate multiple sclerosis from other neurological disorders and significantly improved the prediction of the development of sustained disability in multiple sclerosis. Serum models rivalled those of cerebrospinal fluid, holding promise for a non-invasive approach. The utility of our biomarker models can only be established by robust validation in different and varied cohorts.

In 2002, the UK's National Institute for Clinical Excellence (NICE) concluded that interferon beta and glatiramer acetate would be cost effective as disease-modifying therapies (DMTs) for multiple sclerosis only if the short-term disability benefits reported in clinical trials were maintained. The UK Multiple Sclerosis Risk Sharing Scheme (RSS) was established to assess whether disability progression was consistent with a cost-effectiveness target of £36 000 per quality-adjusted life-year projected over 20 years. We aimed to evaluate the long-term effectiveness and cost-effectiveness of these DMTs by comparing a cohort of patients with relapsing-remitting multiple sclerosis enrolled in the UK RSS with a natural history cohort from British Columbia, Canada. In our clinical cohort we included patients starting a DMT who were enrolled in the UK RSS who had relapsing multiple sclerosis at baseline and had at least one further clinical assessment. In our control cohort we included patients in the British Columbia multiple sclerosis database (BCMS; data collection 1980–96) who met the same eligibility criteria as for the RSS cohort. We compared disability progression at 6 years for RSS patients with untreated progression modelled from BCMS patients using continuous Markov and multilevel models. The primary outcomes were the progression ratio (treated vs untreated) measured both in Expanded Disability Status Scale (EDSS) score and utility. A ratio of less than 100% for EDSS implied slower than expected progression on treatment compared with off treatment; a utility ratio of 62% or less implied that the DMTs were cost effective. 5610 patients starting a DMT were enrolled in the UK RSS between Jan 14, 2002, and July 13, 2005 (72 sites), of whom 4137 were included in our clinical cohort. We included 898 BCMS patients in the control cohort who met the RSS inclusion criteria and had at least one EDSS score after baseline. RSS patients had a mean follow-up of 5·1 years (SD 1·4). Both models showed slower EDSS progression than predicted for untreated controls (Markov model, 75·8% [95% CI 71·4–80·2]; multilevel model, 60·0% [56·6–63·4]). Utility ratios were consistent with cost-effectiveness (Markov model, 58·5% [95% CI 54·2–62·8]; multilevel model, 57·1% [53·0–61·2]). Findings from this large observational study of treatment with interferon beta or glatiramer acetate provide evidence that their effects on disability in patients with relapsing-remitting multiple sclerosis are maintained and cost effective over 6 years. Similar modelling approaches could be applied to other chronic diseases for which long-term controlled trials are not feasible. Health Departments of England, Wales, Scotland, and Northern Ireland, Biogen Idec, Merck Serono, Bayer Schering Pharmaceuticals, Teva Pharmaceuticals Industries, UK National Institute of Health Research's Health Technology Assessment Programme.

The first of several phase 3 trials examining efficacy in relapsing MS has not been able to demonstrate a significant benefit and has also raised important safety concerns. More results are on their way and it will be important to understand whether the safety signals identified are drug- or class-specific and whether other BTKi also fail to reach their endpoints for relapsing MS. However, as reported in preliminary data for another BTKi, it may be that they will have more of a role in progressive disease as hinted by the unraveling of relevant molecular mechanisms and pathways.

BackgroundRelatively few studies have searched for potentially pathogenic antibodies in non-paraneoplastic patients with cerebellar ataxia.Methods and ResultsWe first screened sera from 52 idiopathic ataxia patients for binding of serum IgG antibodies to cerebellar neurons. One strong-binding serum was selected for immunoprecipitation and mass spectrometry, which resulted in the identification of contactin-associated protein 2 (CASPR2) as a major antigen. CASPR2 antibodies were then found by a cell-based assay in 9/88 (10%) ataxia patients, compared to 3/144 (2%) multiple sclerosis or dementia controls (p=0.011). CASPR2 is strongly expressed in the cerebellum, only partly in association with voltage-gated potassium channels.ConclusionsProspective studies are now needed to see whether identification of CASPR2 antibodies has relevance for the diagnosis and treatment of idiopathic cerebellar ataxia.