Sessions and Posters

Associate Professor Ellen Mowry is an Associate Professor of Neurology and Epidemiology at Johns Hopkins University. She completed her undergraduate degree in Biology at Georgetown University, medical school at the University of Rochester, internship and neurology residency at the University of Pennsylvania, a fellowship in multiple sclerosis (MS) and Master’s Degree in clinical research at the University of California, San Francisco. Her research focuses on environmental factors that influence the risk and prognosis of MS as well as on improving outcome measures for use in the clinic and in clinical trials. She is also Principal Investigator (PI) or Co-PI of several investigator-initiated randomized trials, including Vitamin D to Ameliorate Multiple Sclerosis (VIDAMS), intranasal insulin for cognitive impairment in MS, and the Traditional vs. Early Aggressive Therapy for MS (TREAT-MS) trials. Associate Professor Mowry also co-directs the Johns Hopkins MS Precision Medicine Center of Excellence and is Director of Experimental Therapeutics in MS at Johns Hopkins.

Determinants of Therapeutic Lag in Relapsing Multiple Sclerosis

BACKGROUND
Therapeutic lag represents the delay from initiation of therapy to attainment of clinically apparent treatment effect. Understanding of therapeutic lag and its determinants will provide valuable information for personalised treatment choices. 

OBJECTIVE
We aim to explore the associations of patient and disease characteristics with the time to full clinically manifest effect of treatment on relapses. 

METHODS
Data from MSBase were used. Inclusion criteria consisted of patients with relapsing multiple sclerosis (MS), 1-year treatment persistence, minimum 3-year pre-treatment follow-up and yearly review. Studied baseline determinants of therapeutic lag included: age at treatment start, MS onset age, sex, MS phenotype, disease duration, Expanded Disability Status Scale (EDSS), prebaseline EDSS trajectory and annualised relapse rate (ARR). Using relapse incidence, therapeutic lag was calculated by identifying the first post-treatment local minimum of the first derivative in subgroups stratified by patient and disease characteristics. 

RESULTS
5611 patients with 7048 treatment epochs were identified. Time, in years, of therapeutic lag differed by sex (female 0.27, 0.26-0.29 [mean, 95% confidence interval]; male 0.58, 0.49-0.67), age of MS onset (< 35 years 0.25, 0.17-0.37; ³ 35 years 0.56, 0.53-0.58) and pre-baseline ARR (ARR <2 0.28, 0.22-0.39; ARR³2 0.52, 0.43-0.61). Therapeutic lag was shortest in females with MS onset younger than 35 years (0.27, 0.26-0.29) and longest in males with MS onset older than 35 (0.69, 0.55-0.82). In males with MS onset younger than 35, ARR <1 was a significant modifier of therapeutic lag (0.24, 0.18-0.29 vs 0.44, 0.37-0.5). EDSS £ 3 and baseline disease duration <11 years reduced the therapeutic lag in females with younger onset of MS. 

CONCLUSIONS
We utilised a novel method for the quantification of therapeutic lag in different patient groups. Important demographic and clinical determinants of time to treatment effect on relapses are sex, age of MS onset and pre-baseline ARR. Minor contributors are baseline EDSS and disease duration.

Increased ankle muscle coactivation in people with MS with EDSS< 3.5

BACKGROUND

People with MS (PwMS) at early stages of the disease exhibit sub-clinical gait abnormalities. Altered activation patterns of muscles around the ankle joint may be responsible for this deterioration. Increased coactivation of ankle plantar/dorsiflexors may be an early indicator of gait deterioration.

OBJECTIVE

The aim of this study was to compare ankle dorsi/plantarflexor coactivation indexes (COI) between a group of PwMS at early stages and a group of healthy controls over long treadmill walking trials.

METHODS

32 PwMS (EDSS<3.5) and 15 healthy controls walked on a treadmill at 1.2m/s for 5 minutes. Surface EMG was recorded at 1000Hz using 6 Cometa Wave Plus  sensors (Milan, Italy) attached over the tibialis anterior (TA), gastrocnemius (LG) and soleus (SO) muscles. TA/LG and TA/SO COI were calculated over 150 strides for the whole gait cycle and stance and swing phases using a custom-made Matlab script (R2017a).

RESULTS

Overall, PwMS exhibited greater TA/LG and TA/SO COI than controls. These differences were significant for LG/TA during swing (p<0.01) but not during stance. For the whole gait cycle differences approached significant (p=0.05). Although TA/SO COI was greater in PwMS, no significant differences were found between groups.

CONCLUSION

PwMS have greater coactivation of ankle muscles than healthy controls when walking on a treadmill. Increased ankle muscle coactivation indexes may be an indicator of gait deterioration at early stages of the disease and may be a factor contributing to falls.

Effectiveness of a tailored cognitive behavior therapy for depression in those newly diagnosed with multiple sclerosis: preliminary results

BACKGROUND
Those with multiple sclerosis have been found to report high levels of depressive symptoms in the years following diagnosis. There is a lack of research examining the effectiveness of tailored cognitive behavioural therapy for the treatment of depression in those newly diagnosed with multiple sclerosis.

OBJECTIVE
To assess the efficacy of a tailored cognitive behavioural therapy intervention compared to a supportive listening intervention for the treatment of depressive symptoms in those newly diagnosed with multiple sclerosis. Anxiety, fatigue, pain, sleep, QOL, social support, resilience, coping and illness acceptance are also examined.

METHODS
This is an assessor-blinded, active-comparator, parallel-group, stratified clinical trial with balanced randomization. Participants are mild to moderately depressed individuals with MS who are within 5 years of having received a MS diagnosis. Level of depression, anxiety, fatigue, pain, sleep quality, QOL, social support, resilience, coping, illness acceptance, QALYs are measured. Participants are randomly allocated to the 8-week CBT or SL interventions and undertake pre, post and 3-month follow up assessments.

RESULTS
The trial is currently recruiting participants from three MS clinics in Melbourne (Royal Melbourne Hospital, St Vincent’s Hospital, Western Health). Preliminary results of the current trial will be discussed.

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).

Translational perspectives, valley of death

Presentation synopsis: Lessons learned from industry on how to successfully translate your research across the innovator’s “valley of death”. This presentation will cover the R-W-W assessment framework, BioCurate’s perspective on translational potential, red flags and key criteria for success with particular focus on opportunities and guidelines for the development of new therapeutics in multiple sclerosis.

CuII(atsm) as a novel treatment option for progressive multiple sclerosis

BACKGROUND
It is recognised that copper and myelination are intimately connected, where copper alterations induced by cuprizone is commonly utilised as a model of MS.  However, the mechanistic roles for copper in human MS remains poorly understood providing scope for the development of novel therapeutic interventions.

OBJECTIVE
Our aims were to assess copper and the activity of copper-dependent enzymes in human progressive MS-affected CNS tissue and also in two mouse models of MS, then to pre-clinically determine efficacy of the blood-brain barrier permeable and orally bioavailable, copper-containing compound CuII(atsm).

METHODS
Elemental copper levels in CNS tissues from progressive MS cases, cuprizone model mice and EAE model mice were assessed using quantitative, in situ elemental mass spectrometry.  Functionality of cuproenzymes was then interrogated using immunoblotting for protein levels and biochemical assays or liquid chromatography mass spectrometry (LC-MS) for activity levels.  Myelin levels and integrity in cuprizone and EAE mice treated with CuII(atsm) were investigated using immunoblotting and electron microscopy.

RESULTS
We show that multiple important cuproenzymes are disrupted in the human progressive MS spinal cord where this is associated with a perturbation in the natural anatomical distribution and biochemical partitioning of copper.  Many of these changes are recapitulated in the EAE mouse spinal cord and cuprizone mouse brain.  Moreover, treating these mice with CuII(atsm) mitigated these changes, improved disease phenotype, and was protective against impaired myelination in the CNS.

CONCLUSION
CuII(atsm) is in clinical trials for motor neurone disease and Parkinson’s disease, with copper delivery implicated in its therapeutic mechanism.  Based on our evidences for copper alterations in progressive MS and the compound’s protective activity, we propose CuII(atsm) as a potential therapeutic for progressive MS.

Mertk-positive microglia as a potential cellular therapy for multiple sclerosis

KEYWORDS

Mertk, microglia, transplantation, efferocytosis, therapy

BACKGROUND

A key prerequisite of promoting central nervous system remyelination is the clearance of apoptotic/dead cells and myelin debris by resident microglia.  Mertk is a receptor tyrosine kinase expressed on these cells, and is involved in initiating efferocytosis and maintaining an “anti-inflammatory” microglia phenotype.

OBJECTIVES

Firstly, this study aimed to determine the ability of endogenously high-Mertk-expressing neonatal microglia to home from the periphery to demyelinated white matter. Secondly, it aimed to further elucidate Mertk’s role in mediating myelin clearance, and explore the ability of microglia to facilitate myelin repair.

METHODS

Microglia were isolated from glial cultures generated from neonatal brain tissue. Fluorescently-labeled microglia were used to assess migration potential after intravenous and intraperitoneal transplantation into cuprizone-challenged, adult C57Bl/6 mice. Animals were also collected for myelin analysis after 7 days on standard chow following intraparenchymal brain injection. Mertk conditional knockout mice (deletion in CX3CR1-positive cells) and floxed controls were used to generate cultures to assess uptake of fluorescently-tagged myelin.

RESULTS

We observed the persistence and migration of intraparenchymally-transplanted cells in the corpus callosum 3 days after injection. Importantly, these cells maintained high Mertk expression. However, we did not detect cells administered intravenously or intraperitoneally.  We assessed the effect of Mertk deletion on myelin efferocytosis, and observed that this was promoted by the receptor ligand, Gas6. Myelin analysis of animals that recovered for 7 days following intraparenchymal injection is currently ongoing.

CONCLUSIONS

These experiments highlight the need for further interrogation of the route of cell delivery as a focus of future experiments, and provide a basis for understanding the therapeutic ability of intraparenchymally-injected microglia in ameliorating demyelination in the cuprizone model.

FUNDING SOURCES

This study was funded by the Bethlehem Griffiths Research Foundation, Stem Cells Australia and ARSEP Foundation.

NgR-Fc protein delivered by hematopoietic cells enhances neurorepair in a multiple sclerosis model

BACKGROUND
Nogo receptor 1 (NgR1) is the high affinity receptor for the potent myelin-associated inhibitory factors (MAIFs) that make up part of the inflammatory extracellular milieu during experimental autoimmune encephalomyelitis (EAE). NgR1-dependent signalling is associated with axonal degeneration in an animal model of multiple sclerosis (MS).

OBJECTIVE
As MS and EAE exhibit large numbers of inflammatory cell infiltrates within central nervous system (CNS) lesions, in this study, we utilised transplantable haematopoietic stem cells (HSCs) as a cellular delivery method of the NgR(310)ecto-Fc fusion protein.

METHODS
Male bone marrow donor (6-8 weeks old), and 8-12 weeks old female recipient C57Bl/6 wild-type mice were approved for experimental use (AMREP Animal Ethics Committee AEC# E/1532/2015/M). Bone marrow-derived lineage-negative cells were either non-transduced, transduced with either lentivirus (LV)-carrying empty or NgR(310)ecto-Fc vectors. Recipient mice were transplanted with lineage-negative enriched HSCs either non-transduced, transduced with empty or NgR(310)ecto-Fc vectors. Following post-transplantation recovery, recipient mice underwent myelin oligodendrocyte glycoprotein (MOG35-55)-induced EAE.

RESULTS
No immunomodulatory effects of the transplanted, modified HSCs with LV-NgR(310)ecto-Fc on immune cell lineages in EAE-induced recipient female mice were observed. We identified CNS infiltrating macrophages expressing myc-tagged NgR(310)ecto-Fc. These differentiated phagocytes predominated in immune cell infiltrated regions at the peak of clinical disease with extensive demyelination.

Professor Tanuja Chitnis is a Professor of Neurology at Harvard Medical School and Senior Scientist within the Ann Romney Center for Neurologic Diseases at BWH where she created the Translational Neuroimmunology Research Center focused on bringing bench discoveries to clinical trials for multiple sclerosis and related diseases. She is a board-certified neurologist specializing in multiple sclerosis (MS) with a dual appointment at the Brigham and Women’s Hospital (BWH) and Massachusetts General Hospital (MGH) in Boston, Massachusetts, USA. In addition, she serves as the Director of the CLIMB Natural History of MS study at the Partners MS Center, which is located within the Neurosciences Center at BWH. CLIMB follows over 2400 MS patients longitudinally. Here she oversees a team of analysts and postdoctoral fellows working to understand the causes, heterogeneity and response to treatment in MS patients, including immune mechanisms, sex differences and the effects of hormones on MS risk and disease course. Her interest in children with MS led her to start the Partners Pediatric MS Center at MassGeneral Hospital for Children where she serves as the Director. Between 2010-2018, she served as the elected Chair of the International Pediatric MS Study Group, where she has led several initiatives in the study of MS in children including the launch of the first clinical trials in this population, leading to the first FDA-approved therapy for this disease. She has authored over 200 publications and reviews related to MS and demyelinating disorders, including the New England Journal of Medicine, Neurology and Journal of Neuroscience. She also serves on the advisory board and steering committee of several MS-related organizations and studies including ACTRIMS, U.S. Network of Pediatric MS Centers, and the National MS Society. She receives grant funding from the Department of Defense, NIH, National MS Society, Guthy Jackson Charitable Foundation, and is the co-PI of a large study of SysteMS biology in MS sponsored by Verily. Throughout her career, her primary scientific contributions have been in immune mechanisms in MS, sex differences in autoimmune disorders, pediatric MS research and clinical trials, the CLIMB observational cohort study, and Neuromyelitis Optica spectrum disorders (NMO-SD). She is the recipient of several awards including the Joseph Martin Award for Clinical Research in 2019 from the Scientific Advisory Council at MGH, and the 2018 Milestones Award from the National MS Society.

IgG3+ switched memory B cells are elevated in active relapse-remitting multiple sclerosis

BACKGROUND
The success of B cell-depleting therapies including rituximab and more recently ocrelizumab suggests that B cells play a prominent role in the pathogenesis of multiple sclerosis (MS).

OBJECTIVE
Different subsets of B cells exist, but not all are involved in MS pathogenesis. Some suppress autoimmunity and may protect from MS. To understand this process better, we have interrogated the B cell repertoire in MS patients with active and inactive disease states.

METHODS
Peripheral blood mononuclear cells from 7 active and 8 inactive MS patients were stained with our panel of 35 isotope-conjugated antibodies. Mass cytometry, which combines flow cytometry with mass spectrometry, was then used to analyse and phenotype B cell subsets with unparalleled depth.

RESULTS
Active MS disease was associated with a decrease in naïve and an increase in switched memory B cells. Detailed phenotypic analyse revealed that IgG3+ B cells specifically were significantly elevated in active MS disease.

CONCLUSION
Our use of mass cytometry has allowed us to reveal that switched memory B cells, particularly those that express IgG3+, are associated with active MS. This will allow us to design more targeted therapies that inhibit these B cells specifically.

Funding source: Multiple Sclerosis Research Australia

Measuring axonal loss in the clinic using advanced diffusion MRI

BACKGROUND
Axonal loss is a key driver of progressive neurological impairments in people with MS (pwMS). There is significant interest in the development of biomarkers for axonal loss to be used for patient monitoring and prognostication. Axonal fibre density, measured using Diffusion MRI, is a putative in-vivo axonal biomarker for MS, but is yet to be clinically translated.

OBJECTIVE
To determine the sensitivity of longitudinal axonal fibre density measurements acquired as part of a standard radiological MRI protocol for detecting progressive axonal loss in pwMS.

METHODS
Between 2011 and 2014, 59 pwMS underwent 3T MRI (15 with 1 and 25o with 2 year follow-up) as part of their routine clinical monitoring. Patients were imaged using 3D-FLAIR for measuring brain and lesion volumes, and 30-direction diffusion weighted MRI (b-value=3000s/mm2) for measurement of axonal fibre density using the MRtrix 3.0 analysis package. EDSS and symbol digit modality test (SDMT) scores were obtained to measure neurological and cognitive progression.

RESULTS
Patients exhibited loss of axonal fibre density (mean (SD) = -3.44 (0.03)% p/a, p<0.0001)
and brain parenchymal fraction (-0.49 (0.009)% p/a,  p=0.002) with no group-level change in EDSS or SDMT. Loss of axonal fibre density significantly correlated with atrophy (R=0.43, p=0.005). Baseline axonal fibre density correlated with subsequent SDMT worsening (R=0.49, p=0.002) but not with EDSS worsening. Neither baseline nor longitudinal change in brain volume correlated with EDSS or SDMT progression.

CONCLUSION
Axonal fibre density can be measured from data collected within a standard radiological MS study and is nearly 7 times more sensitive to structural brain change in pwMS than brain volume measurements. Patients with greater axonal loss are at risk of more rapid cognitive decline.

Title: Diversification of the T cell repertoire following AHSCT is underpinned by thymic derived naïve T cells in patients with aggressive RRMS.

BACKGROUND
Autologous haematopoietic stem cell transplant is a highly effective immune reconstitution therapy for patients with aggressive RRMS. It is believed that AHSCT results in an immunotolerant state through deletion of pathogenic clones and regeneration of a diversified T cell repertoire, however data to support these postulations is limited.

OBJECTIVE
To understand the mechanisms of action underlying the re-establishment of CNS tolerance induced by AHSCT in MS. This study focuses on T cell receptor (TCR) repertoire of peripheral blood T lymphocytes longitudinally post-AHSCT to define the T cell subsets driving lymphocyte diversification and uses DNA markers of thymic output to determine whether these cells are derived principally from the re-infused graft or develop de-novo from a renewed thymus.

METHODS
This study utilises the stored blood samples of highly active RRMS patients undergoing AHSCT in an ongoing phase 2 clinical trial at St Vincent’s Hospital, Sydney (HREC approval - SVH File No. 10/206). Samples collected pre-AHSCT and 6, 12, 24 and 36 months post-AHSCT were used for high throughput sequencing of T cell receptors in FACS sorted CD45RA+ Tnaive and CD45RO+ Tmemory CD4+ and CD8+ subsets [n=10]. DNA markers of thymic function - Sj:b TREC ratio [n=17] were also studied and correlated with clinical response.

RESULTS
Each T cell phenotype was assessed longitudinally for metrics of diversity, clonal homeostasis and the kinetics of dominant clones. TCR diversity, as assessed by Shannon entropy, falls significantly in all cell phenotypes in the first 12 months following AHSCT. At 24 months post-transplant an increase in diversity is detected in all lymphocyte phenotypes; however, only approximates pre-transplant levels in the CD4+ naïve population. A corresponding increase in the proportion of rare clones at 24 months post-AHSCT was also detected in the CD4+ naive population when assessed by clonal homeostasis metrics. Dominant CD4+ naïve clones detected pre-AHSCT are undetectable post-transplant, whilst dominant CD4+ memory, CD8+ naïve and CD8+ memory populations frequently undergo further expansion. Antigen specificity to common viruses such as EBV has been detected in clonal expansions post-AHSCT. sJ:B TREC ratio, a measure of thymic output accounting for the lymphopenic environment post-AHSCT remains below baseline at 6 and 12 months, increasing to baseline levels at 24 months and remaining at baseline levels at 36 months, independent of patient age. A trend towards lower thymic output was noted at 24 months in patients who relapsed, suggesting a clinical correlate of this marker.

CONCLUSION
This is the first study of MS patients undergoing AHSCT to explore the kinetics of immune reconstitution by T cell phenotype. These results suggests that by 24 months post-AHSCT diversification of the T cell repertoire occurs through de novo thymic output of naïve CD4+ T cells. The expansion of dominant CD4+ memory and CD8+ cells requires further investigation and clinical correlation.

Dr J Massey has been supported by a postgraduate scholarship from MSRA.

Vitamin D genetic variants and relapses in multiple sclerosis: A Mendelian randomization study

BACKGROUND
Observational studies have shown an association between 25-hydroxyvitamin D level and relapse rate in multiple sclerosis. Reverse causation, selection and recall bias impede causal inference assessment. Applying Mendelian randomization, Graves JS et al. (MSJ, 2019) showed a significant association between vitamin D genes and relapse risk in children.

OBJECTIVES
The objective of this study is to replicate in our adult cohort of multiple sclerosis patients the previously shown causal association between hypovitaminosis D and relapse risk.

METHODS
Genetic variants associated with serum 25-hydroxyvitamin D level were derived from the most recent GWAS meta-analysis on serum 25OHD from the SUNLIGHT consortium (Jiang X et al., Nat Commun, 2018) and from a published meta‑analysis for low frequency variants (Manousaki D et al., Am J Hum Genet, 2017). Weighted polygenic scores for vitamin D levels were associated with time to relapse using the Cox proportional hazards model.

RESULTS
In our survival analysis, including 506 MS patients with 1412 relapses captured prior to treatment, 1‑SD increase of weighted vitamin D polygenic score is associated with a lower hazard rate of a relapse occurring (hazard ratio = 0.18 (95% CI = 0.06-0.53), P = 0.002).  Adding covariates age at onset and gender or multiple sclerosis genetic burden did not alter the results. 

CONCLUSION
In a large dataset of N = 506 Belgian MS patients with N = 1412 documented relapses, we observe a significant association of the weighted vitamin D polygenic score with a 82% decreased hazard rate for a relapse occurring.

FUNDING SOURCES
Marijne Vandebergh: Ph.D fellowship of the Research Foundation-Flanders (FWO)

Increased erythrocyte microRNA-183 cluster expression during relapse - A potential new MS-specific marker for disease activity?

BACKGROUND
Erythrocytes may be involved in MS through impaired antioxidant capacity and haemorheological modifications. Erythrocytes harbour microRNAs, which may silence translation. Some microRNAs are transcribed as clusters, exerting increased control over the same pathway. Erythrocyte microRNAs may participate in intercellular communication through extracellular vesicles (EVs).

OBJECTIVE
To identify erythrocyte microRNAs’ diagnostic and disease activity biomarker potential and determine their possible role as a form of intercellular communication in MS through extracellular vesicles.

METHODS
Erythrocyte microRNAs were sequenced from 11 relapsing-remitting MS (RRMS), 12 secondary progressive MS (SPMS), and 22 healthy controls (HCs). Differentially expressed microRNAs were further assessed with a targeted assay in an independent cohort [21 RRMS - remission, 14 RRMS - relapse, 11 SPMS, 25 HCs, 20 migraine patients (MPs)]. Correlations between microRNAs and EDSS, MSSS, ARMSS, and cognitive screens were assessed. Plasma extracellular vesicles were surveyed by flow cytometry.

RESULTS
During relapse miR-183-5p and miR-182-5p, miR-183 cluster members, showed increased expression compared to remission (3.15-fold, p<0.001; 2.44-fold, p<0.001), SPMS, HCs, and MPs. This expression correlated with physical disability. The third miR-183 cluster member, miR-96-5p, correlated with cognitive disability. MiR-183 cluster target genes are enriched in pathways related to transendothelial migration and immune cell activation. Erythrocyte-derived EVs, which may carry microRNAs to recipient cells, were also increased in MS.

CONCLUSIONS
Erythrocyte miR-183 cluster expression was increased during relapse, correlated with MS severity, and may serve as an activity marker. Increased expression appeared to be MS specific. Erythrocyte miR-183 cluster microRNAs might be involved in intercellular communication through EVs, which were upregulated in MS.

FUNDING
This project was funded by a Multiple Sclerosis Research Australia Incubator Grant. K. Groen is funded by a scholarship from the University of Newcastle. V.E. Maltby is funded by fellowships from Multiple Sclerosis Research Australia and the Canadian Institutes of Health Research.

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.

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.

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. 

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.

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.

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.

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.

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.

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.

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.

CONCLUSION
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
MS Research Australia, NHMRC.

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.

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.

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.