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.

Introduction People with multiple sclerosis (pwMS) treated with ocrelizumab mount a T-cell response to vaccination, irrespective of humoral response. Correlates of this pattern of immune response remain unclear. Methods PwMS on ocrelizumab who were seronegative following initial COVID-19 vaccine course provided whole blood samples 2-8 weeks after vaccines 3 (n=30) and 4 (n=15). T-cell responses to SARS- CoV-2 spike protein (IFNγ release assay), anti-spike IgG, and anti-nucleocapsid IgG were measured. Clinical data including COVID-19 PCR/lateral flow test results, and infection severity were recorded. Results 26/30 (86.7%) people showed measurable T-cell responses following COVID-19 vaccine 3, 13/15 (87%) after vaccine 4. 12/30 (40.0%) had detectable anti-spike IgG following vaccine 3. Of the 15 who gave samples at both timepoints, 5/15 (33.3%) were seropositive after vaccine 3 and 4/15 (26.7%) following vaccine 4. Rates of anti-spike IgG at vaccine 3 were 6/8 (75.0%) where there was evidence of prior COVID-19 (nucleocapsid IgG, PCR, or lateral flow test), and 6/22 (27.3%) where there was no evidence of prior infection (chi-squared; p=0.0183). Four pwMS had COVID-19 infection between vaccines 3 and 4, none required hospitalisation. All had a T-cell response at vaccine 3 and two had detectable anti-spike IgG. Conclusions Many pwMS who were seronegative following the initial COVID-19 vaccine course show T- cell responses following booster vaccination. The lack of severe infections is relatively reassuring.

OBJECTIVE:Currently, 233 genetic loci are known to be associated with susceptibility to multiple sclerosis (MS). Two independent pivotal severity genome-wide association studies recently found the first genome-wide significant single-nucleotide variant (SNV; rs10191329A ) and several other suggestive loci associated with overall disability outcomes. It is now important to understand if these findings can influence individual patient management. METHODS:We assessed whether these progression SNVs are associated with detailed clinical phenotypes in a well-characterized prospective cohort of 1,455 MS patients. We used logistic regression, survival analysis, and propensity score matching to predict relevant long-term clinical outcomes. RESULTS:We were unable to detect any association between rs10191329A and a range of clinically relevant outcomes (eg, time to Expanded Disability Status Scale milestones, age-related MS severity score, anatomical localization at onset or during subsequent relapses, annualized relapse rate). In addition, an extremes of outcome case-control analysis using a propensity score matching for genotype detected no association between disease severity and rs10191329A . However, we were able to replicate the association of two suggestive SNVs (rs7289446G and rs868824C ) with the development of fixed disability, albeit with modest effect sizes, and the association of HLA-DRB1*1501 with age at onset. INTERPRETATION:Identification of rs10191329A and other suggestive SNVs are of considerable importance in understanding pathophysiological processes associated with MS severity. However, it is unlikely that individual genotyping can currently be used in a clinical setting to guide disease management. This study shows the importance of independent replication of genome-wide association studies associated with disease progression in neurodegenerative disorders. ANN NEUROL 2024;95:459-470.

As treatment options expand for all disease phases of MS, accurate epidemiological data reflecting contemporary patterns of disease by direct clinical assessment remain key to planning and delivering services. This study describes disease frequency and phenotype in a South Wales population on 01/01/2019 according to McDonald 2017 diagnostic criteria. 831 MS cases were identified in a population of 496,413. Prevalence 167/100,000 (95% CI 156–179); females 236/100,000, males 97/100,000, sex ratio 2.5:1 (F:M), mean age 52.6 (range 17–91). Age specific preva- lence was highest in females aged 50–54 (403/100,000). European population standardised prevalence 189/10,000. Disease course: 55.3% relapsing, 34.8% secondary progressive, and 9.9% primary progressive disease. 76.1% had a disability assessment and 36% an interval MR brain within 12 months of prevalence day. EDSS: <4.0 (45.3%), 4.0–5.5 (11.2%), 6.0–7.5 (31.2%), 8–9.5 (12.3%). MR brain: 73% stable; 22% active (new T2 ± Gd+); the remainder had features of MS but no relevant comparison scan. 39.6% of patients had prior DMT use of which highly effective treatments 17.3%. These data sets establish a trend of increasing prevalence but remain less than recent estimates from the MS Society (179/100,000) for Wales, and offer baseline estimates of disease course classification and activity to plan services. drhawken@outlook.com

BACKGROUND:Multiple sclerosis (MS) is an immune-mediated disease that damages myelin in the central nervous system (CNS). We investigated the profile of CCN3, a known regulator of immune function and a potential mediator of myelin regeneration, in multiple sclerosis in the context of disease state and disease-modifying treatment.METHODS:CCN3 expression was analysed in plasma, immune cells, CSF and brain tissue of MS patient groups and control subjects by ELISA, western blot, qPCR, histology and in situ hybridization.RESULTS:Plasma CCN3 levels were comparable between collective MS cohorts and controls but were significantly higher in progressive versus relapsing-remitting MS and between patients on interferon-β versus natalizumab. Higher body mass index was associated with higher CCN3 levels in controls as reported previously, but this correlation was absent in MS patients. A significant positive correlation was found between CCN3 levels in matched plasma and CSF of MS patients which was absent in a comparator group of idiopathic intracranial hypertension patients. PBMCs and CD4+ T cells significantly upregulated CCN3 mRNA in MS patients versus controls. In the CNS, CCN3 was detected in neurons, astrocytes and blood vessels. Although overall levels of area immunoreactivity were comparable between non-affected, demyelinated and remyelinated tissue, the profile of expression varied dramatically.CONCLUSIONS:This investigation provides the first comprehensive profile of CCN3 expression in MS and provides rationale to determine if CCN3 contributes to neuroimmunological functions in the CNS.

Predicting clinical outcome in multiple sclerosis (MS) remains challenging, and biomarkers capable of providing prognostic information would be valuable in shaping therapeutic decisions. Neurofilament light (NfL) measurements have shown promise in predicting clinical outcomes in MS. We evaluated the relationship between serum NfL (sNfL), measured at diagnosis in 164 people with MS, with contemporary disability, short-term (1-year) and medium-term (5-year) clinical outcomes. Analyses were adjusted for relevant confounders. sNfL concentration at diagnosis was modestly but significantly associated with baseline EDSS score (B=0.264, 95% CI 0.043 to 0.485, p=0.020). However, no significant associations were found between baseline sNfL and the incidence of relapse at 12-months, time to sustained accumulation of disability or 5-year change in EDSS. Dichotomising baseline sNfL according to previously cited cut-offs (> 14.2pg/ml and > 90th percentile for age) did not change these results. sNfL appears to be of limited clinical utility in predicting future irreversible neurological disability, in a largely treated population, and remains insufficiently validated to shape treatment decisions at the time of diagnosis. Further studies exploring the value of sequential sNfL measurement and developing valid, universally accepted cut-offs are needed before sNfL can be incorporated as a prognostic marker in the clinic.tallantyreec@cardiff.ac.uk

BackgroundIdentifying diagnostic and prognostic biomarkers in multiple sclerosis (MS) remains challenging. Cerebrospinal fluid (CSF) is a potentially valuable source.AimTest association between CSF cell counts and (i) neuroinflammatory diagnosis, (ii) early MS disease activity.MethodsCD4+/CD8+ T-cells were counted using flow cytometry in CSF collected from 162 patients. Clinical data were collected for 12–52 months. Cell counts were tested for association with: 1) diagnostic group: MS (n=63), clinically isolated syndrome (CIS, n=11), other inflammatory disorders (n=8) and non-inflammatory disorders (NID, n=75)); 2) time to next relapse/treatment commencement; 3) MS initial course. Area under receiver operator characteristic curve (AUC) was calculated for diagnostic utility.ResultsCSF CD4+ and CD8+ counts were higher in MS vs CIS (e.g. CD4+: p=0.0063, AUC=0.76) and NID (e.g. CD4+: p=1.4×10–6, AUC=0.74). NHS laboratory CSF white cell counts (WCC) were also higher in MS vs CIS (p=0.049, AUC=0.68) and NID (p=8.9×10–10, AUC=0.73). In logistic regression models of MS vs CIS in OCB negative patients, NHS WCC explained the greatest proportion of the variance (16.5%, p=0.03, AUC=0.70), but the optimal model also included CD4+ and CD8+ counts. Cell counts did not predict initial course or clinical outcomes.ConclusionsCSF cell counts could aid diagnosis of MS. CSF cell counts alone did not predict early MS disease activity in this size cohort, but prognostic utility in combination with other biomarkers should be tested in a larger cohort.

Long-term outcomes in multiple sclerosis (MS) are highly varied and treatment with disease-modifying therapies carries significant risks. Finding tissue biomarkers that can predict clinical outcomes would be valuable in individualising treatment decisions for people with MS. Several candidate biomarkers—reflecting inflammation, neurodegeneration and glial pathophysiology—show promise for predicting outcomes. However, many candidates still require validation in cohorts with long-term follow-up and evaluation for their independent contribution in predicting outcome when models are adjusted for known demographic, clinical and radiological predictors. Given the complexity of MS pathophysiology, heterogeneous panels comprising a combination of biomarkers that encompass the various aspects of neurodegenerative, glial and immune pathology seen in MS, may enhance future predictions of outcome.

Background: CD59, a broadly expressed glycosylphosphatidylinositol-anchored protein, is the principal cell inhibitor of complement membrane attack on cells. In the demyelinating disorders, multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD), elevated complement protein levels, including soluble CD59 (sCD59), were reported in cerebrospinal fluid (CSF). Objectives: We compared sCD59 levels in CSF and matched plasma in controls and patients with MS, NMOSD and clinically isolated syndrome (CIS) and investigated the source of CSF sCD59 and whether it was microparticle associated. Methods: sCD59 was quantified using enzyme-linked immunosorbent assay (ELISA; Hycult; HK374-02). Patient and control CSF was subjected to western blotting to characterise anti-CD59-reactive materials. CD59 was localised by immunostaining and in situ hybridisation. Results: CSF sCD59 levels were double those in plasma (CSF, 30.2 ng/mL; plasma, 16.3 ng/mL). Plasma but not CSF sCD59 levels differentiated MS from NMOSD, MS from CIS and NMOSD/CIS from controls. Elimination of microparticles confirmed that CSF sCD59 was not membrane anchored. Conclusion: CSF levels of sCD59 are not a biomarker of demyelinating diseases. High levels of sCD59 in CSF relative to plasma suggest an intrathecal source; CD59 expression in brain parenchyma was low, but expression was strong on choroid plexus (CP) epithelium, immediately adjacent the CSF, suggesting that this is the likely source.

To determine the optimal timing of arterial first pass computed tomography (CT) myocardial perfusion imaging (CTMPI) based on dynamic CTMPI acquisitions.Twenty-five patients (59 ± 8.4 years, 14 male)underwent adenosine-stress dynamic CTMPI on second-generation dual-source CT in shuttle mode (30 s at 100 kV and 300 mAs). Stress perfusion magnetic resonance imaging (MRI) was used as reference standard for differentiation of non-ischemic and ischemic segments. The left ventricle (LV) wall was manually segmented according to the AHA 16-segment model. Hounsfield units (HU) in myocardial segments and ascending (AA) and descending aorta (AD) were monitored over time. Time difference between peak AA and peak AD and peak myocardial enhancement was calculated, as well as the, time delay from fixed HU thresholds of 150 and 250 HU in the AA and AD to a minimal difference of 15 HU between normal and ischemic segments. Furthermore, the duration of the 15 HU difference between ischemic and non-ischemic segments was calculated.Myocardial ischemia was observed by MRI in 10 patients (56.3 ± 9.0 years; 8 male). The delay between the maximum HU in the AA and AD and maximal HU in the non-ischemic segments was 2.8 s [2.2–4.3] and 0.0 s [0.0–2.8], respectively. Differentiation between ischemic and non-ischemic myocardial segments in CT was best during a time window of 8.6 ± 3.8 s. Time delays for AA triggering were 4.5 s [2.2–5.6] and 2.2 s [0–2.8] for the 150 HU and 250 HU thresholds, respectively. While for AD triggering, time delays were 2.4 s [0.0–4.8] and 0.0 s [−2.2–2.6] for the 150 HU and 250 HU thresholds, respectively.In CTMPI, the differentiation between normal and ischemic myocardium is best accomplished during a time interval of 8.6 ± 3.8 s. This time window can be utilized by a test bolus or bolus tracking in the AA or AD using the time delays identified here.

The complement pathway has potential contributions to both white (WM) and grey matter (GM) pathology in Multiple Sclerosis (MS). A quantitative assessment of complement involvement is lacking. Here we describe the use of Tissue MicroArray (TMA) methodology in conjunction with immunohistochemistry to investigate the localization of complement pathway proteins in progressive MS cortical GM and subcortical WM. Antibodies targeting complement proteins C1q, C3b, regulatory proteins C1 inhibitor (C1INH, complement receptor 1 (CR1), clusterin, factor H (FH) and the C5a anaphylatoxin receptor (C5aR) were utilised alongside standard markers of tissue pathology. All stained slides were digitised for quantitative analysis. We found that numbers of cells immunolabelled for HLA-DR, GFAP, C5aR, C1q and C3b were increased in WM lesions (WML) and GM lesions (GML) compared to normal appearing WM (NAWM) and GM (NAGM), respectively. The complement regulators C1INH, CR1, FH and clusterin were more abundant in WM lesions, while the number of C1q+ neurons were increased and the number of C1INH+, clusterin+, FH+ and CR1+ neurons decreased in GM lesions. The number of complement component positive cells (C1q, C3b) correlated with complement regulator expression in WM, but there was no statistical association between complement activation and regulator expression in the GM. We conclude that TMA methodology and quantitative analysis provides evidence of complement dysregulation in MS GML, including an association of the numerical density of C1q+ cells with tissue lesions. Our work confirms that complement activation and dysregulation occur in all cases of progressive MS and suggest that complement may provide potential biomarkers of the disease.

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MS and NMOSD are inflammatory CNS diseases and early manifestations can be similar creating management problems, since MS drugs may be ineffective and/or worsen NMOSD. MR imaging and AQP4-Abs provide diagnostic information in most NMOSD cases, but a minority remain AQP4-Ab-negative; underlining the need for alternative diagnostic biomarkers. Complement (C) activation is a core pathological feature in both. We have investigated whether plasma C analytes can distinguish MS from NMOSD. Plasma from 53 NMOSD, 49 MS and 69 controls was tested in 2 multiplex assays: the first measuring 5 C activation products and the second comprising 5 C proteins. All activation products were significantly elevated in NMOSD compared to control or MS, particularly in AQP4-Ab-positive samples. Four C proteins (C1inh,C1s,C5,FH) were significantly higher in NMOSD (notably AQP4-Ab-positive) compared to MS or controls, whilst one (C3) was significantly lower. Receiver operating characteristic curves for each comparator identified best distinguishing analytes; a model developed from the most predictive gave an area under the curve of 0.938 for NMOSD versus controls and 0.977 for NMOSD versus MS. These data demonstrate NMOSD is characterised by significant C activation and C3 consumption, and a subset of C analytes could provide a supplementary tool for diagnosis.

Background: The symptoms of multiple sclerosis (MS) are caused by damage to myelin and nerve cells in the brain and spinal cord. Inflammation is tightly linked with neurodegeneration, and it is the accumulation of neurodegeneration that underlies increasing neurological disability in progressive MS. Determining pathological mechanisms at play in MS grey matter is therefore a key to our understanding of disease progression.Methods: We analysed complement expression and activation by immunocytochemistry and in situ hybridisation in frozen or formalin-fixed paraffin-embedded post-mortem tissue blocks from 22 progressive MS cases and made comparisons to inflammatory central nervous system disease and non-neurological disease controls.Results: Expression of the transcript for C1qA was noted in neurons and the activation fragment and opsonin C3b-labelled neurons and glia in the MS cortical and deep grey matter. The density of immunostained cells positive for the classical complement pathway protein C1q and the alternative complement pathway activation fragment Bb was significantly increased in cortical grey matter lesions in comparison to control grey matter. The number of cells immunostained for the membrane attack complex was elevated in cortical lesions, indicating complement activation to completion. The numbers of classical (C1-inhibitor) and alternative (factor H) pathway regulator-positive cells were unchanged between MS and controls, whilst complement anaphylatoxin receptor-bearing microglia in the MS cortex were found closely apposed to cortical neurons. Complement immunopositive neurons displayed an altered nuclear morphology, indicative of cell stress/damage, supporting our finding of significant neurodegeneration in cortical grey matter lesions.Conclusions: Complement is activated in the MS cortical grey matter lesions in areas of elevated numbers of complement receptor-positive microglia and suggests that complement over-activation may contribute to the worsening pathology that underlies the irreversible progression of MS.

Introduction Inflammation and complement activation are firmly implicated in the pathology of multiple sclerosis; however, the extent and nature of their involvement in specific pathological processes such as axonal damage, myelin loss and disease progression remains uncertain. This study aims to bring clarity to these questions. Results We describe a detailed immunohistochemical study to localise a strategically selected set of complement proteins, activation products and regulators in brain and spinal cord tissue of 17 patients with progressive multiple sclerosis and 16 control donors, including 9 with central nervous system disease. Active, chronic active and chronic inactive multiple sclerosis plaques (35 in total) and non-plaque areas were examined. Multiple sclerosis plaques were consistently positive for complement proteins (C3, factor B, C1q), activation products (C3b, iC3b, C4d, terminal complement complex) and regulators (factor H, C1-inhibitor, clusterin), suggesting continuing local complement synthesis, activation and regulation despite the absence of other evidence of ongoing inflammation. Complement staining was most apparent in plaque and peri-plaque but also present in normal appearing white matter and cortical areas to a greater extent than in control tissue. C1q staining was present in all plaques suggesting a dominant role for the classical pathway. Cellular staining for complement components was largely restricted to reactive astrocytes, often adjacent to clusters of microglia in close apposition to complement opsonised myelin and damaged axons. Conclusions The findings demonstrate the ubiquity of complement involvement in multiple sclerosis, suggest a pathogenic role for complement contributing to cell, axon and myelin damage and make the case for targeting complement for multiple sclerosis monitoring and therapy.

Multiple sclerosis has a variable phenotypic presentation and subsequent disease course that, although unpredictable at disease onset, is of crucial importance in guiding interventions. Effective and accessible biomarkers are required in order to stratify patients and inform treatment. We examined whether the complement regulator factor H and its Tyr402His polymorphism, recently implicated as biomarkers in other chronic inflammatory central nervous system conditions, might identify or predict specific pathological processes and outcomes in multiple sclerosis. Employing novel assays, we measured factor H and its His402 variant in serum from 350 patients with multiple sclerosis classified according to disease course and relapse status. Serum factor H levels were significantly higher in progressive disease (P < 0.001) compared to controls and relapsing patients, after controlling for variables including disease duration, age, gender, disability and treatment. Serum factor H levels were capable of distinguishing secondary progressive from relapsing remitting disease (excluding patients in clinical relapse) with a sensitivity of 89.41%, specificity of 69.47% and a positive predictive value of 72.38%. Acute relapse was also associated with transiently increased factor H levels (P = 0.009) compared to stable relapsing disease. In clinically stable patients, factor H levels remained constant over 1 year (coefficient of variation percentage = 6.8), however, in patients in transition from relapsing to progressive disease, factor H levels significantly increased over a period of 2 years (P = 0.007). Concentration of the His402 variant in heterozytgotes was significantly higher in secondary progressive (P < 0.01) and primary progressive (P < 0.05) disease, suggesting altered expression or consumption of variants when factor H is upregulated. Serum factor H may be an effective indicator of progression and a practical and accessible biomarker and stratifying tool in determining disease course, providing objective evidence to help guide therapeutic decisions.