Is Diffusion Tensor Imaging sensitive to identify clinical differences between early RRMS and Healthy Controls?

BACKGROUND
MRI is the most common technique to monitor MS patients,but T2-FLAIR intensities only poorly explain some of the clinical symptoms observed. Advanced MRI imaging techniques, diffusion tensor imaging (DTI),could potentially be used to describe some of the deficits by studying water diffusion in disrupted tracts.

OBJECTIVES
To compare DTI parameters (Fractional Anisotropy (FA), Mean Diffusivity (MD), Axial Diffusivity (AD) and Radial Diffusivity (RD)) in relapsing-remitting MS (RRMS) patients with age- and sex-matched healthy controls (HCs), and to correlate these with clinical symptoms and brain volumetric measures.

METHODS
MRI protocols included a 3D T1-MPRAGE, 3D T2-FLAIR and a DTI sequence from RRMS patients(N=37) and HCs(N=20). T-test was applied for independent sample analyses to investigate the significant difference of DTI parameters between RRMS and HCs groups. The correlation between DTI parameters and clinical symptoms was performed using the correlation coefficient(r). Total brain, white matter (WM), grey matter (GM) and ventricular CSF volumes were calculated with FSL-FAST software.

RESULTS 
There was a statistically significant increase in MD (3.6%), RD(4.5%) and AD(2.7%) and a significant decrease in FA (-4.3%) for total brain-WM in RRMS patients compared to HCs (p<0.01). There was also an even greater increase in MD, RD and AD by 6.4%, 7.4% and 7.5% respectively for the WM-lesion s(p<0.05). FA correlated positively with Memory (r=0.43) in total brain-WM and negatively with anxiety(r=-0.42) and depression(r=-0.35) in WM-lesions.

CONCLUSION
Quantitative DTI parameters not only differentiate between RRMS patients and HCs, but are also associated with disability and mental health of  RRMS patients.

Transcriptional regulation of the multiple sclerosis risk gene MERTK in myeloid cells

BACKGROUND
The MERTK gene encodes a receptor tyrosine kinase that is strongly expressed by myeloid cells and is critical for resolving inflammatory outcomes in the CNS. Genetic association studies of MERTK identified rs7422195 as a candidate polymorphism associated with altered gene expression and susceptibility to MS.

OBJECTIVE
This study aimed to define genetic mechanisms that regulate myeloid specific expression of MERTK and contribute to MS pathology. This included identifying a causal mechanism at the established MS risk variant rs7422195 and defining the previously uncharacterised promoter region of the human MERTK gene.  

METHODS
In vitro DNA-protein pulldown experiments were undertaken to underpin a regulatory mechanism at the MS risk variant. DNA-protein complexes were analysed by mass spectrometry to identify candidate binding proteins and validated in vivo with chromatin immunoprecipitation. To characterise the MERTK promoter, the 5’ upstream region of the gene was cloned and assessed for transcriptional activity. Sub-cloning and in silico analysis were undertaken to comprehensively characterise identified promoter elements.

RESULTS
We identified several proteins that bound to the MERTK risk variant in vitro. We confirmed that at least one of these candidates binds to the variant in primary human monocytes. We observed both proximal and distal promoter regions upstream of MERTK that strongly induced transcriptional activity. Further characterisation of the distal promoter revealed several myeloid specific transcriptional elements that may define myeloid expression of MERTK. 

CONCLUSIONS
Genetic and functional characterisation of the MERTK promoter and candidate susceptibility variant rs7422195 furthers our knowledge of its regulation in myeloid cells and provide new targets for therapeutic intervention of gene expression in inflammation and disease.

This study was funded by MSRA and NMSS.

The interaction Multiple Sclerosis risk loci with Epstein-Barr virus phenotypes indicates new therapeutic targets

BACKGROUND
Infection with Epstein-Barr virus (EBV) appears to be necessary for the development of multiple sclerosis (MS), but the mechanism for this remains unknown.

OBJECTIVE
To identify interaction between MS risk loci and EBV phenotypes in infected B cells.
 

METHODS
We tested the association of risk alleles with expression of EBV genes, intrinsic growth rate and EBV DNA copy number in EBV infected B cell lines.  We also tested the association of MS risk gene expression with these EBV molecular traits.

RESULTS
The EBV DNA copy number was associated with significantly more risk alleles for MS than for other diseases or traits. An EBV miRNA was highly associated with this EBV phenotype, and with MS risk genes and alleles. One MS risk gene was associated with all EBV phenotypes. This gene indicates Natural Killer cell control of EBV likely affects MS risk.

CONCLUSION
The interaction between MS risk loci and EBV phenotypes indicate targeting molecules controlling EBV DNA copy number, such as particular EBV miRNAs, may provide therapeutic benefit in MS. Augmenting Natural Killer cell control of infected B cells may also be effective.

Untangling the role of neutrophil extracellular traps in multiple sclerosis

BACKGROUND
Neutrophil granulocytes are the most abundant leukocyte in human blood, however their role in multiple sclerosis is still not well understood. Activated neutrophils can extrude their DNA as highly cytotoxic neutrophil extracellular traps (NETs) whose abundance correlates with disease activity in multiple sclerosis (MS).

OBJECTIVE
Here we investigate a first in class, cheap to produce and well tolerated inhibitor of NETs and its potential effects on MS using the mouse model, experimental autoimmune encephalomyelitis (EAE).

METHODS
Mice subjected to EAE were treated with our NET inhibitors (targeting NET cytotoxicity) and peripheral immune responses and disease severity over a course of 30 days were monitored.

At the cellular level, effects of NETs were tested in vitro on key cell populations related to EAE pathology. In particular, CD4 T cell survival, activation and differentiation into encephalitogenic subsets was assessed using cells isolated from mice and human PBMCs.

RESULTS
NET inhibition significantly decreased disease severity over the entire disease course of EAE.

Treatment of CD4 T cells with NETs promoted T cell activation and differentiation of T cells into the pathogenic Th17 cell subset through induction of the Th17 cell fate program. This effect was blocked using our NET inhibitors.

CONCLUSION
Our results suggest a beneficial effect of NET inhibitors on EAE severity partially through preventing NET induced exacerbation of encephalitogenic Th17 cells supporting its further investigation as a novel treatment in MS.

FUNDING ACKNOWLEDGEMENT
MS Research Australia Project grant

Multiple large omics data implicate risk genes within multiple sclerosis susceptibly loci

BACKGROUND
Despite the success of genome-wide association studies (GWAS) in identifying over 200 susceptibility loci for multiple sclerosis (MS), the potential causal genes and the functional mechanisms of these risk loci remained largely unexplained.

OBJECTIVE
To identify genes and infer potential biological mechanisms underlying MS risk loci using multiple large omics data.

METHODS
Our approach used summary data based Mendelian randomisation, and heterogeneity in dependent instruments tests, to evaluate whether the pleiotropic associations across different omics layers are due to single shared genetic variants. We combined several large, independent, publicly available datasets, including DNA methylation (N=1,980) and gene expression (N=2,765) from whole blood, and MS GWAS (14,802 MS and 26,703 controls). We identified 4 independent genomic loci (tagged by 13 DNA methylation probes for 4 genes: TNFSF14, TBKBP1, BANF1 and ARL6IP4) with consistent genome-wide significant pleiotropic associations across different omics layers (DNA methylation, gene transcription level and MS risk). Within each independent locus, the multiple associations are due to the presence of a single, shared genetic variant. All four genes play roles related to viral infection that may contribute to MS development.

RESULTS
Conclusions We provide in-silico evidence for a link between 4 specific genetic variants and the methylation state and transcription level at 4 nearby genes. This work highlights a potential biological mechanism underlying the complex susceptibility to MS.

Feasibility of hybrid functional electrical stimulation training in persons with advanced multiple sclerosis

BACKGROUND
Chronic demyelination and failed myelin repair contribute to progressive clinical disability in multiple sclerosis (MS) patients. However, this aspect of MS pathobiology is not accurately recapitulated in current animal models. To determine if we could model remyelination failure in the mouse we modified the standard cuprizone paradigm, developing a ‘repeat insult’ model of demyelination.

METHODS
C57BL/6 mice were fed 0.2% cuprizone from either 16 weeks of age for 5 weeks, or from 8 weeks for 3 weeks with multiple, repeated exposures. Brain was collected at necropsy on the day of final cuprizone withdrawal, or at 7 or 21 days following final cuprizone withdrawal. These collection points correspond to 21, 22 and 24 weeks of age, with tissue also collected from age-matched unchallenged controls.  Myelin integrity was assessed by spectral confocal reflected (SCoRe) microscopy in the corpus callosum and somatosensory cortex. Oligodendroglia were assessed by immunostaining.

RESULTS
Repeated cuprizone exposure resulted in less severe demyelination in the corpus callosum compared to the standard 5 weeks cuprizone at an older age (p<0.0001) but did not remyelinate (p=0.96) following 7 days recovery despite oligodendroglial densities returning to healthy levels. Following 21 days recovery, myelin paucity remained following repeated insult, but myelin debris was persistent in both the 5 week and repeat challenged mice. In contrast, there was profound demyelination with no myelin debris in the somatosensory cortex of both 5 week and repeated challenged mice, with the levels of myelin comparable at 7 and 21 days remyelination.

CONCLUSION
From these early results, we conclude the repeated cuprizone administration from early adulthood significantly reduces remyelinating efficiency in the corpus callosum. However, there is also regional differences in demyelination and myelin repair, with an absence of myelin debris in the somatosensory cortex. Overall, our findings suggest that repeat-insult cuprizone demyelination may provide a more accurate model in which to test the efficacy of remyelinating therapies for MS.

FUNDING ACKNOWLEDGEMENT
This work is funded by an MS Research Australia Project Grant to SSM & JLF (18-044). JLF was supported by a 2018 Melbourne Neuroscience Institute Research Fellowship.

Experimental Autoimmune Encephalomyelitis is Exacerbated in Male Mice Deficient in Mertk in Dendritic Cells

BACKGROUND
Dendritic cells (DCs) are professional antigen presenting cells of the innate immune system. DCs influence T cell responses through major histocompatibility complex (MHC) signalling and variations in MHC genes confer the highest risk of developing Multiple Sclerosis (MS). MERTK mutations also confer increased MS risk.

OBJECTIVES
To examine the clinical and immunohistochemical implications of Mertk deletion in CD11c-Cre+/-/Mertkfl/fl and Mertkfl/wt mice using Experimental Autoimmune Encephalomyelitis (EAE). As DCs and microglia both express CD11c, additional animals were subjected to cuprizone challenge, a model which permits the interrogation of microglial responses to demyelination.

METHODS
EAE was induced in adult mice with hetero- or homozygous deletion of Mertk in CD11c+ cells. Animals were killed 17 days after induction, at clinical nadir. After perfusion with 4% paraformaldehyde, spinal cord lumbar expansions were dissected and prepared for immunohistochemical analysis. Additional animals with heterozygous deletion of Mertk in CD11c+ cells were fed cuprizone for three weeks before 4% paraformaldehyde perfusion. Brains were dissected and prepared for immunohistochemical analysis.

RESULTS
EAE is substantially more severe in male mice with heterozygous deletion of Mertk in CD11c+ cells on a mixed C57BL/6 – 129/SvEv background. That clinical phenotype is not present in backcrossed C57BL/6 mice. Homozygous deletion of Mertk induces an even more severe phenotype in male mice regardless of background strain, suggesting a dose effect from Mertk deletion. No clinical or immunohistochemical differences are seen in Mertk deficient animals following cuprizone challenge.

CONCLUSION
Taken together, these findings suggest that Mertk is important in modifying immune responses to central inflammatory demyelination via DC signalling. Background-specific differences in clinical severity may relate to changes in Tyro3 expression, a paralog of Mertk known to be constitutively diminished in 129 background mice.

Effect of ocrelizumab on peripheral immune B lymphocyte subsets in patients with Multiple Sclerosis at 6, 12 and 18 months post therapy

BACKGROUND  
Ocrelizumab has PBS approval for patients with relapsing remitting MS. The mechanism of action of this medication is to suppress ‘overactive’ B cells in multiple sclerosis patients. The dosing interval is 6 monthly, but its duration of effect on B cells is not entirely known.

OBJECTIVE
The objective of this research was to retrospectively measure changes in lymphocyte subsets in patients with multiple sclerosis treated with ocrelizumab, and to decipher if suppression of certain lymphocytes subsets is associated with differing clinical parameters.

METHODS
Patients included approximately 40 adults with multiple sclerosis. Peripheral blood samples were collected prior to commencement of treatment, at 6 months (prior to second dose) and again at 12 months. Data on the clinical outcomes of this treatment was also collected including number of relapses, numbers of new lesions on MRI brain and EDSS score. Statistical analysis of the results was conducted.

RESULTS
Before ocrelizumab administration lymphocyte subsets were within the reference range. At 6 months treatment, there was a significant reduction in lymphocytes: notably CD19+ levels which approached a count of zero. At 12 months there was ongoing suppression of CD19+ levels.

CONCLUSION
The use of ocrelizumab is associated with significant and lasting reduction in B cell lymphocyte subsets, with associated beneficial effects on clinical parameters. Our research provides evidence for theorised mechanism of action of ocrelizumab but it highlights that dosing frequency of 6 monthly may not be warranted as the effect of the drug on B subsets can last as long as 12 months.

Modifiable risk factors associated with depresssion and anxiety in Australian people with multiple sclerosis

BACKGROUND 
Modifiable risk factors are associated with multiple sclerosis (MS) symptoms but their role in mood is unclear. Risk factors for morbidity such as smoking, nutrition, alcohol consumption, physical activity and body mass index (BMI) make up the ‘SNAP’ factors, a construct used in general practice.

OBJECTIVE
To investigate how common the SNAP risk factors are, and assess associations between SNAP and the prevalence and severity of symptoms of depression and anxiety in Australian people with MS.

METHODS
Self-reported data from the Australian Multiple Sclerosis Longitudinal Study included the Hospital Anxiety and Depression Scale (HADS) and lifestyle measurements from 1,500 participants. SNAP score (range 0-5) was the sum of: non-smoking, sufficient fruit and vegetable intake, non-hazardous alcohol consumption, sufficient physical activity, and healthy BMI, using Australian guidelines. Analyses by log-binomial and linear regression were adjusted for confounding.

RESULTS 
Symptoms of depression and anxiety were prevalent in 27% and 40%, respectively; 20% had both. Mean SNAP score was 2.7/5; only 3% scored 5. 10% reported adequate fruit/vegetable intake, and 22% reported a combination of unhealthy BMI, inadequate physical activity and inadequate nutrition. A healthier SNAP score was associated with lower depressive symptom prevalence (prevalence ratio 0.83 (95%CI 0.75,0.92) per unit) and severity (β-0.44 (95%CI -0.64,-0.24)), but not with anxiety.

CONCLUSION
Symptoms of anxiety and depression are common among Australians with MS. Most don’t meet health recommendations regarding nutrition, BMI and physical activity, which are associated with depression. SNAP factors, anxiety and depression all contribute to morbidity, and therefore should be assessed and addressed appropriately.

Cognitive fatigue in multiple sclerosis: A comparison study with chronic fatigue syndrome and healthy individuals

BACKGROUND
Multiple Sclerosis (MS) is often characterised by persistent and debilitating fatigue. However, whether the fatigue is experienced differently from other fatiguing illnesses is not known.

OBJECTIVE
This study compares the pattern of change in self-reported fatigue and cognitive performance over a single testing session in individuals with MS to individuals with chronic fatigue syndrome (CFS) and healthy individuals.

METHODS
51 people with MS, 33 CFS and 37 healthy participants were administered a battery of cognitive tests, which was repeated over a 2.5-hour testing session.  A visual analogue fatigue scale was administered at baseline, and immediately following each battery administration. A self-report fatigue self-efficacy (perceptions of fatigue management) scale and the Hospital Anxiety and Depression Scale were also completed.

RESULTS
Spatial learning in MS/CFS, and processing speed in only CFS, declined over the session. In a sustained attention task, commission errors in MS/CFS, and omission errors and reaction time only in CFS, increased over the session. Self-reported fatigue was highest in the CFS participants. Commission errors in MS, and omission errors in CFS were related to self-reported fatigue. Fatigue self-efficacy and depression/anxiety were differentially related to fatigue across the groups.

CONCLUSIONS
The pattern and experience of fatigue in MS differs from individuals with CFS. The results may help to inform more targeted behavioural treatments for fatigue, and highlights the need to further investigate the etiology and differential experience of fatigue, in these patient groups.

Effects of Neuromuscular Electrostimulation and exercise in the Treatment of Dysphagia in Adults with Multiple Sclerosis: A Randomized Controlled Trial

BACKGROUND 
There are limited treatment options for dysphagia in adults with Multiple Sclerosis. In previous studies, neuromuscular electrostimulation (NMES) showed positive therapeutic effects in treating dysphagia, however these studies had limitations.

OBJECTIVES
The aim of this study was to determine the effectiveness of NMES paired with an effortful swallow in improving or restoring the swallowing function and the quality of life in adults with MS-related dysphagia with an explanatory parallel-group randomized controlled trial.

METHODS 
Twelve adults with MS were randomly assigned to two groups: active electrical stimulation group (n=6) or sham electrical stimulation group (n=6). Randomisation was carried out by a computer system. Both groups were treated twice per week for three weeks. At baseline and two weeks post treatment, fiberoptic endoscopic evaluation of swallowing was conducted to evaluate the effectiveness of the treatment using outcome measures including bolus residue scale, penetration/aspiration scale, and functional oral intake scale and swallowing quality of life questionnaire.

RESULTS
After treatment, participants who received active stimulation showed no yoghurt residue in the bolus residue scale yoghurt subscale. They also showed a significant improvement in the Symptom Frequency domain in the swallowing quality of life questionnaire. Improvements were noted in all other measures; however those improvements were not significant. No adverse effects were reported during or after the treatment.

CONCLUSIONS
The study suggests a potential benefit of NMES in eliminating and or reducing residue of thick fluids in dysphagic adults with mild MS and in improving their quality.

FUNDING ACKNOWLEDGEMENT
This study was supported by MS Research Australia.

Identification of longitudinal trajectories of cognitive change in multiple sclerosis using the MSReactor computerized cognitive battery.

BACKGROUND
40-65% of multiple sclerosis patients experience cognitive deficits involving processing speed, attention and working memory. Practical tools, sensitive to early cognitive change rather than impairment, are critically needed in clinical practice. Computerized tests that can interface with medical records could fill this gap. 

OBJECTIVE
We hypothesize that a serial computerized test could be a sensitive method to detect trajectories of change in select cognitive domains.

METHODS
MSReactor is a web-based, self-administered test to assess psychomotor function, visual attention and working memory. Multiple sclerosis participants completed 6-monthly testing with optional additional home-based testing. Longitudinal reaction times were modeled to determine membership into unobserved latent classes of similar characteristics. Multinomial logistic regression was performed to assess baseline predictors of cognitive trajectory.

RESULTS
551 participants with ≥ 2 psychomotor function tests over a minimum of 2 months and a maximum of 36 months were included. A model with 3 latent classes was chosen as the best fit based on information criterion and posterior probabilities. A ‘declining’ trajectory of 45 (class 1) participants was identified as well as two ‘stable’ trajectories (class 2 and 3). Higher disability (p<0.01), higher age (p<0.01), slower task performance (p<0.001) but not disease duration or therapy use (p>0.05) at baseline conferred increased relative odds of membership in class 1 when compared to the largest class (class 3).

CONCLUSION
Discrete longitudinal trajectories of psychomotor function are identified within 3 years of MSReactor testing. Older age, increased disability levels and a slower baseline performance increased the probability of being classified into a worsening cognitive trajectory. 

Generous support provided the Multiple Sclerosis Research Australia Postgraduate scholarship and Novartis Australia (CFTY720DAU08T).

Concurrent validity of the computerised cognitive battery MSReactor and the Processing Speed Test in relapsing-remitting multiple sclerosis. 

BACKGROUND
Cognitive decline is insidious in patients with multiple sclerosis (MS), even in early stages of the disease. Computerized, web-based, cognitive tests can facilitate remote self-testing but requires validation. MSReactor is a web-based platform that screens psychomotor speed, attention and working memory using a game-like interface.

OBJECTIVE
To determine the concurrent validity of the internet delivered brief computerised cognitive battery,  MSreactor (MSR), with the Processing Speed test (PST). The PST is a validated computerised version of the  the symbol digit modalities test (SDMT).

METHODS
Prospectively- enrolled relapsing-remitting multiple sclerosis (RRMS) patients completed a 10-minute MSR battery and the MSPT during their clinic attendance. Repeat testing occurred at 6 monthly intervals. All participants were enrolled in the MSBase Registry. Pearson’s correlation between the 3 individual tests of the battery (Simple reaction test SiRT, Choice reaction test ChRT and One- back test OBK) with the PST was calculated.

RESULTS
We recruited 218 RRMS patients from 6 tertiary MS centres in Australia. The mean age was 39.7 years (standard deviation 10.5 years), mean expanded disability status scale was 1.5 (standard deviation 1.1) and the  majority were on higher efficacy disease-modifying treatment.  There was a moderate correlation between the PST and the MSR battery; SiRT (Pearson’s r=  -0.46), ChRT (r= -0.50) and OBK (r= -0.43),  p <0.05.

CONCLUSIONS
The MSR battery and PST are both reliable measuring tools of processing speed and psychomotor function. Further studies are required to determine the predictive validity of the MSR and PST in a longitudinally monitored cohort.

Wnt / beta-catenin signaling in oligodendroglia alters inflammatory responses during demyelination
 

BACKGROUND + OBJECTIVE
Recent studies have revealed new perspectives on the possible functions of Oligodendrocyte Precursor Cells in multiple sclerosis (MS). OPCs within post mortem tissue from MS patients, or animal models of disease, express all of the machinery required to modulate immune responses directly. These novel findings open up the possibility that oligodendroglia may present as cellular targets for novel immunomodulatory therapeutics. Our study investigates the role that intrinsic oligodendroglial Wnt-b-catenin signalling plays the regulation of immune responses during demyelination in vivo.

METHODS
Our approach represents significant innovation: 1) We generated a mouse in which beta-catenin is deleted specifically from oligodendroglia and used this tool in a model of demyelination; 2) We combine cell cycle assays with stereology, enabling mathematical modelling to track the changes in total cell numbers; and 3) By combining Spectral Confocal Reflectance Microscopy and immunofluorescence, we devised a novel means to quantify macrophage phagocytic function ex vivo.

RESULTS
In animals where beta-catenin was conditionally deleted from oligodendroglia (cKO), we identified a 50% reduction in the total number of IBA-1 positive macrophages after 3 weeks of dietary cuprizone (ANOVA p< 0.01 n = 3–5/ genotype/group). Importantly, there was no significant genotype effect on oligodendrocyte loss, or OPC production, due to cuprizone demyelination. Excitingly, also observed a significant reduction in myelin debris, in the cKO tissue compared to controls.

CONCLUSION
We provide the first evidence that blocking an oligodendroglial intrinsic signalling pathway leads to a profound reduction in macrophage number and myelin debris during demyelination. Identifying the mechanisms mediating these effects, may reveal novel targets to limit damage and improve capacity for repair in MS.

The effects of Abdominal Functional Electrical Stimulation on bowel and bladder function in Multiple Sclerosis

BACKGROUND
Bowel and bladder problems affect >50% of people with MS. These problems have traditionally been managed using combined physical and pharmacological interventions. However, such solutions are usually only partially effective. A non-invasive method to improve bowel and bladder function for people with MS is needed.

OBJECTIVES
Abdominal Functional Electrical Stimulation (FES) is transcutaneous electrical stimulation of the abdominal muscles. This fully powered study aimed to evaluate whether Abdominal FES can reduce whole gut and colonic transit time and improve bowel and bladder related quality of life for people with MS.

METHODS
Twenty three people with MS who fulfilled the Rome III criteria for functional constipation applied Abdominal FES at home for 1 hour per day, 5 days per week, for 6 weeks. Whole gut and colonic transit time were measured using the SmartPill motility system before and after Abdominal FES training. Quality of life was measured using the Patient Assessment of Constipation Quality of Life and Incontinence Quality of Life questionnaires.

RESULTS
Mean whole gut (pre: 81.3 hours (SD: 28.7) vs. post: 96.1 (53.6), p=0.160) and colonic (pre: 65.1 hours (SD: 31.4) vs. post: 74.8 (51.1), p=0.304) transit times were unchanged following Abdominal FES. There was a significant improvement in mean bowel (pre: 1.78 (SD: 0.64) vs. post: 1.28 (0.54), p=0.001) and bladder (pre: 50.6 (SD: 26.49) vs. post: 64.5 (21.92), p=0.007) related quality of life after the intervention period.

CONCLUSIONS
This study indicates that six weeks of Abdominal FES does not reduce whole gut or colonic transit time for people with MS. However, participants reported a significant improvement in bowel and bladder related quality of life after the intervention.

The prevalence of bowel and bladder problems in multiple sclerosis and its association with fatigue

BACKGROUND
It is estimated that there are 23,700 people living with Multiple Sclerosis (MS) in Australia. Bowel and bladder problems affect more than 50% of people with MS. These problems have a large impact on quality of life and place a significant burden on health systems.

OBJECTIVES
This study aimed to ascertain the prevalence of bladder and bowel problems in an Australian MS cohort and to investigate the relationships between level of disability, bladder and bowel problems, and fatigue.

METHODS
The simplified, 8-item Actionable Bladder Symptom Screening Tool, the Revised Faecal Incontinence Scale and the Modified Fatigue Impact Scale Questionnaires were distributed to clients attending an Australian MS centre. Binary multiple logistic regression and multiple linear regression models were used to investigate relationships between disability, fatigue, and bladder and bowel problems.

RESULTS
Bladder problems were reported by 87 (74.4%) respondents, whilst 66 (48.9%) experienced functional constipation and 43 (31.9%) faecal incontinence. This prevalence of bowel and bladder problems in the Australian MS population is similar to that reported globally. There was a significant correlation between level of disability and: bladder problems (p=0.015), faecal incontinence (p=0.001), fatigue (p<0.001) and constipation (p=0.016). 

CONCLUSION
This study shows, for the first time, a significant correlation between bowel and bladder problems and fatigue. Further investigation into the causal relationships between various MS symptoms may be beneficial in the development of novel therapeutic strategies for people with MS.

In depth analysis of B cells in multiple sclerosis patients after treatment with Cladribine

OBJECTIVE
Early studies suggest a possible mechanism of action is via depletion of CD27+ memory B cells. Whether other B cell subsets are affected is not known. To investigate this, we have performed an in-depth phenotypic analysis of B cell subsets within cladribine-treated MS patients.

METHODS
We used mass cytometry, which combines flow cytometry with mass spectrometry, to interrogate the entire B cell compartment in relapse-remitting MS patients before and after treatment with cladribine.

RESULTS
Our results show that Cladribine-induced remission is associated with decreases in 3 previously unrecognised populations of CD27+ memory B cells that differentially express IgG1 and IgM. These B cells expressed high levels of the activation markers CD80 and CD267 (TACI).

CONCLUSION
Mass cytometry has provided us with unparalleled levels of detail to determine which B cell subsets are affected by Cladribine treatment. These studies will allow us to track efficacy and design new ways of targeting these B cells more specifically.

Imaging the pathophysiology of tremor in MS

BACKGROUND
Tremor is a disabling symptom and is estimated to affect up to half of people with multiple sclerosis (pwMS). The presence of tremor significantly adds to disease disability and treatment is challenging. Underlying structural and functional correlates of tremor in MS are poorly understood.

OBJECTIVE
The objective of this longitudinal neuroimaging was to define the pathophysiological characteristics of tremor in pwMS. We aimed to examine if central networks are affected by intramuscular, upper-limb injections of onabotulinumtoxinA (BoNT-A) compared to placebo in a randomized controlled trial setting.

METHODS
We recruited 43 pwMS with tremor, 27 pwMS without tremor and 15 healthy controls. The tremor group was randomised into receiving either placebo or BoNT-A. Tremor severity was measured at baseline, 6 weeks and 12 weeks using the Bain tremor scores (0 to 10) for overall tremor severity, handwriting, and Archimedes spiral drawing. Structural, and tremor-evoking joystick task functional magnetic resonance imaging was performed at baseline and 6 weeks.

RESULTS
pwMS with tremor have increased atrophy and lesion load within the cerebellum and thalamus compared to pwMS without tremor and healthy controls. Furthermore, tremor affected pwMS have increased activation within the inferior parietal lobule (IPL). Activation within both areas negatively correlated to tremor severity. BoNT-A injections reduced clinical tremor severity and corresponded with decreased IPL activation. The change in IPL activation positively correlated with the reduction in tremor.

CONCLUSION
We showed that the clinical presentation of tremor in pwMS depends on the interaction between cerebello-thalamic injury and functional neuroplasticity. Peripherally administered BoNT-A exerts a neural effect by modulating functional motor integration networks that, in turn, is associated with a positive clinical response to treatment.

Adaptive myelination mediated by changes to oligodendrocyte survival

BACKGROUND
Myelination is a crucial component of normal development. Understanding the processes underlying normal myelination during ageing is essential to understanding the mechanisms by which remyelination (the generation of new myelin in an injury scenario) can be promoted. This knowledge is especially important for demyelinating diseases in which remyelination is a key target, such as multiple sclerosis (MS). Recent studies show that enhanced neural activity promotes myelination, a phenomenon termed ‘adaptive myelination’. This project sought to qualitatively and quantitatively understand the mechanistic action describing adaptive myelination during normal development, finding that adaptive myelination can determine survival efficiency of newly-generated myelinating oligodendrocytes. It also aims to understand whether adaptive myelination can alter rate or extent of demyelination and/or functional recovery and remyelination in an animal model of MS.

Understanding processes underlying myelin addition by oligodendrocytes during normal development is essential to discovering mechanisms by which remyelination (the generation of new myelin in an injury scenario) can be promoted. Recent studies show neural activity as a key determinant of myelination (termed ‘adaptive myelination’).

OBJECTIVE
This project sought to assess whether ‘adaptive myelination’ (enhanced oligodendrocyte integration following neural activity) exerts its effect primarily by influencing new oligodendrocyte generation, or existing oligodendrocyte survival. This will have important implications for how to promote oligodendrocyte integration and functional recovery in demyelinating diseases. (44 words)

METHODS
Changes in production of myelinating oligodendrocytes (OLs) in murine prefrontal cortex (PFC) were measured following periods of sensory enrichment, sensory deprivation or regular housing during juvenile postnatal development (P21-P35; n=5/6 mice/condition). By measuring individual animal’s oligodendrocyte precursor cell (OPC) proliferation rates (via Double-S phase BrdU labelling) and changes in the total number of OLs (via stereology), we were able to mathematically predict oligodendrocyte growth (via linear interpolation models), and death. (70 words)

RESULTS
Counterintuitively, we found sensory enrichment and deprivation significantly increased daily OL production relative to controls (p=0.0008 and p=0.0010, respectively). Survival rates, however, vastly differed between housing groups and only in the enriched condition was production greater than death (p<0.0001), enabling retention of OLs. Interestingly, this did not result in an increased volumetric density of OLs (p=0.4853), as total PFC volume increased in enriched animals (p=0.0143) to accommodate the new cells. (70 words)

CONCLUSIONS
This work demonstrates that new oligodendrocyte production can be uncoupled from successful oligodendrocyte integration. Ultimately, the level of survival will dictate total oligodendrocyte number. This has important consequences for remyelinating therapies, as it indicates targeting survival of newly-generated oligodendrocytes, rather than production, may be crucial. (45 words)

We found enhancing neural activity promotes integration of new oligodendrocytes via increasing the survival efficiency of newly-generated oligodendrocytes.

Homeostatic Regulation May Limit Immune Response to Vitamin D Supplementation, Especially to Cholecalciferol

BACKGROUND
Although serum vitamin D levels and latitude have been strongly associated with autoimmune diseases such as Multiple Sclerosis, several recent meta-analyses indicate that supplementation which increases serum levels of inactive vitamin D (25D3) does not ameliorate disease.

OBJECTIVE
Currently there are no biomarkers to indicate changes in immunological state due to increases in serum 25D3, limiting ability to assess the efficacy of different vitamin D analogues. Here we tested the utility of blood gene expression quantification to assess immune response to vitamin D.

METHODS
We assessed changes in immune cell expression of potentially vitamin D responsive MS risk genes (VDRMS), some of which regulate activation of vitamin D, in a cohort of clinically isolated syndrome (CIS) patients supplemented with cholecalciferol to obtain >80nmol/L 25D3 that were treated +/- UVB phototherapy as part of the PhOCIS trial. VDRMS expression was also determined on whole blood cultures treated with active and inactive vitamin D analogues.

RESULTS
Gene expression was altered by treatment with 25D3 (cholecalciferol) and 1,25D (paricalcitol) on immune cells in vitro, especially CYP24A1. However, expression of these genes in whole blood was not altered on supplementation with cholecalciferol or UVB therapy in CIS patients. VDRMS gene expression in CIS was lower than healthy controls at baseline. Expression of the VDRMS genes was correlated with each other and stable over time, indicating shared homeostatic regulation. 

CONCLUSIONS
Explanations for the equivocal response to vitamin D /UV treatment include; insufficient activation of the precursor due to limiting CYP27B1 expression in disease; and insensitivity to supplementation due to homeostatic constraints. VDRMS expression, especially CYP24A1, may prove useful biomarkers of immunological response to vitamin D.

FUNDING ACKNOWLEDGEMENT
MS Research Australia, NHMRC.

Speech in Multiple Sclerosis: characteristics and its potential as a marker of disease status

BACKGROUND
A shift towards objective measurement of speech has shown promising results to detect the presence of Multiple Sclerosis. However, its potential to track disease progression remains unknown.

OBJECTIVE
In this study, we characterize the cross-sectional relationship between disease severity and speech, through both subjective and objective speech assessments.

METHODS
Speech characteristics from 119 people with Multiple Sclerosis divided in three disease stages were compared with those from 22 healthy controls. Correlations were then calculated between speech outcomes and disability scores, MRI-measured brain volume and lesion load, and self-assessed quality of life. Linear regression was used to fit a model for EDSS scores using speech features as predictors.

RESULTS
Speech impairment was detected in moderately and severely disabled groups. Greater effect sizes were observed through objective analysis in comparison to subjective assessments. Subclinical differences in speech were detected in the mildly disabled subgroup through objective analysis alone. Differences in speech between each disease stage could only be gauged through an objective composite score. Specific speech features correlated with the degree of overall disability, MRI outcomes and quality of life.

CONCLUSION
We have demonstrated the relationship between speech and overall disability, imaging and quality of life outcomes in a diverse cohort of participants. This is the first step in exploring the potential use of automated analysis of speech as biomarker of disease progression and treatment success.

FUNDING ACKNOWLEDGEMENT
This study was funded through a University of Melbourne Postgraduate Scholarship and NHMRC grants.

Sleep disorders and poor sleep are common and associated with fatigue and depression in people with mild to moderately-severe MS

BACKGROUND
Knowledge on the role sleep disorders and poor sleep play in people with MS is increasing. Sleep problems are common and likely contribute to other major disease comorbidities (e.g. fatigue). However, previous investigations into sleep disorders in MS have had crucial methodological limitations.

OBJECTIVES
To define rates of sleep disorders/poor sleep in a consecutively recruited cohort of people with mild to moderately-severe MS. Secondary aims were to investigate relationships between sleep disorders/poor sleep with fatigue and depression and identify potential causes of poor sleep.

METHODS
111 adults (85 females) years with MS and an expanded disability status scale score between 2-6 were consecutively recruited to take part in this sleep sub-study as part of a larger randomised controlled trial (ACTRN12616001053415). Validated sleep and health-related questionnaires, a home sleep study and 1-week of daily-life actigraphy were performed.

RESULTS
52% of the participants had one or more sleep disorders; sleep apnoea (31%), insomnia (29%), restless legs (26%) and 71% reported poor sleep. Poor sleep was an independent predictor of fatigue (OR:3.27 [1.18-9.04] p= 0.022) and insomnia was an independent predictor of depression (OR:2.35 [0.93-5.98] p=0.003). Over a 24h period, participants spent 10.7±1.9 hours lying down and increased time lying was associated with poorer subjective sleep quality (r=0.44 p< 0.01).

CONCLUSIONS
Sleep disorders and poor sleep are common in MS and are associated with fatigue and depression. Strategies to improve sleep may help reduce comorbidity burden. However, simple recommendations to increase sleep opportunity are unlikely to be helpful as this may further worsen perceived sleep quality.

Cost-of-illness analysis of Multiple Sclerosis in Australia in 2017

BACKGROUND
Economic costs of Multiple Sclerosis (MS) in Australia were last estimated in 2010 and are likely to have changed over time due to the introduction of new generation higher efficacy disease modifying therapies (DMTs) and better MS management. 

OBJECTIVES
To estimate the direct and indirect costs of MS from individual and societal perspectives for an updated representative sample of Australians with MS, and to evaluate the relationship between these costs and MS-related disability.

METHODS
Costs were calculated from a sample of 488 Australians with MS who completed both (the baseline and the cost diary) questionnaires of the 2016 Economic Impact Survey (EIS) of the Australian MS longitudinal Study (AMSLS). Costs were broken down by disability severity, classified as no disability (Expanded Disability Status Scale [EDSS] level: 0), mild (EDSS 1–3.5), moderate (EDSS 4–6) and severe (EDSS 6.5–9.5). All costs are presented in 2017 Australian dollars.

RESULTS
Annual total costs of MS per person increased 17% from $58,652 in 2010 to $68,382 in 2017, driven largely by increased costs of DMTs, but offset by decreased costs of lost wages. Costs increased with increasing disability severity: $30,561 (95% confident interval [CI]: $25,672–$35,451), $55,815 (95% CI: $47,503–$64,126), $76,915 (95% CI: $68,866–$84,964), and $114,813 (95% CI: $102,252–$127,374) for no, mild, moderate and severe disability, respectively.

CONCLUSIONS
We provided a snapshot of the nature and extent of costs of MS in Australia and demonstrated that MS is an increasingly substantial economic burden to people with MS and society. Further strategies to improve the lives of people with MS, and to prevent disease onset/progression are recommended.

FUNDING ACKNOWLEDGEMENT
Multiple Sclerosis Research Australia, Research Training Program (RTP) Scholarship.

Risk factors of leaving employment due to multiple sclerosis and changes in risk over the past decades

BACKGROUND
Disease modifying therapies (DMTs) may have contributed positively on employment outcomes for those with relapsing remitting MS (RRMS). However, no study has assessed changes in employment survival in MS over the past decades.

OBJECTIVES
To evaluate risk factors of leaving employment due to MS, and to assess whether the risk of leaving employment has changed over the past decades in Australia and whether the changes were different between people with RRMS and people with primary progressive MS (PPMS).

METHODS
A retrospective cohort study using data from the Australian MS longitudinal study. We included 1,240 participants (diagnosed between 1949- 2015) who were working before MS diagnosis. Information on employment status, reasons for leaving employment, and year of leaving employment were collected. Data were analysed using competing risk survival analysis.

RESULTS
Males, people with PPMS, those with a lower education level and those with a higher age at diagnosis had a higher subdistribution hazard of leaving employment. Compared to the period before 2012, the subdistribution hazard of leaving employment due to MS during 2012 -2016 for RRMS was reduced by 43% (sHR 0.57, 95% CI 0.41 to 0.80), while no significant change was seen for PPMS (sHR 1.31,95% CI: 0.74 to 2.33).

CONCLUSIONS
Males, people with PPMS, and those with a lower education level might need additional assistance to remain in the workforce. The differential changes in risk of leaving employment between people with RRMS and PPMS seem to coincide with the increased usage of high efficacy DMTs.

Depression and pain have the largest impact on the quality of life in people with MS, irrespective of MS phenotype

BACKGROUND
The symptoms that have the largest impact on quality of life (QoL) in people with multiple sclerosis (MS) might be different by MS phenotype (relapsing-remitting MS, RRMS; secondary progressive MS, SPMS; primary progressive MS, PPMS). Knowing these symptoms assists the priority setting in symptom management.

OBJECTIVE
To examine the associations between 13 common MS symptoms (depression, fatigue, walking difficulties, balance difficulties, pain, sexual dysfunction, cognitive symptoms, vision problems, bladder problems, bowel problems, anxiety, sensory symptoms, and spasticity) and QoL in the total sample and stratified by MS phenotype.

METHODS
The study included 1,319 participants from the Australian Multiple Sclerosis Longitudinal Study. QoL was assessed using the Assessment of Quality of Life with eight dimensions (AQoL-8D). Linear regression was used to assess the association between the individual symptoms and AQoL-8D. Stepwise regression was used to identify the symptoms that had the largest impact on AQoL-8D when combining all symptoms in a single multi-variable model.

RESULTS
We found strong associations between individual MS symptoms and AQol-8D. The effect sizes were generally strongest for those with RRMS followed by PPMS and SPMS. In a multivariable model that takes other symptoms into account, depression and pain were most strongly associated with the AQoL-8D irrespective of MS phenotype and together accounted for 54% in RRMS, 40% for SPMS and 47% for PPMS.

CONCLUSION
Improving depression and pain need to become primary intervention targets in order to improve QoL in people with MS, irrespective of MS phenotype.

A systematic review of self-administered medication adherence behaviours in people with multiple sclerosis

BACKGROUND
In multiple sclerosis (MS), suboptimal medication adherence is associated with subsequent discontinuation, which occurs in ~30-50% of people within 2-years. Medication adherence is critical for realisation of pharmacotherapy benefits and reduced healthcare expenditure. Previous systematic reviews have not included oral DMTs, first introduced in 2010.

OBJECTIVE
To undertake a systematic review of adherence and persistence rates for self-administered oral and injectable disease modifying therapies (DMTs) in people with MS.

METHODS
A literature search for publications examining DMT adherence and persistence rates in MS was conducted through databases including PubMed, Web of Science, Scopus and PsycINFO yielding 1,590 records, with 1,150 duplications removed, resulting in 440 abstracts screened. We included peer-reviewed, English written studies for objectively measured self-administered DMTs in samples aged 18+, published between July 1993, and December 2018. Studies reporting combined intravenous and self-administered DMT adherence results were excluded.

RESULTS 
In total, 119 articles were extracted for quantitative synthesis. Preliminary results show 12-month mean adherence (medication possession ratio) for injectable compared to oral DMTs was 71.7% (n = 6) and 83.0% (n = 3), respectively. Mean discontinuation over 12- and 24-months for injectable DMTs were 30.4% (n = 9) and 29.7% (n = 8); and oral DMTs were 25.4% (n = 3) and 16.5% (n = 5), respectively.

CONCLUSIONS
Medication adherence and persistence remains problematic despite the introduction of oral DMTs. Oral DMT improvements are noted for 12-month adherence and 24-month discontinuation. With limited oral DMT adherence and persistence data, studies are hampered by methodological issues, such as inconsistent adherence and persistence definitions.

The effect of lifestyle modification on future quality of life in people with multiple sclerosis: prediction model development and validation

BACKGROUND
Associations between healthy lifestyle factors and improved quality of life in people with multiple sclerosis (MS) are well-established; however, the degree to which lifestyle modification can (predictably) alter quality of life over a set time into the future remains unclear.

OBJECTIVE
To construct and validate a prediction model of 2.5-year follow-up MS-specific quality of life (MSQoL-54) scores using baseline modifiable lifestyle variables as predictors and, subsequently, to estimate the mean change in model-predicted MSQoL-54 scores in the cohort stratified for depression status, fatigue status, and relapse status.

METHODS
Design: Temporally-validated, prediction model

Setting: Self-assessed, using health utility instruments

Participants: International cohort sourced from an MS-society-based social media recruitment drive

Sample size: Estimation subsample, n = 2466; validation subsample, n = 573

Predictors: Baseline modifiable lifestyle factors; socio-demographic and clinical controllers

Outcomes: 2.5-year follow-up MS-specific quality of life score, physical and emotional composites

Statistical analysis: Least absolute shrinkage and selection operator applied to a complete-case, multivariable linear regression model.

RESULTS
Abstinence from smoking and healthy diet were positively-associated with improved future physical and emotional quality of life; supplementation with vitamin D, omega-3 and disease-modifying drugs additionally improved physical quality of life; alcohol moderation and physical activity additionally improved emotional quality of life.

Both physical and emotional quality of life were negatively-associated with the following multiple sclerosis co-morbidities: fatigue, depression and 12-month relapse.

CONCLUSION
Construction and validation of quality of life prediction models for people with multiple sclerosis demonstrate the utility of adopting healthy lifestyle changes for improving the clinical course of their condition.

Activation of the platelet P2X7 receptor releases free mitochondria which can initiate localized vascular inflammation

BACKGROUND
Although many risk factors are known, the initiating cause of multiple sclerosis (MS) is not known.  A recent study of EAE, the mouse model of MS, showed that platelet depletion from day 7 to 15 abolished clinical EAE.  In a case-controlled genetic association study our group has shown that loss-of-function variants of the P2X7 receptor protect against neuroinflammation in MS.

OBJECTIVE
We examined the expression and function of the P2X7 receptor on human platelets as well as studying the circulating microparticles (MP) derived from platelets, since these can mediate inflammatory effects, such as transfusion-related acute lung injury.  Our aim was to identify the MP causing inflammation.

METHODS
Expression of P2X7 on human platelets was assessed on flow cytometry by binding of P2X7 monoclonal antibody as well as by Western analysis of platelet and MP lysates.  Blood was centrifuged to prepare platelet-rich plasma (PRP) and stained with MitoTracker dyes to label mitochondria.  PRP was then incubated for 10min with BzATP, a selective agonist for the P2X7 receptor, followed by centrifugation and flow cytometric analysis on Cytoflex S of supernatant for released mitochondria.  MitoTracker-positive events in platelet-free plasma (PFP) were enumerated with FlowJo software.

RESULTS
The P2X7 receptor was expressed on blood platelets and, to a lesser extent, on the MPs released by BzATP.  Expression of P2X7 on circulating platelets and MPs was confirmed by Western analysis showing immunoreactive bands migrating at the expected 68-70 kDa size.  The number of MitoTracker-positive events was measured in PFP of 15 normal donors with values between 0.1 and 11.3x106/mL with most values less than 1.5x106/mL.  An additional release of mitochondria was seen after 10min incubation of PRP with the agonist BzATP (1-500 uM).

CONCLUSIONS
Our data suggest that the inflammatory effects of platelets are initiated by their P2X7 receptor causing release of free mitochondria, and increased numbers of this organelle may permeabilise the blood-brain barrier, allowing entry of immune cells.

Disability, burden and societal cost in people with MS in Australia

BACKGROUND 
The cost of progressive disability to individuals with MS and society is not fully reported. Australia has introduced a national disability insurance scheme (NDIS) to improve quality of life of people with disability through community and home support; MS specific costs have not been published.

OBJECTIVE
To address this our study A description of disease burden and societal cost in people with multiple sclerosis in Australia (BAC-MS) assesses burden and cost at each Expanded Disability Status Scale (EDSS) step.

METHODS 
People with MS were recruited from John Hunter Hospital MS Clinic, Newcastle, part of the region where the NDIS was first introduced. Participants provided a copy of their NDIS plan and via interview completed the modified Client Services Receipt Inventory, EuroQoL 5-Domain 5-Level, Multiple Sclerosis Impact Scale and Work Productivity and Activity Impairment questionnaires. Resource utilisation was extracted from local health district databases and costs applied.

RESULTS
Interim analysis includes 104 patients. Of the 91 patients eligible to apply to NDIS, a package was held by 20.9% of those with mild disability, 62.5% with moderate and 95.8% with severe disability. No patient with mild or moderate disability had an NDIS package ≥$100,000, whereas in patients with severe disability, 20.8% had $100,000-<$150,000 and 29.2% had ≥$150,000. 4.7% of mild and 20.8% of moderate patients were awaiting assessment outcome.

CONCLUSION 
The value of an NDIS package increases with level of disability in people with MS. It appears that most patients with high needs are being supported by community based services. The appropriateness of service provision is to be determined.

Gender and multiple sclerosis (MS) risk alleles may determine the Epstein-Barr virus infection pattern in B cells and influence MS pathogenesis

BACKGROUND
EBV appears to be necessary for the development of MS. The gender imbalance in MS prevalence is poorly understood. EBV latently infects B cells by interacting with the host genome to alter immune response. Differential response to EBV infection may in part explain the gender imbalance in MS.

OBJECTIVE
We aimed to determine (i) whether MS risk alleles are differentially expressed in EBV-infected B cells depending on the donor gender when cultured in hormone-containing media and, (ii) whether these effects are mediated through oestrogen.

METHODS
Peripheral blood mononuclear cells from 25 males and 22 females were infected with EBV inoculum to provide lymphoblastoid cells (LCLS). LCLs were cultured media containing 10% fetal bovine serum (which contains multiple hormones as well as the oestrogenic phenol red) or cultured in serum free media. Cells were harvested for measurement of gene expression and MS risk genotype for the MS risk genes TRAF3, CD40, and ZC3HAV1.

RESULTS
LCL donor gender affected the nature of the association of risk SNP with gene expression for several MS risk genes, generally with stronger associations seen in females. In particular, ZC3HAV1 expression in females was reduced in those with the CC protective allele compared to carriers (p= 0.005) whereas in males the protective CC allele had increased expression (p=0.0266). Gender and genotype effects were also reflected in the Geovadis LCL cohort.

CONCLUSION
LCLs cultured in serum-containing media express certain MS risk genes in a gender-specific manner. We postulate that host response to EBV infection and the expression of these genes is due to a differential response to sex hormones. This effect is being tested in ongoing experiments.

Repeated demyelination results in impaired myelin repain in the young adult mouse brain

BACKGROUND
Chronic demyelination and failed myelin repair contribute to progressive clinical disability in multiple sclerosis (MS) patients. However, this aspect of MS pathobiology is not accurately recapitulated in current animal models. To determine if we could model remyelination failure in the mouse we modified the standard cuprizone paradigm, developing a ‘repeat insult’ model of demyelination.

METHODS
C57BL/6 mice were fed 0.2% cuprizone from either 16 weeks of age for 5 weeks, or from 8 weeks for 3 weeks with multiple, repeated exposures. Brain was collected at necropsy on the day of final cuprizone withdrawal, or at 7 or 21 days following final cuprizone withdrawal. These collection points correspond to 21, 22 and 24 weeks of age, with tissue also collected from age-matched unchallenged controls.  Myelin integrity was assessed by spectral confocal reflected (SCoRe) microscopy in the corpus callosum and somatosensory cortex. Oligodendroglia were assessed by immunostaining.

RESULTS
Repeated cuprizone exposure resulted in less severe demyelination in the corpus callosum compared to the standard 5 weeks cuprizone at an older age (p<0.0001) but did not remyelinate (p=0.96) following 7 days recovery despite oligodendroglial densities returning to healthy levels. Following 21 days recovery, myelin paucity remained following repeated insult, but myelin debris was persistent in both the 5 week and repeat challenged mice. In contrast, there was profound demyelination with no myelin debris in the somatosensory cortex of both 5 week and repeated challenged mice, with the levels of myelin comparable at 7 and 21 days remyelination.

CONCLUSION
From these early results, we conclude the repeated cuprizone administration from early adulthood significantly reduces remyelinating efficiency in the corpus callosum. However, there is also regional differences in demyelination and myelin repair, with an absence of myelin debris in the somatosensory cortex. Overall, our findings suggest that repeat-insult cuprizone demyelination may provide a more accurate model in which to test the efficacy of remyelinating therapies for MS.

FUNDING ACKNOWLEDGEMENT
This work is funded by an MS Research Australia Project Grant to SSM & JLF (18-044). JLF was supported by a 2018 Melbourne Neuroscience Institute Research Fellowship.

A systematic review of self-administered medication adherence behaviours in people with multiple sclerosis

BACKGROUND 
In multiple sclerosis (MS), suboptimal medication adherence is associated with subsequent discontinuation, which occurs in ~30-50% of people within 2-years. Medication adherence is critical for realisation of pharmacotherapy benefits and reduced healthcare expenditure. Previous systematic reviews have not included oral DMTs, first introduced in 2010.

OBJECTIVE
To undertake a systematic review of adherence and persistence rates for self-administered oral and injectable disease modifying therapies (DMTs) in people with MS.

METHODS
A literature search for publications examining DMT adherence and persistence rates in MS was conducted through databases including PubMed, Web of Science, Scopus and PsycINFO yielding 1,590 records, with 1,150 duplications removed, resulting in 440 abstracts screened. We included peer-reviewed, English written studies for objectively measured self-administered DMTs in samples aged 18+, published between July 1993, and December 2018. Studies reporting combined intravenous and self-administered DMT adherence results were excluded.

RESULTS

In total, 119 articles were extracted for quantitative synthesis. Preliminary results show 12-month mean adherence (medication possession ratio) for injectable compared to oral DMTs was 71.7% (n = 6) and 83.0% (n = 3), respectively. Mean discontinuation over 12- and 24-months for injectable DMTs were 30.4% (n = 9) and 29.7% (n = 8); and oral DMTs were 25.4% (n = 3) and 16.5% (n = 5), respectively.

CONCLUSIONS
Medication adherence and persistence remains problematic despite the introduction of oral DMTs. Oral DMT improvements are noted for 12-month adherence and 24-month discontinuation. With limited oral DMT adherence and persistence data, studies are hampered by methodological issues, such as inconsistent adherence and persistence definitions.

CD39+ T regulatory cell reconstitution in MS patients with different CD39 gene polymorphisms undergoing autologous HSCT: A Preliminary Study

BACKGROUND
Autologous haematopoietic stem cell transplantation (AHSCT) may induce remission in Multiple Sclerosis (MS) patients by regenerating immune-regulatory cells, including antigen-specific CD39+ T regulatory cells (Tregs). CD39+ Tregs play a role in regulating inflammatory responses contributing to MS pathogenesis but are defective in MS patients.

OBJECTIVE 
Variable CD39 expression and Treg function between individuals has been attributed to polymorphism of the CD39 gene. We aimed to determine whether CD39 genetic polymorphism affects CD39+ Treg reconstitution post-AHSCT in MS patients.

METHODS
CD39+ Tregs (CD4+CD25hiCD127loCD39+) were quantified in the patient blood by flow cytometry and CD39 genetic polymorphism was analysed at position rs_10748643 (A-to-G SNP) in MS patients undergoing AHSCT (after informed and written consent according to the declaration of Helsinki; n=25). Statistical analysis was performed using Mann-Whitney U and Wilcoxon’s test.

RESULTS
Between three genotype groups detected (AA, AG and GG) in MS patients, there were significant differences in CD39 antigen density (median fluorescence intensity – MFI) of CD39+ Tregs (mean MFI: AA–1144 vs AG–2119 vs GG–3284; p<0.01) at pre-AHSCT. There was a significant increase in Treg CD39 MFI expression (p<0.05) and proportion of CD39+ Tregs (p<0.01) at 12 months post-AHSCT compared to baseline in the AG cohort only. CD39+ Treg absolute counts (cells/µl) did not change at 12 months post-AHSCT in the AG cohort.

CONCLUSIONS
CD39 gene polymorphism is correlated with Treg CD39 expression and medium CD39 expression was correlated with a higher proportion of CD39+ Tregs post-AHSCT. These changes highlight that CD39 gene polymorphism may determine the survival of CD39+ Tregs post-AHSCT, and perhaps subsequently, functional changes and clinical response towards AHSCT for MS patients.

FUNDING ACKNOWLEDGEMENT
NHMRC, SVH Haematology Research Fund, Reset Australia, Maple-Brown Family Foundation, St Vincent’s Clinic Foundation, John Kirkpatrick Family Foundation, NSW Health, Medich Family Foundation.

Influence of comorbidities on the severity of common symptoms in people with Multiple Sclerosis

BACKGROUND
The symptoms that people with Multiple Sclerosis (MS) experience varies greatly between people and are influenced by various factors. Few studies have examined the influence of comorbidities on the severity of symptoms.

OBJECTIVES
To determine the impact of comorbidities on the severity of common MS symptoms.

METHODS
We used 1,223 participants of the Australian Multiple Sclerosis Longitudinal Study with data on the presence of 30 comorbidities and the severity of 13 symptoms (0-10 scale). Negative binomial regression was used to estimate ratios of mean severity. Dominance analysis was used to determine the relative importance of each comorbidity group in predicting the total variance and the individual comorbidities that mostly influenced symptom severity.

RESULTS
The total number of comorbidities had the strongest effect on pain severity (mean ratio 1.13; 95%CI (1.09-1.17) per comorbidity increase) followed by depression and anxiety. All comorbidity groups explained between 2.22% (vision problems) to 6.35% (pain) of the total variance in symptom severity.  Musculoskeletal disorders were of highest relative importance for 12/13 symptoms, while the diseases of the nervous system were of highest relative importance for cognitive impairment.

CONCLUSIONS
Comorbidities have the largest impact on the severity of pain, depression and anxiety. For most symptoms, musculoskeletal disorders were of highest relative importance. Optimal management of comorbidities, particularly musculoskeletal disorders, could assist with the reduction of symptom severity in people with MS. 

Estimating the magnitudes of effect and the relative importance of comorbidities in predicting health-related quality of life of people with multiple sclerosis

BACKGROUND
The health-related quality of life (HRQoL) of people with Multiple Sclerosis (MS) is affected by the presence of comorbidities but only few studies have examined the impact of common comorbidities on HRQoL in people with MS.

OBJECTIVES
To examine the total impact, effect sizes and the relative importance of comorbidities on psychosocial, physical and overall HRQoL of people with MS.

METHODS
Data on comorbidities and HRQoL were obtained from the 902 participants of the Australian Multiple Sclerosis Longitudinal Study (AMSLS). HRQoL was assessed using the Assessment of Quality of Life-8 Dimensions (AQoL-8D) with scores ranging from 0 (death) to 1 (perfect health). We used linear regression and dominance analysis.

RESULTS
Higher total number of comorbidities were associated with a lower HRQoL (p<0.01). All comorbidities accounted for 18.09% of the variance. Systemic lupus erythematosus (β=-0.16), depression (β=-0.15), hyperthyroidism (β=-0.12) and anxiety (β=-0.11) had the largest impact on overall HRQoL. Depression (β=-0.14) and anxiety (β=-0.10) had the largest impact on psychosocial HRQoL, while systemic lupus erythematosus (β=-0.18), rheumatoid arthritis (β=-0.11) and hyperthyroidism (β=-0.11) had largest impact on physical HRQoL.

CONCLUSION
The identified effect sizes and the relative importance of comorbidities in predicting the HRQoL suggest that aggressive treatment of comorbidities has potential to drastically improve the HRQoL of people with MS.

The prevalence of comorbidities at and after the onset of multiple sclerosis compared to the general population

BACKGROUND

Comorbidities have substantial adverse effects on Multiple Sclerosis (MS), however, few studies have examined changes in the prevalence of comorbidities at critical time points of the disease.

OBJECTIVES

To determine the prevalence of comorbidities before MS symptom onset and at the time of study, to compare these prevalences to the Australian population and to examine the differences on sex and MS onset-type.

METHODS

We assessed the self-reported presence of 30 comorbidities of the 1,518 participants of the Australian MS Longitudinal Study (AMSLS) and used the 2014-15 National Health Survey data from the Australian Bureau of Statistics as our comparator data. Prevalence ratios (PR) (crude, age-and-sex adjusted, sex and MS onset-type stratified) were calculated.

RESULTS

After age-and-sex adjustment, at symptom onset 9/17 comorbidities were significantly more common in people with MS and at the time of study 14/19 were more common, with increasing effect sizes. At symptom onset, effect sizes were strongest for cancer, heart disease, stroke and vascular disease (PR>2.00). At the time of study, they were strongest for anaemia, cancer, anxiety, depression, and migraine (PR>3.00). No significant differences were seen by MS onset-type.

CONCLUSIONS

The increasing number and effect sizes of comorbidities from symptom onset to the time of study course highlight the importance of optimal screening, management and treatment of comorbidities in early MS and throughout the disease course.

CLADIN: CLADribine and INnate Immune responses

BACKGROUND
Cladribine (Mavenclad®) is a purine analog, and an oral treatment for relapsing remitting MS (RRMS). P2X7R is a purinergic receptor expressed innate immune cells, and is thought to play a critical role in neuroinflammation. The mechanism of action of Cladribine on peripheral innate immune cells, in particular monocytes, and its effect on P2X7R, is unclear, and forms the basis of this study.

METHODS 
This will be a Phase IV, multi-centre, 3 year, translational trial in RRMS patients commencing Cladribine. Peripheral innate immune phenotypes (monocyte numbers and activation states) will be measured at various times prior and after commencement of therapy. In addition, and in an in vitro setting we will assess the effect of Cladribine on P2X7R expression and function, as well as quantifying various cytokines / chemokines in the periphery.

RESULTS 
This study has been approved by Alfred Health Human Research Ethics Committee to commence in July 2019. As a premise to this study, retrospectively analysis of peripheral immune cell counts from 44 patients with RRMS, revealed that subsequent to commencement of Cladribine, there is a reduction in peripheral monocyte counts. This will be addressed in a prospective manner as part of CLADIN.

CONCLUSION 
CLADIN will shed light on whether Cladribine has an effect on peripheral innate immune cells, in particular clarifying if the medication is exerting some of its beneficial effects via action on monocytes and alterations of their P2X7Rs. Our retrospective analysis thus far has highlighted that Cladribine is associated with a reduction in peripheral monocyte counts.

Increased default mode network connectivity is related to slowed cognitive information processing in Clinically Isolated Syndrome

BACKGROUND
Increased functional connectivity in clinically isolated syndrome (CIS) is proposed to represent adaptive functional reorganisation, accounting for the absence of cognitive deficit and correlations with neuropsychological performance. Instead, this may reflect the relative insensitivity of conventional neuropsychological measures to the subtle cognitive changes at CIS.

OBJECTIVE
Ocular motor (OM) cognitive assessments have demonstrated deficit in CIS in absence of change in neuropsychological performance. We sought to determine whether the use of OM cognitive assessments allow the elucidation of meaningful relationships with resting state network connectivity in CIS.  

METHODS
18 CIS and 35 controls were compared on OM cognitive tests (inhibition = antisaccades; attention = endogenous cue, working memory = memory guided), neuropsychological tests (symbol digit modalities test, SDMT; California verbal learning test, CVLT; paced auditory serial addition test, PASAT), resting state functional connectivity (default mode network, DMN; dorsal attention network, DAN). For CIS, correlations between DMN/DAN connectivity and cognitive performance (OM and neuropsychological) were completed.

RESULTS
CIS patients demonstrated significantly slowed information processing speed (IPS) on an OM measure of attention (endogenous cue task). For CIS, increased DMN connectivity was associated with slowed IPS on all cognitive OM tasks that measured inhibition, attention and working memory. In contrast, no group differences in  neuropsychological performance (SDMT, CVLT or PAST), or correlations with network connectivity were found.

CONCLUSION
These results suggest that increased connectivity in CIS may not represent an adaptive response that compensates for damage and ameliorates cognitive deficit. Instead, increased connectivity represents a maladaptive response at CIS that results in slowed IPS, only evident when more sensitive measures are used.

Multiple Sclerosis Risk Associated Expression Quantitative Trait Loci in Innate and Adaptive Immune Cell Types.

BACKGROUND
More than 200 single nucleotide polymorphisms (SNPs) have now been associated with Multiple Sclerosis (MS) risk. These SNPs are more commonly found in non-coding regions of DNA, and hence their functions are largely unknown.

OBJECTIVE
We aim to identify MS risk SNPs that function by regulating the expression of nearby immune genes, known as cis expression quantitative trait loci (eQTL).

METHODS
Innate and adaptive immune cell types were purified from untreated MS cases (n=75) and healthy controls (n=99), and transcript expression levels were assayed by microarray. All participants were genotyped on the Illumina Immunochip array. We selected 2720 SNP gene pairs for analysis. Additive linear models were used to test for associations between genotype and gene expression in each cell type. The effect of disease phenotype (ie. MS case/control status) on eQTL was also explored.

1. We report eQTL associations for 63 MS risk loci in both innate and adaptive immune cell types (FDR <0.05), and approximately 40% of these are cell type specific. Interestingly, our data also suggest some MS risk SNPs could have differential regulatory effects in MS cases compared to healthy controls.

CONCLUSIONS
Our studies provide insight into genetically determined immune heterogeneity that contributes to MS susceptibility.

The kynurenine pathway of tryptophan metabolism modulates mouse neural stem cell proliferation

BACKGROUND
The search for molecules which critically regulate neural stem cell (NSC) proliferation is ongoing, underpinning future production of cell lineages for therapy, while helping understand why innate repair in neurodegenerative and neuroinflammatory diseases fails. Our ongoing research has investigated a role of the kynurenine pathway (KP) in healthy metabolism and neurological diseases.

OBJECTIVE
The KP critically regulates bioavailability of the essential amino acid tryptophan, and is induced by interferon treatment. In MS the KP is dysregulated, producing high levels of metabolites including the potent neurotoxin Quinolinic acid. We investigated the hypothesis that modulating the KP by interferon treatment drove changes in NSC proliferation.

METHODS
E14 mouse neurospheres were generated and cultured. Agonists, antagonists or siRNAs to KP enzymes were used to dissect the pathways.

RESULTS
Interferon-gamma (IFN-g) significantly upregulated (4.1 fold) indoleamine-2,3-dioxygenase (IDO-1; the initial rate-limiting enzyme that metabolises Tryptophan) mRNA in NSCs cultured in proliferative conditions, and to a lesser extent with differentiation. NSCs express all KP enzymes and notably, IFN-g also upregulated mRNA for other KP enzymes including Kynureninase. 10IU/mL IFN-g significantly impaired NSC proliferation (*p=0.0323), and alteration of the metabolic state of NSCs including NAD+/NADH ratio (representing cellular energy levels) via Trp depletion (needed for protein biosynthesis). KP metabolites themselves can modulate NSC proliferation, as co-treatment with the neuroprotective Kynurenic Acid partially reversed the reduced proliferation in IFN-g-treated NSCs.

CONCLUSION
This study is the first characterization of KP enzyme expression in NSCs. KP enzymes play a specific role in the biology of NSCs and tryptophan metabolism, and are highly regulated by interferons. Selective KP inhibition could minimize cell death and impaired regeneration during inflammatory episodes and optimize NSC proliferation and differentiation with direct therapeutic applications.

FUNDING ACKNOWLEDGEMENT
The authors acknowledge funding of the Peter Duncan Neurosciences Unit, and from St. Vincent’s Clinic and Ian Potter Foundations.

The kynurenine pathway of tryptophan metabolism is induced by interferon-gamma in human adipose stem cells.

BACKGROUND
Adipose stem cells (ASCs) are multipotent mesenchymal cells, and differentiate into cells including adipocytes, bone and neurons. The Kynurenine pathway (KP) critically regulates bioavailability of the essential amino acid Tryptophan (Trp), and is highly induced by interferon-gamma (IFN-g). In Multiple Sclerosis (MS) the KP is dysregulated, producing high levels of metabolites like potent neurotoxin Quinolinic acid (QUIN).

OBJECTIVE
In bone marrow MSCs, we showed IFN-g significantly reduced proliferation while increasing adipogenic/osteogenic lineage differentiation. Here, we hypothesised modulating the KP using IFN-g would drive changes in ASC proliferation and KP metabolite secretion.

METHODS
ASCs were isolated from healthy donor abdominal lipoaspirates with informed consent, purified and cultured in DMEM/10% FBS for 5 passages prior to image analysis. Secreted KP metabolites were quantified by HPLC/GC-MS.

RESULTS
After 72 hours, 500IU/mL IFN-g significantly depleted extracellular Trp in culture media (1.12±0.14uM vs. Untreated 20.17±0.08uM, p<0.0001, n=3 patient cultures), suggesting significant activity of tryptophan-2,3-dioxygenase (TDO) and/or indoleamine-2,3-dioxygenase-1 (IDO-1). Downstream metabolite Kynurenine was significantly upregulated with IFN-g (32.57±0.36uM vs. Untreated 0.43±0.007uM, p<0.0001); neuroprotective Picolinic acid, and QUIN were minimally altered.

IFN-g caused no significant difference in confluency however cell cycle parameters remain to be evaluated. Phase bright small cells with processes (possible neuroblasts) sharply declined with IFN-g (Time 0, 4.33±0.33 cells/field; no cells at 48/72 hours) vs. Untreated (Time 0, 2.33±0.88 cells/field and 0.67±0.33 cells (72 hours).

CONCLUSION
This is the first KP characterization of ASCs. Future studies will assay KP enzymes and compare to MS patient cells. IFN-g is secreted by e.g. blood T-cells, and could detrimentally affect peripheral stem cell reservoirs like ASCs. Assaying ASCs could allow understanding of the impact of MS on stem cells and regenerative mechanisms, and in turn, give insight into potential CNS alterations.

ACKNOWLEDGEMENT
MDL/VSS/FA/BJB acknowledge funding of the Peter Duncan Neurosciences Unit. NKB/MP acknowledge the Schwartz foundation.

Microglial Mertk is crucial for appropriate myelin compaction and its loss leaves axons vulnerable to demyelination

BACKGROUND
Recent discoveries have shown that microglia play an important role in the development of oligodendrocytes and ultimately in myelination, but the mechanism remains largely unknown. We have been investigating the role of Mertk, a receptor tyrosine kinase expressed on microglia, in this process.

OBJECTIVE
To specifically delete Mertk from microglia in order to investigate the role of this receptor in myelination and myelin damage.

METHODS
We used our recently developed floxed-Mertk mouse line in combination with the cre-driver Cx3Cr1 to specifically delete Mertk from microglia (Mertk cKO). We examined myelin ultrastructure in Mertk cKO mice and WT littermates during development and following cuprizone-induced demyelination. The effect of Mertk deletion upon the density of microglia and oligodendrocytes in the corpus callosum was determined using immunohistochemistry. The expression of myelin genes was determined using quantitative PCR.

RESULTS
Myelin in Mertk cKO mice showed abnormal decompacted morphology, evident as a significant decrease in g-ratio. Myelin alterations were accompanied by a reduction in the expression of myelin genes, and a reduction in microglial density early in development. Whilst the loss of microglial Mertk did not significantly alter the extent of cuprizone-induced demyelination, we observed a significant increase in the g-ratio of myelinated axons in the Mertk cKO mice.

CONCLUSION
The loss of microglial Mertk and resulting changes in myelin structure results in worsened demyelination following cuprizone challenge, indicating that the decompaction of myelin observed during development may leave axons vulnerable to demyelinating events.

Development of a CG4 cell viability assay to assess the bioactivity of Gas6 derivatives

BACKGROUND
Gas6 is a pan-agonist of the TAM (Tyro3, Axl, Mertk) receptor tyrosine kinases that potentiates remyelination. However, with a molecular weight of 80 KD, Gas6 will have limited access to the CNS and needs to be modified to realise its potential therapeutic benefit in progressive MS. 

OBJECTIVE 
To develop a robust bioassay to assess the biological efficacy of derivatives of Gas6 that have superior characteristics comparing the native protein as therapeutic candidates. The rat oligodendroglial cell line, CG4, is a suitable starting point for this purpose as it has unbridled proliferative potential.

METHODS
CG4 cells were seeded on Poly-D-lysine-coated T25 flasks, and cultured in modified Bottenstein-SATO medium. The cells were then differentiated by withdrawal of PDGF and NT3 and plated at a density of ~150 cells/well in a 24 well plate (n=6 per condition). Three culture conditions were assessed; SATO deficient of Insulin, normal SATO and SATO deficient of Insulin supplemented with 100ng/ml rhGas6 (Biotechne, USA). Live/dead cells were counted after 48 hours by using Ethidium homodimer-1/Calcein AM under an inverted florescent microscope, blinded.

RESULTS
In the absence of insulin(a), there was compromised viability (%32.33 ± 3.68) which was partially rescued by Gas6(b) (%44.76 ± 0.49) and further potentiated with insulin in SATO(c) (%86.04 ± 3.64); p<0.05 for (a) vs. (b). The results were analysed using one-way ANOVA with Tukey’s post-hoc correction. The experiment was repeated with similar results.

CONCLUSION
We have established a robust viability assay of a rat oligodendroglial cell line that is useful in determining the bioactivity of derivatives of rhGas6 that we produce in this translational project for developing a novel therapeutic agent based on agonist activity for the TAM receptors.

FUNDING ACKNOWLEDGEMENT
Negar Asadian is supported by the Australian Government Research Training Program.

Characterisation of the EOMES-TBX21low gene expression signature in multiple sclerosis by high-dimensional immunophenotyping of natural killer cells 

BACKGROUND
We previously showed that expression of MS risk genes governing NK cell differentiation is reduced in MS peripheral blood. Natural killer (NK) cells exert immunoregulatory and cytotoxic functions, killing targets implicated in MS pathogenesis such as activated T cells and inflammatory microglia.

OBJECTIVE
The aim of this work was to characterise the dysregulated immunophenotype of NK cells in MS. Immunophenotyping is particularly focussed on genes correlated with reduced expression of the NK global transcriptional regulators, Eomes and Tbet, encoded by MS risk genes EOMES and TBX21.

METHODS
A flow cytometry panel was selected for: 1. canonical phenotyping/ functional molecules;  and antigens: 2. downregulated in MS NK cells (CD244, CD226); 3. MS risk gene-encoded (Eomes, Tbet, CD244, CD226, SLAMF7) or 4. having gene expression levels highly correlated with genes encoding Eomes and Tbet in peripheral blood (perforin, granzyme, SLAMF7, GPR56, CD94). We collected PBMCs from untreated MS patients (n=20) and age- and gender-matched healthy controls (n=20).

RESULTS
A 21-colour flow cytometry panel including 18 NK cell antigens was optimised, including basic phenotype (CD3/CD56/CD16/NKp46) and NK differentiation markers (CD57/CD94), activating and inhibitory receptors (NKG2A/NKG2C/NKG2D), costimulatory receptors (CD244/CD226), killer inhibitory receptors (KIR), transcription factors (Eomes/Tbet), effector molecules (granzyme/perforin) and cytotoxicity-associated (SLAMF7, GPR56) molecules. Immunophenotypic analysis of MS and control samples is in progress and results will be presented.

CONCLUSION
Our development of a high-dimensional immunophenotypic panel allows identification of changes in NK cells in MS including antigen expression levels, subset percentages and potentially of novel subsets. This panel will be available for collaborative studies investigating NK cell changes with disease course and therapeutic response.

FUNDING ACKNOWLEDGEMENT
We thank MS Research Australia, The Trish Foundation and MS Angels for funding this project.

Development of a high-dimensional flow cytometry panel to analyse natural killer cells in multiple sclerosis

BACKGROUND 
Natural killer (NK) cells exert immunoregulatory and cytotoxic functions, with several of their cellular targets implicated in MS pathogenesis, including activated T cells and EBV-infected B cells. NK cell activation is determined by integration of input from a myriad of activating and inhibitory receptors.

OBJECTIVE
Several studies have shown reduced functionality or expression of NK cell effector molecules and transcription factors in MS. Our aim was to develop a high-dimensional flow cytometry panel to characterise dysregulation of NK cells in MS, with particular reference to cytotoxic function and NK differentiation.

METHODS
The NK panel was designed to minimise spectral overlap; with expression level and co-expression of markers considered. Antibodies were titrated and voltages optimised to achieve optimal separation index for each of the antibodies. For chemokine receptor CX3CR1, an additional incubation step at 37°C increased the separation index. Optimisations were performed on an 8-laser flow cytometer (BD Symphony) and differences between compensation methods (spectral versus traditional) analysed.

RESULTS
 A 21-colour single panel including 18 NK antigens was optimised, including basic phenotype and NK differentiation (CD3/CD56/CD16, CD57/CD94), activating and inhibitory receptors (NKG2A/NKG2C/NKG2D), costimulatory receptors (CD244/CD226), killer inhibitory receptors (KIR), transcription factors (Eomes/Tbet), effector molecules (granzyme-B/perforin) and cytotoxicity receptor (NKp46) and cytotoxicity-associated (SLAMF7, GPR56) molecules.

CONCLUSION
This optimised panel will be used for identification of changes in MS in antigen expression levels, NK subset percentages and possibly novel subsets by clustering-based analyses. Future work may include collaborative studies investigating NK cell changes with disease course and response to therapeutic intervention.

FUNDING ACKNOWLEDGEMENT
We thank MS Research Australia, The Trish Foundation and MS Angels for funding this project.

The effect of Dimethyl fumarate treatment on pre-frontal cortex metabolite levels in RRMS using 1H-MR spectroscopy

BACKGROUND
The effect of Dimethyl Fumarate (DMF) in MS is thought to be partly due to its anti-oxidative properties. However, its impact on neurometabolite profiles and anti-inflammatory status of the brain has not been evaluated.

OBJECTIVE
In this study, non-invasive atrophy measures and 1H-MRS was applied to evaluate pre-frontal cortex(PFC) neurometabolite changes in RRMS patients compared to healthy controls (HCs) and evaluate the metabolic impact of DMF treatment longitudinally over 24 months.

METHODS
1H-MRS was undertaken on 20 RRMS patients prior to and up to 24 months post-inception of DMF treatment, and 20 age sex-matched HCs. Spectroscopic data were acquired from PFC using single voxel(3.375ml). To investigate the significant difference between RRMS and HCs groups, T-tests were applied. Changes in metabolite levels during treatment were evaluated using repeated measures ANOVA followed by post hoc testing. Annualised atrophy changes in percentage were assessed using SIENA.

RESULTS
We identified a significant reduction in N-acetylaspartate (-6%,p= 0.05) and increased total choline (+13%,p= 0.02) in RRMS, before starting DMF, compared to HCs. Following treatment, NAA levels significantly increased over 12 months in the RRMS (F= 2.6, p< 0.05), but didn’t significantly change between 1-2yrs. No substantial change was observed in the average annualised percentage of brain volume change between 1st(-0.35±0.14) and 2nd (-0.40±0.12) year of treatment with DMF.

CONCLUSIONS
Our findings suggest that DMF treatment is associated with a modification in the level of N-acetylaspartate in MS. Further studies with longer treatment durations are warranted to investigate the impact of DMF on metabolic changes in the MS brain and their association with disease activity.

Dietary responses to a multiple sclerosis diagnosis: a qualitative study

BACKGROUND
There is insufficient evidence to support a special therapeutic diet to slow progression of multiple sclerosis (MS). Several studies have reported dietary changes made by people with MS, but there has been very limited investigation into perceptions around diet in those recently diagnosed.

OBJECTIVE
We explored attitudes and experiences regarding diet and dietary advice after a recent diagnosis of MS, in people living in Western Australia.

METHODS
Eleven adults with MS (mean time since diagnosis 8 months) participated in semi-structured interviews focusing on responses to diet since MS diagnosis. Interviews were transcribed verbatim, and data were coded and analysed using grounded theory principles.

RESULTS
Participants were mostly female (82%), were diagnosed with relapsing-remitting MS (82%), and the mean age was 47 years (standard deviation, 13). Three theme responses emerged: 1) the perceived incompatibility between the lack of/non-specific dietary advice given at the time of diagnosis and disease seriousness; 2) extensive personal research and information seeking with difficulty judging credibility of information; and 3) self-experimentation with diet to either cure MS or control symptoms.

CONCLUSION
Dietary information provided to people with MS after diagnosis lacks consistency and clarity. Healthcare professionals should address the special diets advertised for MS, and clearly convey reasons behind recommending general healthy eating guidelines. This could reduce information-seeking from less reputable sources.

FUNDING ACKNOWLEDGEMENTS
Curtin University School of Public Health provided funding for this study.

Epigenetic Age Acceleration in Multiple Sclerosis Patients

BACKGROUND
DNA methylation changes with age and age-related markers have been used to establish an “epigenetic clock”, which can accurately predict a person’s chronological age.  Epigenetic age acceleration (EAA) has been previously demonstrated using blood samples from patients with Parkinson’s disease and Downs’ syndrome.

OBJECTIVE
The aim of this study was to investigate if EAA exists in MS in three separate cohorts (Newcastle, Norway and Sweden). Furthermore we wanted to investigate if EAA was associated with disease outcomes in MS patients.

METHODS
DNA methylation data was collected from the CD4+T cells of 53 MS patients and 57 healthy controls (HC) (Newcastle cohort), 47 RRMS patients and 40 HC (Norway cohort) and 36 RRMS patients and 6 HC (Sweden cohort).  EAA was calculated using the Horvath epigenetic clock. Correlations between EAA and Expanded Disability Status Scale (EDSS) were calculated with Pearson’s correlation analysis.

RESULTS
Results show that in each cohort there is no EAA in the MS patients relative to their respective HC cohorts (Newcastle EAA 1.13 SEM 0.92 P=0.22; Norway: EAA -1.6, SEM 1.15 P=0.17; Sweden EAA -0.58, SEM 2.41, P=0.81).  However, further investigation of the Newcastle cohort, identified a moderate positive correlation between EAA and EDSS (r=0.34, P=0.009).

CONCLUSIONS
Although MS patients do not appear to have overall EAA; the correlation between EAA and EDSS suggests that EAA may be linked to increased severity in MS patients. Future analyses will focus on other cell types and correlations between EAA and EDSS in all cohorts.

DISCLOSURES
Professor J Lechner-Scott's institution receives non-directed funding, as well as honoraria for presentations and membership on advisory boards from Sanofi Genzyme, Biogen, Merck, Teva, Roche, and Novartis Australia.

V.E.Maltby has received honoraria for presentations from Merck and Biogen.

Multiple Sclerosis Risk Gene Biomarkers for Vitamin D Response

BACKGROUND
Despite environmental, cellular and genetic evidence indicating the importance of vitamin D in Multiple Sclerosis (MS) susceptibility, supplementation with cholecalciferol has not been beneficial in therapy. Further, there is no assay to assess cellular response to supplementation or alternative forms of vitamin D pathway stimulation.

OBJECTIVE
We aimed to design an assay to determine if vitamin D responsive MS risk genes expression in immune cells is altered by culture with vitamin D in homeostatic and inflammatory conditions.

METHODS
Peripheral blood mononuclear cells from controls were cultured with and without Calcifediol, and with and without stimulation with TNF-a, IFN-b, CD40L, and a combination of antiCD3 and antiCD28. Expression of the MS risk genes CYP24A1, CYP27B1, ZMIZ1, EOMES, PTGER4 was determined in each condition.

RESULTS
CYP27B1 and CYP24A1 increased expression significantly in a number of conditions. CYP27B1 catalyses addition of the second hydroxyl group needed to activate vitamin D. CYP24A1, responsible for inactivation of activated vitamin D, increased expression in conditions where CYP27B1 expression increased, either significantly or by trend. This was most notable where TNFA was the stimulant. Expression of the other risk genes were also affected by vitamin D in specific conditions.

CONCLUSION
These data support this protocol as a method for assessing immunological response to different forms of vitamin D, and agents stimulating the vitamin D pathway, in clinical trials and on therapy.

Deletion Of TrkB Neurotrophin Receptor In Neurons Leads To Functional Deficits

BACKGROUND
Despite the important roles of neurons in regulating normal CNS myelination, it remains unclear how neuronal signals regulate myelin repair and functional recovery following demyelination. BDNF acts through TrkB receptor to promote myelination. However, the exact effect of BDNF-neuronal TrkB signaling in remyelination remains unknown.

OBJECTIVE
We hypothesize that neuronally expressed TrkB receptor promotes myelin repair and functional recovery following a central demyelinating insult. Therefore, we aim to determine the relative contribution of neuronal TrkB to the extent of white and gray remyelination and functional recovery.

METHODS
To do this, we specifically deleted the BDNF receptor TrkB in adult cortical neurons via generating an inducible neuronal-specific TrkB conditional knockout mice (TrkB icKO, TrkB fl/fl : Thy1-EGFP-CreERT2). To induced brain demyelination, murine cuprizone model of demyelination and remyelination was employed and a functional test known as ledged beam test was used to assess balance and coordination.

RESULTS
Following 4 and 5 weeks of oil or tamoxifen injection, the TrkB icKO showed significantly greater number of foot faults compared to the oil control group in the ledged beam test (p<0.0001). Similar results were observed in cuprizone challenged groups. The cuprizone-challenged TrkB icKO performed significantly worse than cuprizone-challenged oil control at both the peak of demyelination (p<0.0001) and during recovery (p=0.0031).

CONCLUSION
The preliminary data suggest that removal of neuronal TrkB results in functional deficit. We are currently examining the extent of myelin pathology to determine if TrkB expression in adult neurons regulates the innate capacity of remyelination and whether this correlates with the functional changes. 

Prevalence of self-reported expressive and receptive languag problems in MS and impact on quality of life: An international cross-sectional pilot study

BACKGROUND
It is only recently that deficits in the area of language have been considered a potential clinical manifestation of MS. Language impairment can place persons with MS (PwMS) at risk of restricted participation in everyday activities that require communication, including vocational, social, and educational settings.

OBJECTIVE
This study investigated the prevalence of self-reported language impairment in MS using a MS-specific patient-reported outcome measure (PROM), and its relationship with health-related quality of life (HRQoL).

METHODS
In this cross-sectional study, 160 PwMS completed the four language items of the Speech pathology-specific questionnaire for persons with MS (SMS) and the HRQoL questionnaire, the 12-Item Short Form Survey (SF-12). Participants were recruited internationally through professional MS bodies and support groups and completed all questionnaires online.

RESULTS
A large number of participants reported language impairment (75%). 65.7% reported word finding difficulties and 41% reported receptive language problems. Statistical analyses showed that age, sex, MS subtype, age at diagnosis, disease duration, educational attainment, and country of residence were not associated with the presence of self-reported language impairment. PwMS with language impairment scored lower than those without language impairment on the SF-12 mental and physical component summary scores. Only one participant received speech therapy.

CONCLUSION
Language functions can be compromised in PwMS regardless of clinical/demographic characteristics and can impact HRQoL. Frontline clinicians need to be aware of potential language impairment in PwMS and make prompt referrals to speech pathologists. Further investigation into the nature and underlying neural mechanisms of language impairment in MS is needed to guide evidence-based management.

The TrkB agonist LM22A-4 increases oligodendroglial populations and remyelination in the brain

BACKGROUND 
There is a strong clinical need to identify remyelinating therapies to prevent progression of disability in central demyelinating diseases. We have shown that the neurotrophin brain-derived neurotrophic factor (BDNF) and structural peptide mimic TDP6 promotes myelin repair by acting through oligodendrocyte expressed TrkB receptors.

OBJECTIVE
To compare the influence that TDP6 and another known chemical TrkB agonist, LM22A-4, exert upon remyelination in the cuprizone model of toxic central demyelination.

METHODS
Wild-type and transgenic mice with conditional deletion of TrkB from maturing oligodendrocytes were subjected to cuprizone-induced demyelination for 5 weeks.  At peak of disease, mice were subjected to intra-cerebral ventricular administration of either TDP6, LM22A-4 or vehicle.  After 1 week treatment, mice were killed, brain dissected, and the corpus callosum analysed for both myelin and oligodendroglia.

RESULTS
In wild type mice, LM22A-4 and TDP6 enhance myelin repair to a similar extent, increasing levels of myelin basic protein and myelin sheath thickness.  Whereas TDP6 only increased the density of post-mitotic oligodendrocytes, LM22A-4 increased the density of both oligodendrocyte precursor cells and post-mitotic oligodendrocytes. These effects of LM22A-4 were TrkB specific, as it exerted no effect on myelin repair in transgenic mice carrying a TrkB-deletion in oligodendrocytes.

CONCLUSION
These data suggest that LM22A-4 is a TrkB agonist that can promote myelin repair in vivo.  This study further supports the development of therapeutics that target TrkB signalling to promote myelin repair in central demyelinating disease.

Narrowband UVB phototherapy alters peripheral blood immune cell frequencies in people with clinically isolated syndrome

BACKGROUND 
UV radiation exposure has immunosuppressive effects in humans. Low environmental UV radiation exposure is associated with multiple sclerosis risk. Narrowband UVB phototherapy is an established treatment for psoriasis, and is under investigation in people with clinically isolated syndrome (CIS) at risk of developing MS.

OBJECTIVE 
To determine whether systemic leukocyte phenotypes in blood are significantly altered by a short course of narrowband UVB phototherapy in people with CIS.

METHODS 
20 participants with CIS were recruited and half received narrowband UVB phototherapy in 24 sessions over 2 months. Peripheral blood mononuclear cells (PBMC) were collected at baseline, and 1, 2, 3, 6 and 12 months after enrolment. Multicolour flow cytometry was used to identify T cell, B cell, monocyte, dendritic cell, and natural killer cell subsets. The effects of phototherapy on the frequencies of PBMCs were examined using linear regression.

RESULTS 
Significant differences in the frequencies of PBMC phenotypes were detected between treated and untreated individuals during phototherapy, including in subsets of B cells, monocytes and T cells. No effects were detected after phototherapy treatment ceased. The largest treatment effects were detected in B cell subsets, where phototherapy was associated with significantly higher naïve B cell frequency and significantly lower frequency of switched memory B cells.

CONCLUSION
Narrowband UVB phototherapy may contribute to immune suppression through regulation of systemic leukocyte phenotypes in people with CIS.

FUNDING ACKNOWLEDGEMENT
This work was initially funded by a National Health and Medical Research Council Project Grant (ID 1067209) and subsequently by Multiple Sclerosis Western Australia (MSWA).

Latitude continues to be significantly associated with the prevalence of multiple sclerosis: an updated meta-analysis

BACKGROUND
Previous studies have demonstrated a strong latitudinal gradient in multiple sclerosis (MS) prevalence. We previously demonstrated a positive latitudinal gradient in 2011 using studies published until 2010.

OBJECTIVES
We conducted a meta-analysis of MS prevalence including studies published since our 2011 review, seeking to assess the latitudinal gradient and whether it has changed since our previous study.

METHODS
Studies published prior to December 2018 were located via EMBASE, ISI and PubMed, using standardised search terms; data was extracted from peer-reviewed studies and these studies added to those from our previous analysis. Where age-specific data were available, prevalence estimates were age/sex-standardised to the 2009 European population. Prevalence estimates were adjusted for study prevalence year and case ascertainment methods. The latitude association with MS prevalence was assessed by meta-regression.

RESULTS
94 studies met inclusion criteria, yielding 230 new prevalence points and 880 altogether. There was a significant positive gradient in time-corrected MS prevalence with increasing latitude (5.27/100,000 per degree latitude), attenuating slightly to 4.34/100,000 on age-standardisation and persisting on adjustment. The age-standardised gradient was significantly enhanced from our previous study, regardless of adjustment. The Scandinavian and Atlantic Coast/Central Europe regions showed significant changes in regional gradient, but other regional gradients were similar.

CONCLUSIONS
This new meta-analysis confirms that MS prevalence is still strongly positively associated with increasing latitude and that the gradient is increasing, suggesting that potentially modifiable environmental factors like sun exposure are still strongly associated with MS risk.

Change in Multiple Sclerosis Prevalence Over Time in Australia 2010-2017 Utilising Disease Modifying Therapy Prescription Data

OBJECTIVE
Determine the prevalence of multiple sclerosis (MS) in Australia in 2017 using MS-specific disease-modifying therapy (DMT) prescription data and estimate the change in prevalence from 2010.

METHODS
DMT prescriptions were extracted from Australia’s Pharmaceutical Benefits Scheme (PBS) data for January-December 2017. Percentages of MS patients using DMTs (DMT penetrance) were calculated using data from the Australian MS Longitudinal Study. Prevalence was estimated by dividing total number of monthly prescriptions by 12 (except alemtuzumab), adjusted for DMT penetrance and Australian population estimates. Prevalences in Australian states/territories were age-standardised to the Australian population. Comparisons with 2010 prevalence data was performed using Poisson regression.

RESULTS
DMT penetrance was 64%, yielding an estimated number of people with MS in Australia in 2017 of 25,607 (95%CI: 24,874-26,478), a significant increase since 2010 (p<0.001). The prevalence increased significantly from 95.6/100,000 (2010) to 103.7/100,000 (2017), with 2017 estimates highest in Tasmania (138.7/100,000; 95%CI: 137.2-140.1) and lowest in Queensland (74.6/100,000; 95%CI: 73.5-75.6). Using the median latitudes for each state/territory, the overall latitudinal variation in MS prevalence was an increase of 3.0% per degree-latitude.

CONCLUSIONS
Consistent with global trends, Australia’s MS prevalence has increased, this probably reflecting decreased mortality, increased longevity and increased incidence.

FUNDING ACKNOWLEDGEMENT
This work was supported by funding from MS Research Australia.

Perceived Cognitive Impairment is Significantly Associated with Perceived Sexual Dysfunction in People Living with MS

BACKGROUND 
There is no evidence available about the relationship between perceived cognitive impairment and perceived sexual dysfunction.

OBJECTIVES
To explore the prospective relationship between cognitive and sexual dysfunction over 2.5 years in people with MS.

METHODS
Data were derived from the HOLISM international online cohort. Cognitive function and sexual function were assessed by the relevant subscores of the MSQOL-54. Determinants of perceived sexual dysfunction (PSD) assessed by log-binomial regression, adjusting for age, sex, fatigue, depression, marital status, physical activities, comorbidity and MS severity scale.

RESULTS
2,281 participants provided MSQOL data at baseline, of whom 555 had incident PSD at follow-up. The prevalence of PSD was 45.6%, and cognitive dysfunction was 45.4%. Incident PSD was significantly higher among those with baseline cognitive impairment (RR=1.56), this association higher among those with perceived cognitive impairment (RR=1.74) and among those who became cognitively impaired during follow-up (RR=2.09). These associations were solely evident among females, and among non-married/partnered participants.

CONCLUSION
These results indicate that cognitive dysfunction is an independent risk factor for perceived sexual dysfunction in people with MS. This may represent an additional dimension whereby MS may adversely affect quality of life in this population.

FUNDING ACKNOWLEDGEMENT
The study was funded by the Bloom Foundation, Wal Pisciotta, and the Horne Family Charitable Trust. Funding was received from The Royal Australian and New Zealand College of Psychiatrists, New Investigator Grant (Dr Keryn Taylor).

Mastery significantly associated with physical and mental health-related quality of life in two independent international samples of people with MS

BACKGROUND
Mastery is the sense of being in control of one’s life. Improvement in mastery may help to improve quality of life, the pathway potentially being through stress modulation or greater utilisation of healthy lifestyle behaviours known to improve outcomes in MS. 

OBJECTIVES
To explore the association between mastery and health-related quality of life (HRQOL) in people with multiple sclerosis.

METHODS
Two cross-sectional samples of adults with MS (n=1,401, n=573), recruited through convenience sampling, completed an online survey which measured mastery using the Pearlin Mastery Scale (PMS; range 14-28), physical and mental HRQOL (range 0-100) via the physical and mental composite scores of the MSQOL-54. 

RESULTS
Mastery score was associated with significantly higher physical and mental HRQOL in both samples, such that a one-point increase in the PMS was associated with a 1.75 (p<0.001) and 1.80 (p<0.001) rise in physical HRQOL in the two samples, and a 1.54 (p<0.001) and 1.66 (p<0.001) increase in mental HRQOL in the two samples, all associations highly robust to adjustment.

CONCLUSION
These results indicate mastery is associated with a better quality of life, both physical and mental. Efforts to improve patients’ sense of self control and agency may have benefits for their quality of life.

FUNDING ACKNOWLEDGEMENT
The study was funded by the Bloom Foundation, Wal Pisciotta, and the Horne Family Charitable Trust. Funding was received from The Royal Australian and New Zealand College of Psychiatrists, New Investigator Grant (Dr Keryn Taylor).

Associations of demographic and clinical factors with positive depression-screen over 2.5-years in an international prospective cohort of people living with multiple sclerosis

BACKGROUND 
Depression is common in multiple sclerosis (MS), estimates ranging up to 50% in some studies. Determinants of depression and change in depression state would inform clinical practice and care in this population.

OBJECTIVES
We examined the relationship of demographic and clinical factors with screening positive for depression, as well as prospective associations of these factors with positive depression-screen trajectory over 2.5 years of follow-up of people with multiple sclerosis (MS).

METHODS
Positive depression-screen was assessed by the Patient Health Questionnaire-2 and Patient Health Questionnaire-9 at baseline and 2.5-year review, respectively. The prospective associations of demographic and clinical factors at baseline with positive depression-screen at follow-up were assessed using multivariable regression models, adjusted for age, sex, disability, fatigue and antidepressant use.

RESULTS
Baseline overweight/obese BMI, comorbidity number, fatigue, and disability were associated with depression and higher PHQ-9 at 2.5-year review, as well as newly positive screen at follow-up. Baseline married/partnered state, employment, and education were associated with significantly lower frequencies of depression. Other features of clinical course, including MS type, relapse number, and disease trajectory were not significantly associated with screening outcomes after controlling for disability and fatigue.

CONCLUSIONS
In this, the largest prospective cohort study of depressive symptoms in people living with MS, we have substantiated several accepted predictors of a positive depression screen and positive depression-screen trajectory, particularly fatigue.

FUNDING ACKNOWLEDGEMENT
The study was funded by the Bloom Foundation, Wal Pisciotta, and the Horne Family Charitable Trust. Funding was received from The Royal Australian and New Zealand College of Psychiatrists, New Investigator Grant (Dr Keryn Taylor).

Longitudinal associations of modifiable lifestyle factors with positive depression-screen over 2.5-years in an international cohort of people living with multiple sclerosis

BACKGROUND
Depression is common and has the most significant impact on quality of life for many people with multiple sclerosis (MS). A preventive management approach via modification of lifestyle risk factors holds potential benefits.

OBJECTIVES
We examined the relationship of modifiable lifestyle factors and depression risk and the change in depression over 2.5 years.

METHODS
Sample recruited using online platforms. 2,224 (88.9%) at baseline and 1,309 (93.4%) at 2.5 year follow up completed the necessary survey data. Survey data included depression risk, measured by the Patient Health Questionnaire-2 at baseline and Patient Health Questionnaire-9 at 2.5-year follow-up. Multivariable regression models assessed the relationships between lifestyle factors and depression risk, adjusted for sex, age, fatigue, disability, antidepressant medication use, and baseline depression score, as appropriate.

RESULTS
Alcohol intake, non-smoking, diet quality, vitamin D and omega-3 supplementation, exercise, and meditation were associated with lower frequencies of positive depression-screen. Moderate alcohol intake was associated with greater likelihood of becoming depression-free and a lower likelihood of becoming depressed at 2.5-year follow-up. Smoking, diet, physical activity, and vitamin D and omega-3 supplementation were not independently associated with a change in depression. 

CONCLUSION
In a large prospective cohort study of people with MS and depression, these results suggest a role for some lifestyle factors in depression risk. Further studies should explore the impact of lifestyle and depression in people living with MS. 

FUNDING ACKNOWLEDGEMENT
The study was funded by the Bloom Foundation, Wal Pisciotta, and the Horne Family Charitable Trust. Funding was received from The Royal Australian and New Zealand College of Psychiatrists, New Investigator Grant (Dr Keryn Taylor).

Prudent dietary pattern predicts lower relapse hazard in early multiple sclerosis

BACKGROUND
Dietary patterns and their relationship with clinical course have not been well-studied in early multiple sclerosis (MS).

OBJECTIVES
Examine dietary patterns (DP) in people at hazard of MS in the five years following symptom onset and assess relationships with clinical outcomes.

METHODS
Dietary intake was measured by Food Frequency Questionnaire, this adjusted for energy intake. DP-scores were identified using iterated principal factor analysis. Longitudinal outcomes were MS conversion & relapse hazards and annualised disability progression. Fatigue was assessed at five-year review. All analyses adjusted for age, sex, study site, and energy intake; further adjustments for BMI, smoking, and supplement use were also undertaken.

RESULTS
Three DPs, ‘Western, ‘Mixed’, and ‘Prudent’, were evident, explaining 47%, 32% and 28% of the variance in dietary intake, respectively. Baseline Western DP-score was significantly higher overweight participants, current smokers, and those engaged in physical labour, but lower among females and older participants. Mixed DP-score was lower among current smokers and those who had completed higher education. Prudent DP-score was significantly higher among older participants, those who were more physically active, and those who completed university education, but lower among unemployed participants. Current smokers at baseline had significantly greater increase in Western DP-score but lower increase in Prudent DP-score, while those who were obese at baseline had significantly lower increase in Mixed DP-score but greater increase in Prudent DP-score. DP-scores were not associated with MS conversion hazard. Continuous baseline Prudent DP-score was associated with significantly lower relapse risk (HR=0.81, p=0.045), persisting on further adjustment for BMI, smoking, and omega-3/6 supplement use (aHR=0.77, p=0.022). This association was driven almost entirely (86.2%) by wholegrain, non-fried fish, non-fried potato, and fresh fruit consumption. Baseline Prudent DP-score showed a significant positive association with subsequent disability progression, with those above the 25th percentile having 0.20-points higher progression (p=0.002), robust to adjustment. No DP-scores were associated with fatigue at 5-year review.

CONCLUSIONS
Diet quality improved over 5 years of follow-up. Healthier diet pattern scores were prospectively associated with lower relapse risk, but with greater disability progression. 

FUNDING ACKNOWLEDGEMENT
The Ausimmune and AusLong Studies were funded by the National Multiple Sclerosis Society of the United States of America (award RG3364A1/2) and the National Health and Medical Research Council of Australia (APP316901 and 224215).

Determinants of self-reported adherence to popular diets recommended for people living with multiple sclerosis

BACKGROUND
A range of diets have been recommended for people living with MS, ranging from low or no-meat Swank-inspired diets, to the modified Palaeolithic Wahls diet. While the clinical efficacy of these diets to modulate MS progression is uncertain, the popularity of these diets amongst people living with MS is manifest.

OBJECTIVES
This project will evaluate the distribution and determinants of self-reported adherence to the most popular diets recommended for people with MS, including the Swank, McDougall, Overcoming MS (OMS), Ashton-Embry Best Bet, Wahls Modified Palaeolithic, or Other diets.

METHODS
Data derived from the 5-year review of the HOLISM international cohort study. Self-reported adherence to diets were queried, ranging 1-5. Adherence was restricted to ≥12months adherence, and then dichotomised: those reporting 4-5/5 adherence defined 2nd-most adherent, 3-5/5 adherence defined 3rd-most adherent. Determinants of adherence were evaluated by log-binomial regression, adjusted for age, sex, socioeconomic status (SES), education, and fatigue. Due to low numbers, only the OMS, Swank and Wahls diets were quantitatively evaluated.

RESULTS
458/952 (48.1%) reported following MS-specific diets for ≥12months; 61.8% followed OMS, 12.7% Swank, 3.5% Wahls, 0.9% Ashton-Embry, 0.9% McDougal, 5.7% other. OMS adherence (2nd-most) was more common among males, higher SES, lower disability, less fatigue, and fewer comorbidities. Wahls adherence (2nd-most) was more common among participants with progressive MS or greater disability. Swank adherence (2nd-most) did not vary by any parameter. Analogous results were found using the 3rd-most adherent definition.

CONCLUSIONS
These results indicate that MS-specific diet adherence is common in this patient population, but uptake is highly heterogenous between demographic and clinical subgroups. Further study of diet uptake among MS patients is indicated, particularly as the efficacy of these diets in MS is yet uncertain.

FUNDING ACKNOWLEDGEMENT 
Bloom Foundation, Horne Family Charitable Trust,  and Michael (Mick) Martin and Elizabeth Schefferle.

Using the SF-6D to generate health state utilities in an international cohort of people living with multiple sclerosis

BACKGROUND
While a number of studies have examined the impacts of multiple sclerosis (MS) on quality of life (QoL), generic instruments have been developed to estimate the global impact on health-related QoL, namely health state utilities (HSU) valued on a scale 0-1.

OBJECTIVES
To derive SF-6D HSU values from the SF-36 component of the MSQOL-54 in the Health Outcomes and Lifestyle In a Sample of people with MS (HOLISM) international cohort at baseline, and describe the distribution and determinants thereof.

METHODS
QoL was evaluated among participants with diagnosed MS in the HOLISM cohort at baseline using the MSQOL-54, which includes the SF-36 and 18 MS-specific questions. HSUs were generated using the SF-6D algorithm for the Version 1 US SF-36 for participants with sufficient data to generate a HSU (n=2,185/2,466 (88.6%)). Determinants of HSU were evaluated by linear regression, adjusted for age, sex, MS type, disability, fatigue, and prescription antidepressant medication use.

RESULTS
The mean HSU in the sample was 0.67 (SD: 0.13; range=0.30-1.00). Employment, SES, married/partnered-status, larger social network-size, physical activity, vitamin D and omega-3 supplement use, and alcohol consumption were associated with higher HSU, and BMI and tobacco smoking with lower HSU, but age, sex, and education were not associated. Severe disability (P-MSSS) and clinically significant fatigue (FSS) were each associated with 11%-lower HSU, and positive depression screen (PHQ-2) with 10%-lower HSU.

CONCLUSIONS
We have described HSUs for the largest sample of people with MS thus far studied, showing clinically meaningful differences in HSU for a range of demographic, lifestyle, and clinical characteristics. This will inform our understanding of the determinants of HSU and QoL in MS.

FUNDING ACKNOWLEDGEMENT 
Bloom Foundation, Horne Family Charitable Trust, and Michael (Mick) Martin, Elizabeth Schefferle and an anonymous donor.

The long-term effects of disease modifying therapies on disability in people living with multiple sclerosis: a systematic review and meta-analysis

BACKGROUND
Disease modifying therapies (DMT) are a common medication class for treating people living with MS. However, although treatment with DMT can extend over more than a decade, little is known about their long-term effects.

OBJECTIVE
Here, we systematically review long-term (≥4 years) studies on the effect of DMT on disability progression and relapse in people living with MS.

METHODS 
We searched the EMBASE and Medline databases in January 2018, using search terms that included DMT and relevant outcome measures. Two authors screened all resulting studies and evaluated the risk of bias of included studies using the ROBINS-I tool for non-randomised studies. Where there was sufficient data, we performed meta-analyses using RevMan 5. Studies that could not be included in a meta-analysis were included in data synthesis.

RESULTS 
Our search returned 7,766 unique articles for review. After screening, 18 articles were included. Follow-up in these studies ranged from a mean of 3.9 years to a median of 17.8 years. Fifteen (83.3%) of the included studies had a moderate risk of bias and three (16.7%) had a serious risk of bias. Meta-analysis showed that DMT significantly reduced the risk of EDSS 6.0 and SPMS compared to no treatment.

CONCLUSION 
There is some evidence that long-term treatment with interferon beta reduces the risk of EDSS 6.0 and SPMS compared to no treatment or placebo. More work is needed on the effect of second generation DMT and the relative effect of DMT on health outcomes.

Building a massive open online course (MOOC) about multiple sclerosis for a general audience: A process evaluation

BACKGROUND 
There is often a disconnect between research discovery and knowledge translation. This gap can make it difficult for end users to access the information they need to make informed decisions and can cause frustration for researchers that are eager to share their work.

OBJECTIVE 
Here, we present the results of a process evaluation of the development of a knowledge translation intervention: a massive open online course (MOOC) about MS aimed at a general audience, the Understanding Multiple Sclerosis online course.

METHODS 
We developed an initial course outline from materials used by the Wicking Dementia Research & Education Centre for their MOOCs. We carried out an initial community consultation in a focus group of 21 MS community members. Based on the results of this consultation, we developed the course materials (videos, text, images, animations, quizzes and discussion prompts), working collaboratively with a wide range of experts. Finally, we conducted a pilot study.

RESULTS
The subject areas focus group participants most commonly identified as important were disease management, symptoms, and support. They preferred videos to other modes of information delivery and emphasised the need for clear communication and the inclusion of MS community perspectives. Focus group facilitators felt that the process was effective and rewarding. The course was well received; 93% of pilot study participants were satisfied or very satisfied with the course overall.

CONCLUSION 
We would recommend our approach for constructing health-related online courses aimed at a general audience. In particular, we found the close partnership we established between researchers, service providers, consumers, and clinicians to be highly effective and beneficial for the finalised intervention.

Assessing long-term impacts of disease modifying therapies: preliminary results from the Comparing Australia and New Zealand MS Populations (COMPANZ) Study

BACKGROUND 
Disease modifying therapies (DMT) are a major medication class for people with relapsing MS. However, our knowledge about these medications is largely limited to their short-term effects due to the financial and logistical constraints of studying their long-term effects using traditional methods.

OBJECTIVE 
The Comparing Australia and New Zealand MS Populations (COMPANZ) project was an ecological, observational cohort study that compared populations with similar demographics, but different levels of access to DMT (Australia subsidised DMT 6 years before New Zealand (NZ)), to determine the long-term effects of DMT.

METHODS 
We utilised convenience samples from the Australian MS Longitudinal Study (AMSLS) and the NZ MS Prevalence Study and compared the treated and untreated participants. Our primary outcome was the Multiple Sclerosis Severity Score (MSSS). Our primary predictors were the use of DMT and the number of months between diagnosis and first DMT treatment. Relationships were assessed with back-transformed linear regression models.

RESULTS 
We recruited 328 Australian subjects (72.9% of the eligible cohort), 93.9% of whom were exposed to DMT, and 256 NZ (51.6% of the eligible cohort), 50.8% of whom were exposed to DMT. In our preliminary analysis, DMT use significantly affected EDSS. However, despite numerical differences, there were no other significant effects of the primary predictor variables.

CONCLUSIONS 
This study provides some preliminary evidence for a protective role for DMT therapy at 10-20 years post-diagnosis. However, the effect was not large. These results highlight the difficulty of obtaining meaningful data on long-term outcomes outside of prospective longitudinal cohort studies or clinical trials.

Vitamin D modulation on P2X7 receptor in Multiple Sclerosis

BACKGROUND
Multiple Sclerosis (MS) is a demyelinating disease of the central nervous system. Due to its neuroinflammatory nature, a link between ionotrophic P2X7 receptor (P2X7R) and disease development is proposed. Previous studies have shown the association of vitamin D deficiency and increased relapse rate in MS.

OBJECTIVE
Previous research has shown that inhibition of the proinflammatory P2X7R channel function in chronic kidney disease patients through vitamin D supplementation. Similar studies on MS are lacking. Our research aims to investigate whether vitamin D modulates P2X7R expression and function on monocytes from MS patients.

METHODS
Peripheral monocytes were isolated from blood of MS patients (n=5) and healthy volunteers (n=7) using monocyte enrichment and density gradient centrifugation. After isolation, monocytes were incubated with 1,25(OH)2D3 for 72 hours in a 37 Celsius incubator. P2X7R channel and pore function were investigated using confocal microscopy with fluorescence dyes. Immunohistochemistry using specific antibodies for P2X7R and CD11b (marker of activated monocytes) was used to investigate P2X7R expression on monocytes.

RESULTS
Results showed increased P2X7R expression on monocytes from MS patients compared to healthy controls (P<0.0001). Vitamin D treatment reduced P2X7R expression on monocytes from MS patients (P=0.05). Regarding P2X7R channel function, the application of vitamin D to monocytes reduced channel function in both MS patients (P=0.04) and heathy controls (P=0.0002). However, vitamin D treatment did not significantly reduce P2X7R pore function.

CONCLUSION
Preliminary findings of our study showed potential therapeutic benefits of vitamin D in MS through modulation of P2X7R expressed on human monocytes. Our results also provided a possible mechanism of action of vitamin D in MS. Further investigations are required to verify our findings.

Vitamin D levels in Australian Inpatients – levels according to medical specialty, year, age, gender, and season

BACKGROUND
One billion people worldwide are thought to have deficient levels of vitamin D and Vitamin D has been found to have immunomodulatory roles in a number of inflammatory conditions including Multiple Sclerosis (MS). Low circulating 25-hydroxyvitamin D (25[OH]D) is associated with a higher MS risk.

OBJECTIVES 
The aim of this study was to evaluate Vitamin D levels in patients from a tertiary hospital in Victoria, Australia.

METHODS
>30,000 individuals from a tertiary heath centre in Vic Australia who had their Vitamin D tested in the last 5 years were included in the study. The individuals were divided according to medical specialty, and demographics. For those from Neurology and those with diagnosis of MS, the level of Vitamin was correlated with clinical parameters such as disease activity.

RESULTS
We found that males were deficient in Vitamin D compared to females. Patients under neurology specialty had a higher levels compared to patients within other specialties. Vitamin D levels were higher in 2014 compared to 2017. Winter was associated was lower Vitamin D levels compared to summer. In the MS cohort there was an association between Vitamin D levels and disease activity.

CONCLUSION 
Older age, female gender and neurology patients had higher vitamin D levels contrary to previous reports globally. Our study reveals that despite the role of supplementation, the pharmacology and biology of Vitamin D is more complex than previously thought. Further research is warranted to establish the role of Vitamin D in health and disease state, and the various contributors to total serum levels.

The International women in multiple sclerosis network (iWiMS)

The International Women in Multiple Sclerosis (iWiMS) network unites clinicians and researchers committed to advancing scientific discoveries and improving care for people with MS and related disorders. Founded by women and supportive of all, iWiMS advocates for meritocracy, diversity, parity, and ingenuity in MS research and patient care.

Dr Jokubaitis will describe the iWiMS network and its efforts to-date to promote equity, diversity and parity in our field.

The Researching the Effectiveness of a nutraceuticaL Intervention for multiplE sclerosis-related Fatigue (RELIEF) Trial: a multi-center randomized controlled trial protocol

BACKGROUND
Fatigue and depression are highly significant and major causes of impairment in multiple sclerosis. Over half of people with MS consider fatigue their worst symptom. Depression is frequently comorbid with fatigue. Unfortunately, existing medications used to treat MS-related fatigue also have low efficacy. Recent work indicates that the kynurenine pathway and mitochondrial dysfunction may be central in MS pathogenesis. Single mitochondrial agents have demonstrated efficacy in reducing these symptoms in preliminary randomized controlled trials in MS. However, combined therapy for MS has not been assessed.

OBJECTIVE
The Researching the Effectiveness of a nutraceuticaL Intervention for multiplE sclerosis-related Fatigue (RELIEF) Trial aims to investigate a combined mitochondrial support therapy in people with multiple sclerosis.

METHODS
The RELIEF trial is a 20-week multi-centre randomized double-blind placebo-controlled trial. One hundred and fifty participants with relapse remitting multiple sclerosis will be randomly assigned to receive a placebo or a combined mitochondrial therapy intervention for 16-week with a 20-week follow up timepoint. During this period, data regarding fatigue and depression will be measured as well as biological measures (kynurenine pathway metabolomics, microbiome, antioxidant status, and genomics).

RESULTS
The trial is ongoing and is expected to be completed by late 2020.

CONCLUSION
The RELIEF trial aims to demonstrate the effectiveness of a highly innovative intervention for fatigue in multiple sclerosis, a setting where no efficacious therapies exist. This trial may provide a new intervention against depression in MS, with a therapy that is targeted directly to improve reduced mitochondrial biogenesis in MS.

FUNDING ACKNOWLEDGEMENT
NHMRC MRFF and MS Research Australia

The effect of emerging nutraceutical interventions for clinical and biological outcomes in multiple sclerosis: a systematic review

BACKGROUND
Due to the considerable burden of multiple sclerosis (MS)-related symptoms and the need to identify effective interventions to prevent disease progression, various nutraceutical interventions have been trialed as adjunctive treatments.

OBJECTIVE
The aim of this review was to investigate the efficacy and safety of nutraceutical interventions for clinical and biological outcomes in people with MS.

METHOD
In accordance with PRISMA reporting guidelines, a systematic literature search was conducted using three electronic literature databases. Risk of bias was assessed using the Jadad scale.

RESULTS
Thirty-seven randomized controlled trials, investigating fourteen nutraceuticals, were included in the review. Trials that investigated alpha lipoic acid (n=4/6), ginkgo biloba (n=3/5), vitamin A (n=2/2), biotin (n=1/2), carnitine (n=1/2), green tea (n=1/2), coenzyme Q10 (n=1/1), probiotics (n=1/1), curcumin (n=1/1), andrographis paniculata (n=1/1), ginseng (n=1/1),  and lemon verbena (n=1/1) were reported to improve biological (e.g. MRI brain volume change, antioxidant capacity) and/or clinical (e.g. fatigue, depression, Expanded Disability Status Scale) outcomes in multiple sclerosis compared to control. However, most trials were relatively small (average total study sample n=55) and there were few replicate studies per nutraceutical to validate the reported results. Risk of bias across most studies was low, with 31 studies receiving a score between 4 and 5 (out of 5) on the Jadad Scale.

CONCLUSION
The existing literature provides preliminary support for the use of several nutraceutical interventions in MS. However, sufficiently powered long-term trials are required to expand the currently limited literature and to investigate unexplored nutraceuticals that may target relevant pathways involved in MS such as mitochondrial dysfunction and the gut microbiome.

Relative validity of dietary assessment methods used to determine food group and nutrient intakes in the international HOLISM cohort

BACKGROUND
Multiple sclerosis (MS) affects 2.5 million people and their families, globally. The aetiology for MS is unclear, though lifestyle factors, including nutrition, have been implicated. Unfortunately, the evidence-base for nutrition and MS is limited, because of a lack of systematic measurement.

OBJECTIVE
The aim of the study was to validate the Diet Habits Questionnaire (DHQ) of persons with MS against a gold standard dietary assessment methodology, 24-hour recall as a sub-study of the HOLISM cohort for expanding the DHQ.

METHODS
Participants from Australia (n=14), New Zealand (n=8), the United Kingdom (n=22) and the United States (n=12) completed the DHQ as per the HOLISM protocol. Repeated 24-hour recalls using the Automated Self-administered Assessment-24 tool were used to model usual dietary intakes. The Australian Dietary Guidelines food composition database was applied to calculate the usual intakes of food groups. The DHQ scores of key nutrients and food groups were statistically compared with usual intakes.

RESULTS
The median total score of the DHQ was 90 (IQR 79.25-94.00). There was a moderate agreement for fibre (r=0.524, p=0.000) and omega-3 fatty acids (r=0.565, p=0.000) between the DHQ scores and usual dietary intake. This was not seen for total fat (r=0.055, p=0.686). The median DHQ scores of fruits and vegetables of the participants from Australia and New Zealand were greater than 4, however, they only met the dietary guidelines recommendations for serves of vegetables.

CONCLUSION
Validated dietary assessment methods are vital to cohort studies that consider lifestyle factors such as nutrition. While relative validity is a useful indicator of the accuracy of a tool, the use of biomarkers are better suited to adequately validate these tools particularly in a population that is heavily influenced by health eating messages.

Motivation to engage in and promote exercise is driven by availability and education; perspectives of the multiple slerosis community

BACKGROUND
Exercise training is one of the best approaches for managing the consequences of multiple sclerosis (MS). Many Australians with MS do not achieve recommended levels of exercise to gain health benefits.

OBJECTIVES
We sought to understand the experiences of members of the MS community who lived or worked in regional or remote Australia in relation to exercise delivered by healthcare professionals.

METHOD
We undertook a qualitative research study and invited members of the MS community (i.e., persons with MS, carers, healthcare professionals and healthcare managers)  to participate in focus groups and interviews. Participants lived in, or were responsible for patients who lived in outer regional or remote Australia. Data was analysed using interpretative descriptive methodology and we generated themes and subthemes to understand the experiences of the entire MS community. 

RESULTS
We interviewed 28 people with MS, 8 carers, 12 healthcare professionals and 16 supervisors/managers of healthcare service. Overall, the MS community would be more motivated to participate in exercise services if 1) they were knowledgeable and comfortable with exercise, 2) they were provided with training on, and access to exercise, and 3) there was good communication between people with MS and their healthcare providers in relation to exercise.

CONCLUSIONS
Rurality is only one factor influencing motivation to exercise; education on exercise appears of high importance. Education on exercise for MS is required throughout the MS community and the patient-provider relationship remains an influential juncture to encourage exercise participation.

FUNDING ACKNOWLEDGEMENT
Multiple Sclerosis Research Australia

Proprioception and muscular performance in people with multiple sclerosis

BACKGROUND
Mobility deficits and falls can be disabling for people with multiple sclerosis (MS). Impairments in proprioception at the ankle and/or motor performance of the ankle plantarflexor (calf) muscles can contribute to gait and balance deficits, but have been little investigated in people with MS.

OBJECTIVE
To identify how proprioception and motor performance at the ankle are impaired in people with MS by comparing the performance of people with MS (n = 30, Expanded Disability Status Score range 2 ‑ 6) and aged- and sex-matched healthy controls (n = 30).

METHODS
Participants rested their foot on a motorised footplate with the knee straight to test 3 aspects of ankle proprioception; threshold to detection of passive movement, proprioceptive reaction time, and joint position sense. To test muscular performance, participants performed isometric, maximal voluntary contractions of the plantarflexor muscles. Strength and voluntary activation (neural drive to the muscles) were tested with brief maximal contractions before and after a sustained fatiguing maximal contraction.

RESULTS
Reaction time was slower for people with MS (control mean±SD = 0.26±0.1 s, MS = 0.37±0.2 s, p = 0.001), but other proprioceptive measures were unimpaired. Plantarflexors were weaker (controls = 101.1±34 Nm, MS = 75.9±35.3 Nm, p = 0.005) and less activated (controls = 90.2±12.5%, MS = 75.6±23.7%, p = 0.008) in brief contractions. Voluntary activation fell more during the fatiguing contraction in people with MS (p = 0.0001).

CONCLUSION
The inability to react quickly to perturbations under the foot and weaker calf muscles with reduced neural drive may contribute to impaired walking ability and an increased fall risk in people with MS.

FUNDING ACKNOWLEDGEMENT
NHMRC Program grant “Motor Impairment” funded this study.