Posters

Epstein-Barr virus infection dysregulates the MS risk miRNA expressions in B cells. 

BACKGROUND 

Although over 200 risk genetic loci have been identified for Multiple Sclerosis (MS), the causes of MS largely remain unknown. However, Epstein-Barr virus (EBV) infection has identified as necessary but not sufficient to cause MS. 

OBJECTIVE 

To interrogate the host response to EBV infection that contributes to MS, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. 

METHODS

We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host-EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. Lastly, we tested the genotype effect of the MS risk SNP rs10271373 on expression of ZC3HAV1, an MS risk gene with antiviral functions.

RESULTS  

We found that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells, which is consistent with increased interferon response, promoting viral mRNA degradation, and repressing viral replication process via ZC3HAV1 and so decreased immune evasion by EBV. 

CONCLUSION 

Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly.

PEGylation of Gas6 for therapeutic use in demyelinating disease.

BACKGROUND

Gas6 is a TAM (Tyro3, Axl and Mertk) receptor ligand and is an attractive candidate in potentiating remyelination. However, it has a molecular weight of 75kD  limiting its access to the CNS and therefore its potential as a therapeutic agent in multiple sclerosis.

Objective: To modify Gas6 to increase its half-life by adopting an NHS-PEGylation strategy and to assess the bioactivity of native Gas6 and the synthesised derivatives via a viability assay using the rat oligodendroglial cell line, CG4. The efficacy of PEGylated-Cas6 will then be assessed in-vivo. 

METHODS

PEGylation was carried out by reacting 5 kD NHS-PEG (Creative pegworks, USA) with Gas6. The identity of the final products was confirmed by barium iodide staining followed by western blotting. To check for bioactivity, CG4 cells were cultured on Poly-D-Lysine coated wells at a density of ~150 cells/well in different culture conditions. Live/dead cells were counted after 48 hours using Ethidium homodimer/Calcein AM under an inverted florescent microscope, blinded. 

RESULTS

Successful NHS-PEGylation of Gas6 was confirmed by western blotting. In the viability assay, in the absence of insulin (control) there was compromised viability (34.6% viable cells ± 1.4 SE), whereas viability was potentiated by un-modified/native Gas6 (46.7% viable cells ± 1.4 SE). 5kD-PEGylated versions of Gas6 had similar efficacy in promoting the survival of CG4 cells (46.0% viable cells ± 1.4 SE). This effect was abolished in the presence of Axl-Fc which binds soluble Gas6 but remained unchanged in the presence of the irrelevant receptor, IgG- Fc. 

CONCLUSION

NHS-Pegylated versions of the Gas6 protein have been produced, and a robust viability assay established, which is useful in determining the bioactivity of Gas6 derivatives. This is a starting point to compare the efficacy of modified and un-modified Gas6 in promoting remyelination in animal models of MS. 

ACKNOWLEDGEMENT

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

Tyro3 in myelin repair and axonal conductivity

BACKGROUND
Secondary progressive multiple sclerosis is characterised by demyelination, neurodegeneration and inexorable decline in functional capacity. Axonal dysfunction and degeneration is the key determinant of disability progression, secondary to myelin loss. The lack of therapies that target the progressive MS is a significant unmet need. 

OBJECTIVE

To investigate a therapeutic role for the receptor tyrosine kinase Tyro3 in myelin repair, as well as consequences of Tyro3 loss on axonal conductivity.

METHODS

We have studied the role of Tyro3 in myelination using mice deficient in this receptor. We employed electron microscopy to determine the number of myelinated axons as well as myelin ultrastructure at key developmental times. We have also employed the cuprizone model of demyelination to investigate the influence of Tyro3 upon the course of demyelination and subsequent remyelination. Conduction velocity in white matter was assessed both experimentally and mathematically. 

RESULTS

Tyro3 deficiency resulted in delayed myelination and a chronic reduction in myelin thickness. Myelin changes were correlated with disruptions to paranodal structure. Loss of Tyro3 was associated with an accelerated demyelination in cuprizone-challenged mice and thinner myelin following remyelination. No alterations were observed to experimentally measured compound action potential velocities. Mathematical modelling of the observed disruptions to myelin showed changes in single axon conduction but not compound action potentials. 

CONCLUSION

Tyro3 contributes to multiple aspects of the structure of the myelinated axon. Unexpectedly, experimental assessment of action potential conduction in the Tyro3 deficient mouse did not identify significant electrophysiological changes. Our results highlight the importance of adopting an experimental approach to assessing functional outcomes.

A heterozygote point mutation in the transcription factor IRF4 protects from developing ascending paralysis in a Multiple Sclerosis mouse model. 

BACKGROUND

Experimental autoimmune encephalomyelitis (EAE) is a commonly used mouse model of multiple sclerosis, causing ascending paralysis. Mice completely lacking IRF4 (IRF4del) are protected in this model. This protection is due to a defect in the differentiation of the IL-17 producing T helper 17 (Th17) cells.

OBJECTIVE

A newly described point mutation in the DNA binding domain of IRF4 leads to protein expression changes in multiple hematopoietic cell types. We here investigate the effects of this mutation in the context of EAE. 

METHODS

EAE was induced in mice with functional IRF4 (wildtype), mice lacking one or both copies of the Irf4 allele, as well as mice carrying the point mutation on one or both alleles. These mice were scored daily for disease progression, and blood immune cell phenotypes were monitored throughout disease using flow cytometry. In addition, naïve T cells from all 5 genotype groups were isolated and polarised in Th17 cell inducing conditions. 

RESULTS

The complete lack of IRF4 protects from EAE while the presence of one Irf4 allele is able to revert this protection leading to ascending paralysis equal to the wildtype control. Interestingly, one allelic point mutation of Irf4 was enough to offer complete protection. These mice displayed differential development and function of innate cell populations but produced IL17 producing Th17 cells in T cell polarisation assays.

Conclusion (45/45)

Mice with a point mutation in IRF4 are able to produce Th17 cells capable of producing IL-17, but are still protected in EAE. Thus the IRF4 point mutation provides protection in EAE in a pathway most likely independent of Th17 cells, but potentially linked to innate cell populations. 

FUNDING

Fenner Merit Scholarship in Medical Sciences, Australian Government Research Training Program

Whole-blood methylation as a prognostic biomarker for multiple sclerosis.

BACKGROUND

The lack of prognostic biomarkers in multiple sclerosis makes it difficult for clinicians to optimise care. Variation in disease severity is incompletely explained by genetics, suggesting genetic and environmental interactions are involved. DNA methylation is one epigenetic mechanism by which gene-environment interactions can be assessed. 

OBJECTIVES

To date, no epigenetic predictors of severity have been identified. We aimed to identify whole blood and immune cell specific DNA methylation patterns associated with mild and severe relapse-onset multiple sclerosis, and to further test the utility of DNA methylation as a prognostic biomarker. 

METHODS

We compared whole-blood methylation patterns between 119 and 116 relapse-onset females with mild and severe disease, respectively. Severity was determined with longitudinal Age-Related Multiple Sclerosis Severity (ARMSS) scores using MSBase Registry data. Methylation was measured with the Illumina EPIC array, and pre-processed and analysed using the ChAMP package. Cell-type specific analysis was performed with the EpiDISH package. A predictive model was built with glmnet. FDR<0.05 was used to assess significance.

RESULTS

We identified associations between whole-blood methylation and disease severity. 548 differentially methylation positions (DMPs) were identified; 230 DMPs were hypermethylated, and 318 were hypomethylated in the severe group. Sensitivity analyses demonstrated no major effects of covariates on methylation. No cell-specific DMPs were identified. We showed that whole-blood methylation could accurately predict categorical disease severity using methylation levels at 890 CpG sites (AUC=0.91).

CONCLUSION

Whole-blood DNA methylation patterns can be used to accurately predict disease severity in females with relapse-onset multiple sclerosis. DNA methylation has potential as a prognostic biomarker for multiple sclerosis

FUNDING SOURCES

MSRA Project Grant (18-0424) 
RMH Home Lottery Grant MH2013-055
MSBase Foundation Project Grant 
Charity Works for MS Project Grant 
Pennycook Foundation Grant 2018 
Monash University Project Grant

Assessing health-related quality-of-life in Australian Multiple Sclerosis Longitudinal Study participants using six multi-attribute utility instruments: implications for healthcare decision-making.

BACKGROUND

Despite important discoveries/advances in treating multiple sclerosis (MS), people with MS (pwMS) can experience delays in accessing new treatments if decision-makers lack robust health economic evidence including health-related quality-of-life (HRQoL) benefits of the intervention. Health state utilities (HSU) are a HRQoL input for cost-utility analysis. 

OBJECTIVE

Several multi-attribute utility instruments (MAUIs) are available from which HSUs can be derived, but the most appropriate MAUI for use in MS has not been identified. We aimed to determine the preferentially sensitive MAUI(s) that capture the full impact of MS on HRQoL. 

METHODS

Participants in this study came from a comprehensive HRQoL survey (mid-2020) of the Australian MS Longitudinal Study. The survey included six MAUIs (EQ-5D-5L/EQ-5D5LPsychosocial, SF-6D versions 1 and 2, AQoL-8D and PropR), and sociodemographic, Covid19-related and subjective wellbeing data. HSUs were generated from Australian value sets. Ceiling and floor effects were investigated. Bland-Altman plots and Shannon’s Indices were examined. Minimal important differences and population norms were sourced from the literature. 

RESULTS

N=1,683 pwMS completed the survey (67% response). HSUs were derived for >97% of respondents. Mean age 58.6 years, 80% female, 19% reported severe disease and 63% had relapsing-remitting MS. Mean (SD) HSUs ranged from 0.45±0.29 (SF-6Dv1) to 0.63±0.22 (AQoL-8D). EQ-5D-5L revealed the highest ceiling (HSU=1.0; n=157,10%) and floor (HSU≤0; n=113,7%) effects. PwMS with EQ-5D-5L HSU≤0/HSU=1 reported mean HSUs of 0.37/0.91, 0.35/0.90, and 0.08/0.85 for the AQoL-8D, EQ-5D5LPsychosocial and SF-6D, respectively. 

CONCLUSION

While the EQ-5D is the most commonly cited MAUI (in 85% of health technology assessment guidelines), our preliminary comparison results suggest the EQ-5D-5L is not preferentially sensitive in assessing the complex HRQoL domains for pwMS. 

FUNDING ACKNOWLEDGEMENTS

Dr Julie Campbell is supported by a MS Research Australia, Postdoctoral Research Fellowship, Grant Number 19-0702.

Investigating health-related quality of life impacts for participants of the Australian MS Longitudinal Study using the new EQ-5D-5LPsychosocial instrument.

BACKGROUND

Several multi-attribute utility instruments (MAUIs) are available to derive health state utilities (HSUs) for use in cost-utility analysis (CUA), with the EQ-5D-5L being most common. However, the EQ-5D-5L’s ability in capturing and assessing the complex psychosocial health aspects of people with chronic diseases is lacking. 

OBJECTIVE

Many healthcare resourcing decisions are based on CUA. An EQ-5D-5L psychosocial bolt-on has been developed to address the limited description of psychosocial health in the EQ-5D-5L. We aimed to investigate the health-related quality of life (HRQoL) impacts of multiple sclerosis (MS) using the new EQ-5D-5LPsychosocial. 

METHODS

We conducted a comprehensive HRQoL survey among Australian MS Longitudinal Study participants. Data were collected for the EQ-5D-5L and AQoL-8D MAUIs, along with MS-related disease severity and sociodemographic data. HSUs were generated using Australian tariffs for the EQ-5D-5L and EQ-5D-5LPsychosocial bolt-on (where the bolt-on adopts four questions from the AQoL-8D MAUI). Mean (standard deviation [SD]) HSUs were estimated and stratified by disability severity (none, mild, moderate, severe).

RESULTS

N=1683 completed the survey. Mean age 58.6 years, 80% female, and 55% were in the moderate or severe disability category. Mean HSUs were EQ-5D-5L=0.58±0.32 and EQ-5D-5LPsychosocial=0.62±0.29. Mean EQ-5D-5L/EQ-5D-5LPsychosocial HSUs across disability categories were: no disability 0.85/0.79; mild 0.71/0.66; moderate 0.52/0.53; severe 0.18/0.50. N=157 (10%) reported perfect health (HSU=1.0) on the EQ-5D-5L, however, reported a mean HSU of 0.90 on the EQ-5D-5LPsychosocial. Ceiling effect reduced to <1% (n=7) for the EQ-5D-5LPsychosocial.

CONCLUSION

The EQ-5D5LPsychosocial MAUI had better sensitivity than the worldwide prevalent EQ-5D-5L as people living with MS in Australia approached HSUs of 1.0 on the EQ-5D-5L. This has implications for the choice of MAUI for heath technology assessment guidelines that prescribe or recommend the EQ-5D-5L MAUI. 

FUNDING ACKNOWLEDGEMENT

Dr Julie Campbell is supported by a MS Research Australia, Postdoctoral Research Fellowship, Grant Number 19-0702.

Diagnostic power of rapid and objective visual testing of multiple sclerosis patients.

BACKGROUND

MRI scanning tends to reflect the history of acute inflammation rather than the stage of progressive disease, and hence is poorly correlated with EDSS scores. Some variants of visual function testing may be more related to progressive disease. We explore two new forms here. 

OBJECTIVE

To examine the diagnostic power of two new forms of rapid objective pupil perimetry in patients with multiple sclerosis (MS). We compared persons with different EDSS scores, and those with and without Optic Neuritis (ON) as a marker for acute inflammation in the visual system.   

METHODS

We tested 44 MS patients, 60.7 ± 10.1 y, and 40 normal controls 60.2 ± 12.5 y (mean ± SD). We used the W12 and W20 test variants of the Konan objectiveFIELD Analyser. These presented 12 or 20 stimuli/eye across ±30 degrees, testing both eyes concurrently in 80 seconds. We calculated Receiver Operator Characteristic %AUCs for the mean of the worst 4 per-region response delay asymmetries for different EDSS groups. 

RESULTS

%AUCs increased across our 3 EDSS groups: <2.5; 2.5 to <4.5; >=4.5, reaching 94.4 ± 3.16 for EDSS-3. W20 was better for EDSS-1 patients:  81.1 ± 6.69 vs. 75.0 ± 8.33. A sub-analysis compared subjects who had experienced ON (N=30) and those who had not (no-ON, N=14). %AUC for No-ON and ON were: W12, 86.9 ± 5.24 vs 86.0 ± 4.46; and W20, 87.2 ± 5.45 vs 86.8 ± 4.36. 

CONCLUSION

The relatively large %AUCs for EDSS-1 suggest an ability to monitor early-stage disease. The similarity of ON vs. no-ON results suggest that the tests are more biased towards disease progression rather than the history of acute inflammation, like our earlier data: ODI: 10.1002/ana.20504, 10.1177/1352458513512708.

Going beyond tokenistic: the experience of being a consumer involved in research about multiple sclerosis.  

BACKGROUND

The ANU Our Health in Our Hands (OHIOH) personalised medicine program has incorporated people living with MS in its Health Experience Team and the broader research program, with the aim of enhancing the quality, relevance, credibility and legitimacy of research.  

OBJECTIVE

To describe people living with MS’ experiences of being involved in research about MS. 

METHODS

Using story telling techniques, four Health Experience Team members living with MS were asked to create self-filmed vignettes reflecting on their involvement in the OHIOH program. Each team member reflected on one of four projects they had contributed to. 

RESULTS

Health Experience Team members living with MS felt supported and that their contributions were valued. Examples described included contributions to research question development, qualitative data analysis, preparation of peer reviewed papers, creation of a toolkit for collaboration between people living with MS and researchers, and production of conference presentations. As co-researchers team members were named as investigators on grant applications and as co-authors on peer-reviewed papers. 
 

CONCLUSION

For PwMS, being involved in research can be empowering and provide a meaningful way to contribute to both the research community and the broader MS community. Further evaluation of this model of consumer involvement may elucidate detailed impacts on the research and those involved.

ACKNOWLEDGEMENT

This research was funded by and has been delivered in partnership with Our Health in Our Hands (OHIOH), a strategic initiative of the Australian National University, which aims to transform health care by developing new personalized health technologies and solutions in collaboration with patients, clinicians and health-care providers.

Knowledge translation in the international MS community: an evaluation of the Understanding Multiple Sclerosis free online course.

BACKGROUND

Translating knowledge to the wider multiple sclerosis (MS) community is essential to promote informed decision-making and awareness. The Understanding MS massive open online course (MOOC) is a freely available six-week course covering topics from the underlying biology of MS to its impact on everyday life. 

OBJECTIVE

Here, we present a summary of what we have learned about knowledge translation in the MS community after two years of administering the course. This includes an examination of enrolees; who doesn’t complete it and why; course completer feedback; and the impact of course participation. 

METHOD

We will discuss several course evaluation studies. These include studies of participant engagement and satisfaction (N=3,518); pre/post assessments of outcomes of interest (knowledge, health literacy, self-efficacy, resilience, quality of life and MS symptoms; N=560); qualitative evaluations of discussion board free text data (N=701); and cross-sectional studies of reasons for noncompletion (N=397). Consequently, we employed a range of analytical techniques, including textual analysis, linear regression, correlation, chi-square and t-tests.

RESULTS

Over five enrolments, >20,000 people have enrolled in the Understanding MS MOOC from >125 countries, with a mean 43% completion rate. Participants, particularly those without a university degree and caregivers, are satisfied with the course (97%) and report that the information is applicable to their lives (63%). Noncompletion is primarily due to time constraints and limitations. Participating in the MOOC increases MS-related knowledge, health literacy, self-efficacy and quality of life. 

CONCLUSION

There is an appetite internationally for freely available online MS knowledge translation from reputable sources. The Understanding MS MOOC is a fit-for-purpose MS knowledge translation intervention. Our experience demonstrates that burden (time required) and accessibility should be primary considerations for similar interventions.

Sensory enrichment during early development transiently enhances oligodendrocyte survival.

BACKGROUND

The addition of new myelin sheaths in the central nervous system (CNS) is responsive to neural activity, with increases in activity increasing myelin sheath length, the density of myelinating oligodendrocytes, as well as enhancing the proliferation of their precursors, oligodendrocyte precursor cells (OPCs).

OBJECTIVE

We aimed to assess whether early sensory enrichment promotes long-term myelin deposition via increases in neural activity. We also sought to quantify the varying contributions of OPC proliferation, OPC maturation into a myelinating oligodendrocyte, and myelin sheath changes responsible for enrichment-induced myelin change. 

METHOD

Experimental mice were housed in enriched environments for 2 weeks during early postnatal development, with control mice in regular housing. To assess OPC proliferation, mice were double-injected with 2 thymidine-analogues before collection, enabling quantification of rates of S-phase entry and cell cycle length. Mice were also fed EdU in the drinking water throughout enrichment to assess OPC differentiation and survival. Total number of oligodendrocyte-lineage cells were quantified with stereology. 

RESULTS

Sensory enrichment for two weeks during early murine development significantly increased prefrontal cortical volume and mature oligodendrocyte number. This was alongside an increase in OPC proliferation, however modelling revealed that it was increased oligodendrocyte survival that contributed most to this change. Surprisingly, this change did not last with the removal of the environmental stimulus, and enriched mice subsequently lost oligodendrocytes upon return to standard housing. 

CONCLUSION

This work demonstrates that a non-invasive change to environment can promote new oligodendrocyte production and oligodendrocyte survival, yet that these effects may be transient. This knowledge provides an important basis to explore clinically-relevant strategies aimed at increasing oligodendrocyte survival in the context of MS. 

FUNDING

Australian Postgraduate Research Award – Graduate Research program

Delineating the role of monocarboxylate transporter 8 (MCT8) in the context of neuroinflammation-mediated oligodendrogliopathy. 

ABSTRACT

Oligodendrocytes are the cells that developmentally myelinate axons within the mammalian central nervous system (CNS). The integrity of CNS neurons and their axons require the structural and metabolic support of oligodendrocytes to regulate the continual electrical demands of the complex integral circulatory. Thyroid hormone (T3) is an important mitogen during oligodendrocyte maturation, whereby its intracellular accumulation for the extracellular space is facilitated by its active transport through the plasma membrane using the monocarboxylate transporter 8 (MCT8; encoded by slc16a2). The deprivation of TH in CNS may lead to stalled differentiation or apoptosis of oligodendrocytes. In this study, we demonstrated the in vivo expression of MCT8 on oligodendrocytes in the developing brain and spinal cords of normal mice. We then analyzed how this transporter is modulated in oligodendrocytes and CNS in the following animal models: neuroinflammation-mediated demyelination (experimental autoimmune encephalomyelitis (EAE)) and neurotoxicant-induced demyelination (cuprizone model).  Furthermore, we investigated its oligodendroglial expression in human neurodegenerative brain sections.  The main results are the following: (i) MCT8 expression is maintained in cells residing within the subventricular zone throughout development, suggesting its role during their maintenance; (ii) MCT8 expression on immune cells in naïve and EAE-induced animals may suggest a potential immunomodulatory activity; (iii) oligodendroglial MCT8 expression is significantly reduced in models of demyelination; (iv) potentially altered in neurodegeneration. Collectively, the data suggest that a deprivation of T3 transport into oligodendrocytes may exist during neuroinflammatory conditions such as EAE-induced animals and MS, potentially providing a new avenue of investigation, when searching for novel therapeutics to drive neuroprotection and neurorepair during progressive MS.

Illness Perceptions and Coping in the Relationship between Personality, Depression, Anxiety and Illness Acceptance in Multiple Sclerosis. 

BACKGROUND

Clinical levels of depression and anxiety are highly prevalent among individuals with multiple sclerosis (MS) and have deleterious biopsychological outcomes. Despite this, the mechanisms that contribute to the development and maintenance of depression, anxiety and positive adjustment outcomes are largely unknown. 

OBJECTIVE

Assess the utility of Leventhal’s Common-Sense Model in predicting psychological adjustment outcomes depression, anxiety and illness acceptance in individuals with MS. This study focuses on the relations between personality traits neuroticism and optimism, illness perceptions, coping strategies and adjustment outcomes utilising a cross-sectional design.

METHODS

Individuals with MS (n= 428) completed online questionnaires and were mostly female (86.7%), located in Australia (45%) or the United States (32%) and reported a relapsing remitting course of MS (83.4%). Measures included the NEO-Five Factor Inventory-3; Life Orientation Test Revised; Depression Anxiety and Stress Scale; Acceptance of Chronic Health Conditions Scale; Revised Illness Perceptions Questionnaire; and the BRIEF COPE. Mediation analyses were analysed through PROCESS macro for SPSS.

RESULTS

Perceptions of illness coherence, control, timeline and emotional representations and avoidant coping were significant simple and parallel mediators in the relationships between traits and anxiety and illness acceptance. Neuroticism partially predicted depressive and anxiety levels through the serial effects of negative emotional representations on high avoidant coping, whereas the serial effects of less negative emotional representations on lower avoidant coping mediated the relationship between high optimism and low depressive levels.

CONCLUSION

Illness perceptions and avoidant coping are important mechanisms involved in depression, anxiety and illness acceptance and may represent targets for psychological intervention in individuals with MS.

Variability of the response to immunotherapy among sub-groups of patients with multiple sclerosis. 

BACKGROUND

Our understanding of demographic and clinical modifiers of the effectiveness of MS therapies is limited.   

OBJECTIVES

To assess whether the response to disease modifying therapies (DMT) in multiple sclerosis (MS) varies by previous disease activity (annualized relapse rate, presence of new MRI lesions), disability, age, MS duration or disease phenotype.   

METHOD

Patients with clinically isolated syndrome or definite MS, follow-up ≥1 year, ≥3 EDSS scores with ≥1 score recorded per year and a minimum dataset of clinical and demographic characteristics were included in this study (n=61,810). Marginal structural models (MSMs) were used to compare the hazards of 12-month confirmed worsening and improvement of disability, and the incidence of relapses between treated and untreated periods. MSMs were continuously re-adjusted for patient age, sex, pregnancy, date, time from first symptom, prior relapse history, treatment history, disability and MRI activity.   

RESULTS

Among 24,344 patients with relapsing MS, those on DMTs experienced 48% reduction in relapse incidence, 46% lower risk of disability worsening and 32% greater chance of disability improvement. The effect of DMTs on EDSS worsening and improvement and the risk of relapses was attenuated with more severe baseline disability. The effect of DMTs on suppressing relapses declined with higher prior relapse rate and in patients with prior cerebral MRI activity. Among 1985 participants with progressive MS, an overall evidence for beneficial effects of treatment was not found on disability and on relapses. While the main determinant of the effect of DMTs on disability was MS phenotype, the most important determinant of the effect on relapses was the baseline relapse activity.  

CONCLUSION

DMTs were associated with reduction in relapse frequency and progression of disability, and increased chance of disability improvement.  DMTs were more effective among patients with lower disability, lower prior relapse rate, absence of recent MRI activity and relapsing MS phenotype. 

Comparison of multiple disease modifying therapies in multiple sclerosis with marginal structural models. 

BACKGROUND

Because of methodological challenges comparisons of multiple treatments in multiple sclerosis cohorts have been limited to pairwise and triple comparisons. There is a need to implement an extension of marginal structural models (MSM) that would allow simultaneous comparisons of multiple treatments across a broad spectrum of MS patients. 

OBJECTIVES

Compare: 1) the Average Treatment Effect (ATE), the effect a treatment would have had if the entire study population had been treated with this treatment vs. another treatment; 2) the Average Treatment Effect Among the Treated (ATT), comparison an observed effect of a treatment with a counterfactual (not observed) effect of another treatment in the same population. 

METHOD

We selected patients from the MSBase registry with Clinically Isolated Syndrome and Relapsing-Remitting MS followed for ≥1 year, with ≥3 visits, ≥1 visit per year and exposed to a MS therapy. MSMs were used to compare cumulative hazards of 6-month confirmed worsening and improvement of disability, and the incidence of relapses between treatments. MSMs were continuously re-adjusted for patient age, sex, pregnancy, date, time from first symptom, prior relapse history, treatment history, and MRI activity. 

RESULTS

Among 20754 patients, we compared ATE of glatiramer acetate, interferon b, natalizumab, fingolimod, dimethyl fumarate, and teriflunomide. A reduction of relapse frequency was most prominent on natalizumab, followed by fingolimod (42% and 17% respectively relative to glatiramer acetate). Natalizumab was associated with greater chances of disability improvement than any other treatments (24% compared to glatiramer acetate) and lower risks of disability worsening compared to any other treatment. The ATT pairwise comparisons confirmed the superiority of the effectiveness of natalizumab and fingolimod over other included therapies. 

CONCLUSION

Compared to other DMTs natalizumab and fingolimod were associated with superior reduction in relapse frequency than glatiramer acetate, interferon b, teriflunomide and dimethyl fumarate. In addition, we found a superior effect of natalizumab on reducing the rate of disability worsening and improving disability. This study demonstrates the capacity of ATE and ATT MSM to evaluate the effectiveness of multiple MS therapies simultaneously.

A systematic review of self-concept change in Multiple Sclerosis.

BACKGROUND

Self-concept is increasingly being acknowledged as an important predictor of outcomes in those with neurological illnesses. The notion of self-concept and its relevance to people with multiple sclerosis (MS) is poorly understood, notably the consequential shift in self-concept due to disease progression and disease-related events. 

OBJECTIVES

The current paper systematically reviewed and synthesised quantitative and qualitative studies investigating self-concept in MS to identify studies reporting on self-concept measurement, self-concept change, and the impact of major disease-related events on self-concept. 

METHOD

PsycINFO, MEDLINE (PubMed), CINAHL, Scopus, and Web of Science were systematically searched, with the Mixed Methods Appraisal Tool (MMAT) used to appraise the quality of the identified articles. A total of 29 studies (10 quantitative, 19 qualitative) were identified. 

RESULTS

Results from quantitative studies suggest that MS may be responsible for some degree of self-concept change following diagnosis and throughout the disease course. A noticeable limitation of this literature however is the lack of a standardised (and MS-specific) assessment of self-concept. The results from qualitative studies illustrate the complex process of self-concept change that may be involved in people with MS, and that are catalysed by MS-related events (e.g., work withdrawal). 

CONCLUSION

These findings highlight the need for future research to examine the nature of changing self-concept more thoroughly in MS and the need for a standardised tool to measure self-concept change that is relevant for MS populations.

Adoption of multiple lifestyle behaviours is associated with improved quality of life in people with multiple sclerosis. 

BACKGROUND
Multiple sclerosis (MS) is an incurable neurological disorder, presenting with heterogenous symptoms. Symptoms can impact health and daily functioning, reducing quality of life (QoL). Adoption of modifiable lifestyle behaviours has been associated with improved QoL. However, little is known about which behaviours have greatest impact. 

OBJECTIVE
To assess associations between the adoption of both individual and multiple modifiable lifestyle behaviours and physical and mental QoL, and whether adoption of multiple behaviours are associated with change in QoL over time. 

METHOD
Data from participants who completed an online survey at baseline and 7.5-years were analysed. Lifestyle behaviours included non-smoking, diet, vitamin D and omega-3 supplementation, physical activity, and meditation practice. QoL was assessed by the MSQOL-54. Linear regression was used to examine cross-sectional associations between individual behaviours and QoL, and linear mixed effects models to examine prospective associations of number of behaviours and change in QoL over 7.5-years. 

RESULTS
Individually, diet, non-smoking, and physical exercise were cross-sectionally associated with physical and mental QoL, and meditation with mental QoL. Non-smoking showed strongest associations (8.2/100 mental and 6.9/100 physical QoL). Dose-dependent positive associations were observed with adoption of 2-6 behaviours; maximum effect with six behaviours (16.7/100 mental and 17.7/100 physical QoL). No prospective associations were found between adoption of number of lifestyle behaviours at baseline and change in QoL over 7.5-years. 

CONCLUSION
Individually, diet, non-smoking, physical exercise, and meditation practice were associated with higher QoL. A dose-dependent association was identified with increasing number of lifestyle behaviours adopted and improved QoL. Further investigation, in a diverse MS population, is needed to assess prospective benefits. 

FUNDING
This research was generously funded by philanthropic gifts from Mr Wal Pisciotta, and anonymous donors.

Repeated short-term demyelination events impair oligodendrocyte survival and result in remyelination failure in the mouse brain. 

BACKGROUND

Preventing the progression of neurological disability experienced by people living with Multiple Sclerosis (MS) is a key goal. To achieve it, we are searching for new therapeutics that promote myelin repair and prevent nerve damage. Critical to this, is testing the efficacy of potential remyelinating therapeutics in mouse models. However, none of the existing animal models used, exhibit the spontaneous failed myelin repair that characterises MS. 

OBEJCTIVE

To overcome this, we modified the cuprizone model of de- and re-myelination to more closely mimic the relapsing-remitting myelin loss that can occur in MS, by repeatedly exposing female mice to short-term 0.2% cuprizone demyelination followed by 1-week withdrawal and remyelination. 

METHOD

We examined the extent of myelin loss and repair using Spectral Confocal Reflectance (SCoRe) microscopy to detect compact myelin and immunohistochemistry, immediately after the final cuprizone exposure, 7- and 21-days later. 

RESULTS

This revealed that although some remyelination occurred 21-days after the final demyelination event, the extent of myelination never returned to the healthy levels. Examining the oligodendrocytes, the cells that make myelin, we found that the immature oligodendrocyte progenitor cells (OPCs) did not deviate from healthy levels at any stage. However, the mature oligodendrocytes required for myelin repair, transiently returned to healthy levels 7-days after the final demyelination event, but this was not sustained at 21-days recovery. Importantly, this pattern of oligodendrocyte repopulation was not observed in the cuprizone control group which received a single 5-week exposure of cuprizone and represents the conventional model used for testing remyelinating therapies. In these mice, mature oligodendrocytes returned to healthy levels 7-days after cuprizone demyelination, and these levels were sustained at 21-days recovery. 

CONCLUSION

Collectively, these results indicate that repeated short-term demyelination appears to result in impaired oligodendrocyte survival and limited myelin repair. Repeated cuprizone exposure represents a new tool to effectively test remyelinating drug candidates in a mouse model that is more like MS. 

FUNDING

This work was funded by an MS Research Australia / Trish Foundation Project Grant (#18-055) to SSM and JLF.

A rare case of concurrent anti-NMDA-receptor encephalitis and first presentation Multiple Sclerosis in the setting of adalimumab treatment for psoriatic arthritis. 

BACKGROUND

Anti-N-methyl-D aspartate receptor encephalitis (anti-NMDA-R) is an autoimmune disorder characterized by severe neuropsychiatric symptoms secondary to antibodies against the NMDA receptor. Typical MRI findings include mesial temporal and frontal lobe T2 hyperintensities and leptomeningeal contrast enhancement. Anti-NMDA receptor encephalitis is not associated with demyelinating lesions however overlapping demyelinating disorders developing have been reported. This paper describes a patient presenting with features of concurrent anti NMDA-receptor autoimmune encephalitis and first presentation of Multiple Sclerosis (MS). 

CASE REPORT

A 32-year-old-woman with a history of psoriatic arthritis on adalimumab presented with visual disturbances and left hand paraesthesias 7 weeks post-partum. MRI demonstrated enhancing periventricular, callososeptal and juxta-cortical supratentorial white matter lesions characteristic of demyelination and a left temporal lesion with circular enhancement of unclear etiology. Oligoclonal bands were detected in CSF but not in serum. 1 month after initial presentation, she developed erratic and disorganized behavior requiring psychiatric admission with a provisional diagnosis of postpartum psychosis.  While as an inpatient she had a generalised seizure. Both serum and CSF fluid anti-NMDA-R antibodies were positive on two separate occasions warranting the diagnosis of anti NMDAR mediated autoimmune encephalitis. She was treated with intravenous immunoglobulin (IVIg) and her psychosis resolved. Repeat MRI showed dissemination of the demyelinating lesions in space and time, fulfilling  McDonald criteria for multiple sclerosis. The left temporal lesion had decreased in size and no longer enhanced.

CONCLUSIONS

We present a rare case of both MS and anti NMDA-receptor autoimmune encephalitis in a female postpartum previously treated with adalimumab for psoriatic arthritis. The overlap raises the possibility of a pathogenetic link between the two syndromes, potentially related to an  induced immune aberrancy in the setting of exogenous TNF-α blockade.

Gene expression differences identified in iPSC-derived astrocytes from people with MS.
 

BACKGROUND
Intrinsic dysfunction of central nervous system (CNS) cells may contribute to MS development and progression. Within the CNS, astrocytes support neurotransmission, maintain blood-brain-barrier integrity and regulate inflammation. Their function can be compromised by MS, but these cells could also contribute to disease initiation.

OBJECTIVE
o compare the gene expression of unstimulated, serum-free induced pluripotent stem cell (iPSC) -derived astrocytes generated from MS-affected and unaffected individuals, using a previously published dataset.  

METHOD
Published 125bp single-end RNA-seq data from Perriot et al. (2018) was obtained from the Sequence Read Archive. The data consisted of reads from iPSC-derived astrocytes from 4 people with MS and 3 unaffected controls. After quality control processing, gene expression was quantified using Salmon. Differential gene expression analysis was performed in R using DESeq2 (v1.34), comparing MS and unaffected control iPSC-derived astrocytes.  

RESULTS
Six differentially expressed genes (DEGs) were detected between unstimulated MS and control iPSC-derived astrocytes. The most significant DEG was Glutamate Ionotropic Receptor NMDA Type Subunit 2A (GRIN2A). GRIN2A was upregulated in MS astrocytes (LFC = 3.46, Padj = 0.0027) and has previously been linked to epilepsy and speech disorders. Polymorphisms in GRIN2A have previously been associated with changes in grey and white matter brain volume in people with MS. 

CONCLUSION
There are inherent gene expression differences between serum-free iPSC-derived astrocytes from people with and without MS. Significantly DEGs relate to ion channel, transmembrane transporter, and cytoskeletal functions. 

FUNDING
Medical Research Future Fund, Research Training Program Scholarship

Can oligodendrocyte-derived methylation signatures in cell-free DNA help inform treatment decisions and prevent irreversible neurological damage? 

BACKGROUND

Epigenetics, including methylation, enable tissue-specific gene expression. For instance, MOG is only expressed by oligodendrocytes, where it is hypomethylated compared to all other tissues. With advances in cell-free (cf)DNA analysis, tissue-specific damage can now be characterised through serum cfDNA methylation signatures. 

OBJECTIVE

To investigate whether serum oligodendrocyte-derived cfDNA may serve as a specific diagnostic and monitoring biomarker in multiple sclerosis (MS), which can inform treatment decisions to prevent irreversible disability accumulation. 

METHOD

cfDNA will be extracted, bisulphite converted, and subjected to digital-droplet PCR using methylation-specific primers and probes for three genomic regions that are specifically hypomethylated in oligodendrocytes. Digital droplet is highly sensitive and can detect as little as five copies of unmethylated DNA in the input sample. To determine whether cfDNA is more specific than emerging serum biomarkers, neurofilament light chain (NfL) will be measured on the Simoa platform. 

RESULTS

Serum and cerebrospinal fluid (CSF) were collected from 10 newly diagnosed MS and 10 idiopathic intracranial hypertension (IIH) patients. Oligodendrocyte cfDNA and NfL level will be assessed in the CSF of both groups to determine the specificity of oligodendrocyte-derived cfDNA compared to NfL. Next, matched serum samples will be used to correlate CSF cfDNA with peripheral blood levels. Serum cfDNA levels will be correlated with disease severity and activity. 

CONLCUSIONS

Findings will highlight the biomarker potential of oligodendrocyte cfDNA. Using more specific biomarkers to support MS diagnoses may enable faster diagnoses and earlier treatment initiation. Similarly, earlier detection of disease activity, may enable timely intervention; thus, prevent or reduce the accumulation of disability.

An interactive step training system to reduce falls in people with multiple sclerosis: a multi-centre randomised controlled trial.

BACKGROUND

0% of people with MS (PwMS) fall at least once within a 3-month period. Cognitive-motor step training is a safe and feasible way to improve stepping, balance and functional performance in PwMS. However, whether this intervention can prevent falls in PwMS has not been tested. 

OBJECTIVE

To determine the effects of a six-month home-based step training program versus usual care on falls in adults with MS (³ 18 years of age) with Expanded Disability Status Scale score of 2.0-6.0. 

METHOD

Single-blind two-arm parallel multi-centre randomised-controlled trial. The intervention group received the smart±step system and were instructed to exercise for 120 minutes per week for six months. The smart±step system requires participants to make appropriately timed and accurate steps from both legs to play TV-projected exergames that require fast responses, selective attention, response inhibition, working memory, visuospatial processing and task-switching. The control group was advised to continue their usual exercises and activities. 

RESULTS

462 PwMS (79% women) participated. The rate of falls did not differ between intervention and control groups over the first 6 months: IRR (95%CI) =0.96 (0.69 to 1.34) (p=0.816), or over 12 months: IRR (95%CI) = 0.97 (0.73 to 1.29) (p=0.876). Sub-group analyses (e.g. past falls; disability) did not yield any further differences between groups. Participants who trained 60+min/week showed more benefits (IRR (95%CI): 0.69 (0.44 to 1.10), p=0.123). 

CONCLUSION

 six-month home-based cognitive-motor step training program did not reduce the rate of falls in PwMS. Future research should aim to maximise adherence to exercise-based fall prevention interventions

United they stand, divided they fall: Machine Learning accurately predicts multiple sclerosis severity where GWAS fails.

BACKGROUND

One of the fundamental, unanswered questions with respect to Multiple Sclerosis (MS), is the source of the underlying disease heterogeneity observed clinically. To-date genetic studies of MS severity have been largely unsuccessful in identifying genetic variants that may explain this heterogeneity in clinical course.

OBJECTIVE

To replicate previously published putative genetic markers of severity. To identify novel single nucleotide variants (SNVs) associated with MS severity. Finally, to determine whether machine learning approaches may be more powerful in finding genetic associations with severity than traditional genome-wide association study (GWAS) approaches. 

METHODS

We genotyped individuals of European ancestry with relapse-onset MS, enrolled in the MSBase Registry, using the Illumina MegaEx array; and imputed against the Haplotype Reference Consortium panel. Fixed-effects meta-analyses tested associations with three MS severity phenotypes: longitudinal age-related MS severity scale (ARMSS), longitudinal MS severity scale, and age at onset. Gradient boosted tree machine learning was used to look for non-linear associations between genetic variants and phenotypes of interest.    

RESULTS

1,813 individuals passed quality control procedures. We replicated one putative non-HLA severity variant rs868824, intronic to IMMP2L, associated with age at onset (b=-1.0935; p=4.31x10-4). No novel SNVs were associated with our longitudinal severity phenotypes (p>5x10-8). Using 62k variants with p<0.01, gradient boosted trees predicted severity based on our longitudinal ARMSS phenotype with 85% accuracy in a testing set that the training algorithm had never seen (70-30% split).

CONCLUSION

Using machine learning we were able to accurately classify individuals as having mild or severe MS where GWAS failed. Our results demonstrate that MS severity is not under strong genetic control. That like MS risk, many variants collectively contribute miniscule amounts to disease severity.

Using Amide Proton Transfer to investigate and characterize lesions and normal-appearing white matter in Multiple Sclerosis. 

BACKGROUND

Autoimmune attacks in MS disease cause inflammatory demyelination, leading to axonal loss and destruction of myelin. Amide proton transfer (APT) imaging is a novel type of MRI that can detect the interaction between macromolecular protons, found in the intra/extra-cellular myelin debris proteins and surrounding tissue water. 

OBJECTIVE

We aim to explore the difference of APT signal between MS lesions and NAWM to detect the breakdown products of myelin proteins previously described in histological studies. 

METHOD

Five MS patients diagnosed with relapsing-remitting multiple sclerosis (RRMS) according to the McDonald criteria were recruited. All MRIs were performed on a 3T scanner using a 64-channel coil. For assessment of MS lesions, 3D T1-MPRAGE and T2-FLAIR were used. The APT sequence was performed using a 3D snapshot-GRE. Mean APT signal was computed from identical-size 2 MS lesions vs 2 NAWM regions of interest (ROIs) per subject using Olea Sphere 3.0 software. 

RESULTS

Mean APT signal in MS lesion ROIs increased by an average of 790% compared to NAWM ROIs. This high APT signal in MS lesions is supportive of the presence of mobile myelin proteins decomposition and accumulation from demyelination process. 

CONCLUSION

The APT technique is novel and has the potential to be a powerful and more sensitive tool for investigating the pathophysiology of MS. Increasing APT signal in MS lesions supports MS post-mortem immunochemistry and histological studies.

ACKNOWLEDGEMENT

This research was kindly supported by MS Research Australia. The first author was supported by a PhD scholarship with an annual grant from University of Jeddah, Saudi Arabia. 

Biomarkers of depression in individuals with multiple sclerosis and depression: a systematic review and meta-analysis.

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A longitudinal investigation of biomarkers of depression in individuals newly diagnosed with multiple sclerosis.

Choroid plexus volume predicts expansion of chronic lesions and brain atrophy

BACKGROUND AND OBJECTIVES
The expansion of long-standing multiple sclerosis (MS) lesions and an enlargement of choroid plexus are linked to chronic inflammation and microglial activation. In the current study, we investigated the association between plexus volume and subsequent lesion expansion in patients with relapsing remitting MS.

METHOD
Pre- and post-gadolinium 3D-T1, 3D FLAIR and diffusion tensor images were acquired from 49 patients with relapsing-remitting MS. Choroid plexus volume and lesion activity were analysed between baseline and 48 months.

RESULTS
Plexus volume remained stable during follow-up period. There was a strong correlation between baseline plexus volume and subsequent rate of chronic lesion expansion (r=0.77, p<0.001), which was stronger in close proximity to CSF. Furthermore, baseline plexus volume was also associated with change of Mean Diffusivity (MD) inside expanding area (r=0.55, p<0.001). There was, however, no correlation between baseline plexus volume and volume of new lesions. A cut-off of 98 x 10-5 plexus/TIV ratio predicted future lesion expansion with a sensitivity of 85% and specificity of 76%. Plexus volume larger than a cut-off was associated with >8-fold increased risk of chronic lesion expansion. Furthermore, baseline plexus volume significantly correlated with change of MD in lesional core during the study period (r=0.67, p<0.001) and with central brain atrophy (r=0.57, p<0.001).  

CONCLUSION
Our data demonstrate that baseline plexus volume predicts subsequent expansion of chronic periventricular MS lesions and associated tissue damage.

Acceptability study of ELEVIDA, an online intervention for fatigue in multiple sclerosis.

BACKGROUND

Fatigue is one of the most common and disabling symptoms reported by people with multiple sclerosis (MS), yet effective management is limited. A German version of ELEVIDA, based on the principles of cognitive behaviour therapy (CBT), has shown to be effective in reducing MS-related fatigue. 

OBJECTIVE

To pilot test the acceptability, feasibility and suitability of use in Australia of an English version of ELEVIDA.

METHOD

We recruited volunteers with MS aged 18 years and over who self-reported at least mild fatigue (scoring ≥43 on the Fatigue Scale for Motor and Cognitive Functions scale), mild or no self-reported cognitive difficulties (scoring ≤32 on the Multiple Sclerosis Neuropsychological Screening Questionnaire) and some mobility (EDSS<8). Participants were asked to ‘think aloud’ and provide feedback when completing the nine online conversations. 

RESULTS

Fifteen people living with MS-related fatigue completed the ELEVIDA program (mean age=58.87 years, SD=10.52, 10 females, 5 males, 9 with relapsing MS, 6 with progressive MS). Over 90% of the participants rated the ELEVIDA program as acceptable, good or very good; 80% of participants said they would recommend ELEVIDA to others living with MS-related fatigue, and almost 70% of participants found the ELEVIDA program helpful for treating their fatigue. 

CONCLUSION

The English version of ELEVIDA is acceptable, feasible and helpful for people living with MS-related fatigue. Participant ratings and feedback from this study will contribute to the optimisation of the English version of ELEVIDA for larger studies aimed at improving MS-related fatigue in Australia.

BACKGROUND

Difficulties with sleep are common in people with MS (pwMS), and self-reported sleep quality is reduced in pwMS compared to healthy individuals. 

OBJECTIVE

To examine the relationship between sleep-related measures and 13 common MS symptoms, and to determine the independent effect of sleep-related measures on health-related quality of life in a cross-sectional analysis of data 1717 pwMS from the Australian MS Longitudinal Study. 

METHODS

Sleep quality was assessed in the MS Nurses and Sleep questionnaire (2020) using the Pittsburgh Sleep Quality Index (PSQI), daytime sleepiness the Epworth Sleepiness Scale, severity of restless legs the International Restless Legs Syndrome Study Group Rating Scale, insomnia using the Menzies MS Insomnia Index. Quality of life was assessed by the Assessment of Quality of Life questionnaire (AQoL-8D). We analysed data using hierarchical cluster analysis and multivariable linear regression. 

RESULTS

67% of pwMS had poor sleep (PSQI>5). Poor sleep was associated with obesity, greater disability, worse MS symptom scores. MS symptoms clustered into 7 clusters, with sleep-related measures clustering separately. Three clusters were independently associated with PSQI score (‘pain and sensory symptoms’, ‘anxiety and depression’, ‘fatigue and cognitive symptoms’). All measures of sleep quality were associated with AQoL-8D utility scores independent of symptoms and increasing variance explained (Rsq=58.6% to 65.1%). 

CONCLUSION

Sleep quality and dysfunction cluster independently of common MS symptoms despite being correlated. Poor sleep has substantial deleterious effects on health-related quality of life independent of other MS symptoms. 

FUNDING

The Australian MS Longitudinal Study is funded by MS Research Australia.

Dendritic cell therapy for multiple sclerosis based on promoting Mertk signalling. 

In multiple sclerosis (MS), dendritic cells (DCs) present autoantigens to and activate autoreactive lymphocytes, leading to central nervous system demyelination. Many current treatments are broadly immunosuppressive. A more targeted therapeutic strategy is to re-establish immune tolerance by exposing DCs to tolerogenic stimuli with disease-specific autoantigens. 

I aim to develop tolerogenic DCs by promoting signalling via Mertk, a tyrosine kinase receptor involved in immune tolerance. I will also investigate how Mertk, a susceptibility and severity gene dependent on HLA-DRB1*1501 status, influences presentation of the recently identified MS autoantigen RASGRP2 by HLA-DRB1*1501. 

Peptides derived from RASGRP2 predicted to bind to HLA-DRB1*1501 were synthesised and incubated with HLA-DRB1*1501-expressing antigen presenting cells. HLA-DR-bound peptides were eluted and identified by mass spectrometry. Monocytes isolated from peripheral blood mononuclear cells of blood donors and patients with MS were differentiated to DCs, which were characterised by flow cytometry and endocytosis functional assay. Effect of various tolerogenic stimuli on DC Mertk and cell surface marker expression was assessed. 

One RASGRP2-derived HLA-DR bound peptide with strong affinity for HLA-DRB1*1501 was identified by mass spectrometry. DCs with good viability in serum-free medium, appropriate morphology, expression of mature DC markers including HLA-DR, CD83, CD40 and CD80 and capacity for receptor-mediated endocytosis could be differentiated. The tolerogenic stimulus dexamethasone, but not vitamin D3 or TGF-β, upregulated DC Mertk and downregulated HLA-DR, CD83 and CD80 expression, characterising a more immature and tolerogenic phenotype. 

DCs with a tolerogenic phenotype and Mertk expression could be induced by dexamethasone. The identified HLA-DRB1*1501-bound RASGRP2 peptide will be used to stimulate MS-specific immune tolerance. Further work includes activating the Mertk pathway with its ligand Gas6 and assessing tolerogenic DC effects on CD4+ T-cells.

Toxin-induced demyelination alters PV immunoreactivity and increases the proportion of mature dendritic spines in the adult mouse cortex. 

BACKGROUND
In MS, myelin loss alters neuronal signal conduction and fidelity, increasing vulnerability to damage, and eventually leading to neuronal death. However, it is unknown whether myelin loss alters synaptic connections or how such changes contribute to MS symptoms or neurodegeneration.

OBJECTIVE
To characterise the effect of toxin-induced demyelination on parvalbumin (PV) inhibitory interneurons and synaptic connections in the cortex of adult mice.

METHOD
To induce demyelination, Thy1-YFPH mice received 0.2% (w/w) cuprizone diet for 5-weeks. Myelin basic protein (MBP) and parvalbumin (PV) were detected by performing immunohistochemistry, and extent of demyelination and number of PV+ inhibitory interneurons was quantified. Following confocal imaging and 3D reconstruction of the layer V excitatory pyramidal neurons dendrites labelled by YFP+, in the parietal association area, we quantified the density and morphology of dendritic spines (excitatory post-synaptic compartment). 

RESULTS
Demyelination in the corpus callosum was evidenced by significant reduction in MBP-labelling, relative to control mice. In layer IV of the parietal association area, demyelination significantly increased the number of immunoreactive PV+ inhibitory interneurons.  Demyelination did not alter total spine density, but significantly increased the proportion of mature, mushroom shaped dendritic spines (synaptic connections) on pyramidal neuron apical dendrites. Correspondingly the proportion of immature thin spines was decreased.

CONCLUSION
These data display that demyelination in the corpus callosum and cortex of adult mice may alter the balance of excitatory/inhibitory neuron signalling in a way that could alter neuronal response to glutamatergic signalling, potentially rendering them vulnerable to excitotoxic injury.

FUNDING
MS Australia, NHMRC, UTAS (PhD Scholarship)

Smoking, infectious mononucleosis, early-life exposures and risk of progressive-onset Multiple Sclerosis: a case-control study. 

BACKGROUND

Risk factors in Multiple Sclerosis (MS) have been identified in samples that contain predominantly cases with relapse-onset MS (ROMS). It is unknown whether the risk factors differ in people with a progressive-onset MS (POMS). 

OBJECTIVE

Examine the association between smoking, infectious mononucleosis, early life exposures and POMS and compare the magnitude of association with POMS. 

METHOD

The case-control study included 147 POMS cases, 264 ROMS cases and 558 community controls. Questionnaires were used to collect information on smoking, infections, breastfeeding and vitamin use in early life. Logistic regression was used to examine associations. Odds ratios (ORs) were adjusted for age, sex and latitude band. 

RESULTS

Compared with controls, POMS cases were more likely to have smoked ≥20 pack-years (AOR 2.83, 1.53-5.24) and ROMS cases were more likely to have ever been a smoker (AOR 1.48, 1.09-2.01). POMS cases (AOR 1.75, 1.05-2.93) and ROMS cases (AOR 1.74, 1.21-2.49) were both more likely to have had infectious mononucleosis. In childhood, POMS cases were less likely to use vitamin supplements (AOR 0.52, 0.30-0.91) or being breastfed (AOR 0.49, 0.31-0.79). 

CONCLUSION

POMS and ROMS share some commonly known risk factors. Some protective factors were identified in POMS that were not observed in ROMS.

An attenuated murine EAE variant for evaluating Multiple Sclerosis therapies.

BACKGROUND

The classic murine EAE model used extensively in pre-clinical drug evaluation for MS exhibits rapid disease progression with a narrow time window of about 4 days for drug affect to be accessed. More appropriate models are required for improved drug evaluation. 

OBJECTIVE

This project describes the development of a novel EAE variant characterized by slow disease progression, more suitable for detailed drug evaluation. Hallmarks of inflammatory demyelination must be present for this model to be an acceptable MS model. 

METHOD

The three main reagents used in EAE induction are, Freud’s adjuvant, myelin oligodendrocyte glycoprotein (MOG35-55) and pertussis toxin. A range of protocols were evaluated by titration of each of these components. Effects of change in dosage of each component were determined by clinical profiling as well as quantification of lesion load, axonal and neuronal loss. 

RESULTS

The mice in the optimised protocol can be kept up to 35 days before human endpoint is reached. This is about 20 days longer than the standard MOG35-55 EAE protocol used routinely in the laboratory. At the peak of disease, day 14, mean clinical score of the variant group is 0.2 compared to 1.9 in the standard group with evident lesion damage by demyelination and axonal injury at endpoint. 

CONCLUSION

The results derived suggest that the new variant is suitable to be used in a therapeutic drug trial. Currently a drug trial is being carried out using this variant.

Novel Plasma and Brain Proteins implicated in Multiple Sclerosis Susceptibility as Potential Targets for Drug Repurposing. 

BACKGROUND
Understanding how variations in the plasma and brain proteome contribute to multiple sclerosis (MS) susceptibility can provide important insights to guide drug repurposing and therapeutic development for MS.  

OBJECTIVE
This proteome-wide analysis aims to identify plasma and brain proteins associated with MS risk, followed by assessing differential gene expression patterns in relevant immune and brain cell types for the associated proteins.  

METHOD
Integrating plasma proteomics (n=3,301) and brain proteomics (n=376 discovery; n=152 replication) into MS genome-wide association studies (n=14,802 cases and 26,703 controls), we employed summary-based methods to identify candidate proteins involved in MS susceptibility. We further examined differential gene expression across five immune cell types (n=73 cases and 97 controls) for MS-associated plasma proteins, and across twenty brain cell clusters (17,799 nuclei from n=4 cases and 5 controls) for MS-associated brain proteins.  

RESULTS
We identified 39 novel proteins associated with MS risk. Based on five identified plasma proteins, we found consistent patterns of differential TAPBPL upregulation and MAPK3 downregulation in immune cells from MS patients. Among the 34 candidate brain proteins, 18 were replicated in a smaller cohort and ten showed robust differential gene expression in functional subclusters of brain cells. Further, we highlighted existing approved therapeutics that target the associated proteins.  

CONCLUSION 
The associated proteins present a robust set of candidate biomarkers and therapeutic targets for MS. Findings from this study highlight the complexity of disease-associated proteins with cell-specific transcriptional profiles, providing useful insights that could inform future studies of drug repurposing and novel therapeutic development. 

ACKNOWLEDGEMENTS 
YZ was supported by a MS Research Australia postdoctoral fellowship as well as an NHMRC investigator grant L1 (GNT1173155). We also thank the investigators who have made their datasets freely available to undertake research of this type.

Non-pharmacological methods for reducing fatigue in Multiple Sclerosis (FAMUS). 

BACKGROUND

Fatigue is one of the most difficult symptoms to manage in patients with MS (pwMS). Most studies on fatigue use self-report surveys to determine primary outcomes. These are subjective but are the only option, because measuring the physiological mechanism behind fatigue is unknown.  

OBJECTIVE

Our primary objective is to compare a Mindfulness Based Stress Reduction (MBSR) program to a light exercise program in reducing fatigue in pwMS over an 8 week program. 

METHOD

We will recruit subjects to three study arms: MBSR intervention (pwMS), MSBR intervention (healthy control (HCs)) and a pilates intervention (comparator program). Participants will perform surveys for fatigue, mental health, physical activity levels, cognitive assessment, and sleep quality as well undergo advanced Magnetic Resonance Imaging (MRI) scans. Specifically, resting state functional (rsf) MRI will be performed. All procedures will be performed before and after the intervention. 

RESULTS

We have recruited 7 pwMS to the Pilates program, 6 pwMS to the MBSR program and 7 HCs to the MBSR program. All baseline assessments have been performed and the programs are underway. 

CONCLUSION

This study we will use a combination of surveys and advanced MRI to assess fatigues levels in pwMS. We hope to provide an insight into the physiological mechanisms behind fatigue and provide evidence for whether exercise or MBSR programs can help improve fatigue in pwMS.

The modulation of the kynurenine pathway of tryptophan metabolism by Monomethyl fumarate in mouse subventricular zone neural stem cells. 

BACKGROUND
Our research showed Kynurenine pathway (KP), the canonical route of tryptophan metabolism, is dysregulated in MS, producing increasing metabolite levels paralleling disease severity. KP enzymes including rate-limiting Indoleamine-2,3-diooxygenase-1/IDO-1 are activated by interferon-gamma, produced by activated immune cells. Nanomolar Quinolinic acid (QUIN) potently kills oligodendrocytes/neurons. 


OBJECTIVE
How drugs affect the brain neural stem cell (NSC) niche underpins understanding mechanisms to overcome roadblocks in stem cell-mediated repair, particularly in secondary-progressive MS. We investigated if monomethyl fumarate (MMF; active metabolite of MS drug Tecfidera) reduces the impact of KP activation on NSCs. 

METHOD
Mouse SVZ neurospheres were dissociated at passage 4 and plated for experiment on poly-L-ornithine/laminin-coated dishes. After trypsinisation, apoptosis and other cell health parameters were analysed on a Muse cytometer. A p value <0.5 was regarded statistically significant. 

RESULTS
15mM MMF significantly reduced oxidative stress from interferon-gamma treatment at 24-72 hour timepoints, and caspase expression (24 hours). MMF and interferon-gamma also separately (non-synergistically), significantly reduced cell proliferation. 30mM MMF did not potentiate the effects. Surprisingly, the KP was constitutively active without interferon-gamma, suggesting activity of tryptophan-2,3-dioxygenase (TDO2) enzyme. Moreover, secreted metabolites were not further increased with interferon-gamma treatment, suggesting interferon-gamma-responsive gene IDO-1 is not expressed. Substantial levels (approximately 2,200nM) of 3-Hydroxy-Anthranillic Acid, a QUIN precursor were detected, a known antioxidant, while QUIN was undetectable. 3-Hydroxy-Anthranilic Acid accumulated over time in MMF-treated cultures versus other treatments (declined), suggesting positive KP modulation. 

CONCLUSION
Collectively, these data show MMF is protective toward NSCs and may help reverse the deleterious effects of interferon-gamma or QUIN during neuroinflammation, possibly by enhancing 3-hydroxy-Anthranilic acid production. Enhancing NSC health and survival is an important aspect to consider in novel MS therapeutic strategies. 

FUNDING
We acknowledge funding of the Peter Duncan Neuroscience Research Unit at St. Vincent’s Centre for Applied Medical Research and funding from Biogen Idec Pty Ltd for the completion of this study.

Laquinimod as a novel regulator of Kynurenine Pathway in Neural Stem Cells and Monocytes. 

BACKGROUND

Our research uncovered that Kynurenine pathway (KP), the canonical route of amino acid tryptophan metabolism, is dysregulated in MS, producing increasing levels of metabolites corresponding to disease severity. KP enzymes including rate-limiting IDO-1 are activated by interferon-gamma. Nanomolar Quinolinic acid (QUIN) metabolite potently kills oligodendrocytes/neurons. 

OBJECTIVE

Laquinimod is a drug that for relapsing-remitting MS showed clinical efficacy in reducing brain atrophy/relapse risk, suggesting a neuroprotective action. It’s effects on the KP are currently unknown. We aimed to investigate if Laquinimod reduces KP dysregulation and elevated QUIN levels in two relevant celltypes. 

METHOD

Laquinimod was tested on both cultured human monocytes and cultured mouse subventricular zone mouse neural stem cells. Monocytes were treated with/without interferon-gamma. SVZ neurospheres were dissociated and plated for experiment on poly-L-ornithine/laminin-coated dishes. Apoptosis and cell health parameters were analysed on a Muse cytometer. A p value <0.5 was regarded as statistically significant. 

RESULTS

Laquinimod reduced monocyte basal IDO-1 expression by 18.44+6.1% versus control. In interferon-gamma-treated monocytes, Laquinimod increased levels of neuroprotective Kynurenic acid and significantly increased it in 24-hour pre-treated monocytes prior to 72 hours interferon-gamma. Laquinimod significantly reduced neurotoxic QUIN in interferon-gamma-treated monocytes. Apoptosis was reduced in mouse SVZ cells treated with Laquinimod and QUIN, restoring to control. Cell confluence declined with Laquinimod but cell cycle parameters unaltered, suggesting alteration in cell-size. 

CONCLUSION

Monocytes highly upregulate IDO-1 with IFN-gamma, secrete QUIN, cross the blood-brain barrier and localise to lesions. QUIN may further compromise oligodendrocyte survival and neuronal atrophy. Laquinimod was protective on neural stem cells. Enhancing stem cell survival and modulating monocytes underpins possible novel MS therapeutic strategies. 

FUNDING

We acknowledge funding of the Peter Duncan Neuroscience Research Unit at St. Vincent’s Centre for Applied Medical Research.

A 10-year longitudinal study of rapid and objective visual function testing of multiple sclerosis patients. 

BACKGROUND

Our older work on sparse multifocal visual evoked potentials (mfVEPs, DOI:10.1002/ana.20504) and related pupillographic methods (DOI:10.1177/1352458513512708) suggested that they provided information related to progression of MS. Here we re-examine patients tested 10 years earlier with one of those pupillographic methods of functional testing. 

OBJECTIVE

To examine the ability of an older test available on the FDA-cleared Konan objectiveFIELD Analyser (OFA) to predict MS progression over 10 years.  

METHOD

In 2009 we tested 85 MS patients with an older variant of the OFA test (DOI: 10.1177/1352458513512708). In 2019 we retested 46 of those patients, and reassessed their EDSS scores. The OFA tested 44 stimulus regions/eye across ±30 degrees, concurrently testing both eyes in 8 minutes. Logistic regression of OFA results on EDSS progression was examined using the mean of the worst three per-region delays/eye. 

RESULT

Delays produced log-odds of 18.0/second of delay (p=0.045). One SD above the mean patient delay (across regions) in 2009 was 40 ms. For this relatively common delay the odds of progression were exp(18.0*0.04) =  2.05x,  SD = 1.43x and 2.95x. That also means that patients with 40 ms quicker than average responses were 2.05x less likely to progress. The logistic model also included changes in sensitivity, which were not significant.  

CONCLUSION

he old OFA test predicted progression/non-progression 10 years later. Newer OFA tests have better signal quality and reduced test time, and despite being a visual test provide similar performance in the absence of optic neuritis (e.g. abstract by CF Carle at this meeting).

Disease reactivation after cessation of disease-modifying therapy in relapsing-remitting multiple sclerosis

BACKGROUND

Multiple sclerosis treatment interruptions are common and leave patients vulnerable to breakthrough disease activity.  The safe duration of the ‘switchover time’ for different therapies is not known. 

OJECTIVES

To evaluate the rate of return of disease activity after cessation of multiple sclerosis (MS) disease-modifying therapy.

METHOD

This was a retrospective cohort study from two large observational MS registries: MSBase and OFSEP.  Patients with relapsing-remitting MS who ceased a disease-modifying therapy and were followed up for the subsequent 12-months were included.  The primary study outcome was annualised relapse rate in the 12 months after disease-modifying therapy discontinuation stratified by patients who did, and did not, commence a subsequent therapy.  Predictors of relapse and disability accumulation after treatment discontinuation were a secondary outcome. 

RESULTS

18 029 eligible treatment discontinuation epochs were identified.  Rates of relapse started to increase 2-months after natalizumab cessation.  Commencement of a subsequent therapy within 2-4 months reduced the magnitude of disease reactivation.  After discontinuation of fingolimod, rates of relapse increased overall, and stabilised faster in patients who started a new therapy within 1-2 months.  Predictors of relapse were higher relapse rate in the year before cessation, female sex, younger age and higher EDSS.  

CONCLUSION

Understanding the rate of disease reactivation after discontinuing different MS immunotherapies will help guide optimal wash-out times for therapeutic agents during treatment sequencing. 

Cladribine: a multicentre, LOng-term efficacy and Biomarker Australian Study (CLOBAS). 

BACKGROUND

Cladribine tablets are an oral therapy for treatment of relapsing Multiple Sclerosis (MS). The dosing schedule is 2 courses given a year apart, which is effective for up to 4 years in 75% of people with MS (pwMS) (based on annualised relapse rate). 

OBJECTIVE

The primary objective is the safety and efficacy of cladribine tablets over a 6-year period according to No Evidence of Disease Activity 3. Secondary objectives are safety and efficacy of additional courses, and evaluation of biomarkers as indicators of the need for re-dosing. 

METHODS

150 pwMS will be recruited from multiple centres to participant in a six-year trial. Subjects will receive treatment according to the current schedule. After year 3, patients will have the option for re-dosing, if clinically indicated, or to switch therapy. Throughout the study we will assess blood-based biomarkers including lymphocyte subsets, serum neurofilament light chain, DNA methylation, MRI findings (brain volume/lesion load) and cognitive performance. 

RESULTS

At the time of submission 135 pwMS have been recruited to the trial. Lymphocyte counts one month post dose 1 were significantly lower in patients who had prior therapy than treatment naïve patients (1200 cells/mm3 versus 1500 cells/mm3, p=0.02). We find a significant decrease in B cells by month 3. While the total CD19 population increased to pre-treatment levels by month 12, the memory B population does not recover. 

CONCLUSION

This will be the first efficacy trial of cladribine which offers re-initiation of therapy in year 3 based on disease activity. We expect to identify biomarkers that can be used to predict treatment efficacy or the need for future re-initiation of cladribine in pwMS.

Haematopoietic stem cell transplantation for Multiple Sclerosis results in deletion of auto-reactive public CD4+ T-cell clones. 

BACKGROUND

Multiple sclerosis is an inflammatory disease of the central nervous system, where autoreactive T cells are central to disease pathogenesis. 

OBJECTIVE

This study sought to analyse the kinetics of immune reconstitution for 36 months following autologous haematopoietic stem cell transplantation for MS. 

METHODS

Changes in T cell populations in 19 Multiple Sclerosis patients to 36 months were analysed by a multicolour flow cytometry panel, DNA markers of thymic output and high-throughput TCR RNA sequencing. 

RESULTS

Through T cell reconstitution analysis, we identified progressive ablation of memory T cell clones, which coincided with early lymphopenic-induced proliferation of CD4+ T-cells and delayed recovery of a thymically-derived naïve T cell repertoire that did not reach pre-AHSCT diversity. Significantly, the reconstituted diverse CD4+ memory population included far fewer public CD4+ clones with a high probability of negative thymic selection and potential auto-reactivity. Twelve of forty public CD4+ clones unique to HLA-DRB1*15:01-positive patients could not be detected post AHSCT, with re-emergence of one clone (CASSQELTDTQYF) correlating with disease relapse. 

CONCLUSION

AHSCT appears to induce sustained periods of disease remission with dynamic changes in clonal T cell repertoire out to 36 months post-transplant, including the deletion of putative auto-reactive clones.

Using signatures of natural selection to focus the search for causal genetic variants in multiple sclerosis.

BACKGROUND
Multiple sclerosis prevalence shows a heterogeneous geographical pattern, with higher prevalence in populations of European ancestry, as well as increasing with distance from the equator within those populations. This pattern has likely been shaped by both natural selection and neutral genetic drift. 

OBJECTIVE
Identifying genes that have undergone selection at MS risk loci will improve our understanding of the causative mechanisms behind the disease. We will use population genomics to identify functional variation that has been subject to natural selection at loci associated with MS risk. 

METHOD

We carried out genome-wide scans for natural selection using cross-population extended haplotype homozygosity in population genomic data. MS-related selection was localised by targeting genes prioritised in the large genome-wide association study carried out by the International Multiple Sclerosis Genetics Consortium. 

RESULTS
Strong signatures of natural selection in European and Asian populations were identified in several MS risk genes. Further analysis of these genes is underway to identify likely causes of selection and mechanisms by which they may contribute to MS risk. 

CONCLUSION
This approach allows us to narrow down candidate genes and pinpoint a small number of top causal candidates and mechanisms. This may enable more informed targeting of the molecular mechanisms behind the disease.

Establishing MS Stem - a repository of well characterised induced pluripotent stem cell lines from people with Multiple Sclerosis.

Multiple sclerosis (MS) is a complex autoimmune and neurodegenerative disease with no clear cause and no known cure. MS consumers indicate that a new treatment to protect and repair the central nervous system (CNS) is their greatest unmet need. We aim to achieve this by establishing a biobank of induced pluripotent stem cells (iPSCs) from people with primary progressive and secondary progressive MS that can be used by researchers to identify signalling pathways that precipitate MS pathology. We have quality-controlled procedures for collecting blood from people with MS and reprogramming the blood cells into iPSCs using foot-print free methods.  Blood has been collected from >50 people with MS and reprogramming has commenced. Each iPSC line generated has been well-characterised: they express key pluripotency markers, Oct4, SSEA4, Tra-1-60 and Tra-1-81; they can be differentiated into all three germ layers, ectoderm, mesoderm and endoderm; are karyotypically normal, and morphologically maintain a high nuclear to cytoplasm ratio.  Some of these iPSC lines are now being differentiated into various neural cell types for further study.

Description of Multiple Sclerosis cohorts from the Middle East between 2009 and 2019. 

BACKGROUND

Worldwide multiple sclerosis (MS) landscape is evolving in many regions including the Middle East. The Middle East is located between 12° and 42° of latitude and 35° and 54° of longitude and varying altitudes with various countries that fall within this region. 

OBJECTIVE

Registries such as MSBase enable researchers to aggregate worldwide data; however, precise interpretation of the data depends on correct understanding of the structure of the individual cohorts contributed to these registries. We aimed to provide a clinicodemographic overview of the cohorts of patients contributed to MSBase in the Middle East.  

METHOD

The MSBase registry was interrogated for patients with MS or clinically isolated syndrome from the Middle East with data captured between 2009 and 2019. With focus on the most recent years, we explored the demographic, clinical characteristics, and treatment exposures and reported the results using descriptive statistics. 13,356 patients from 17 centres in 8 countries (Egypt, Iran, Kuwait, Lebanon, Oman, Saudi Arabia, Turkey and the United Arab Emirates (UAE)) were included. 

RESULTS

The cohort was young (median=36, quartiles:29-45) with early MS (median<10 years, quartiles: 3-12 years). Median EDSS ranged from 0-3 except for Saudi Arabia where the median EDSS was 4 (quartiles: 1.5-6.5). The median relapse frequency was highest in Lebanon (1.03, 95% CI: 0.94-1.16) and Egypt (1.02, 95% CI: 0.89-1.24) and lowest in Saudi Arabia (0.70, 95% CI: 0.58-0.95) and Kuwait (0.75, 95% CI: 0.71-0.80). Platform injectable therapies were mostly utilized in Egypt, Iran and Turkey (86%, 79% and 53% respectively), while oral therapies and monoclonal antibodies were more commonly used in Kuwait, Lebanon and the UAE (87.2%, 67.3% and 58.7% respectively).

CONCLUSION

Patients from the Middle East captured in a large multinational registry are representative of the general MS population. The therapies used in these countries, however, are highly variable. Further studies that include rural and non-academic practices are needed to enhance our understanding of the MS cohorts in the Middle East.

Effect of multiple sclerosis disease modifying therapies, natalizumab, ocrelizumab, fingolimod and cladribine, on peripheral immune cell counts. 

BACKGROUND

Varying effects on peripheral immune cell counts after DMT have been shown. Alterations in cell counts are generally on target effects in keeping with the DMT mechanism of action. Variations in other cells counts are possible off target effects that may be of significance. 

OBJECTIVE

Monitor peripheral immune cell counts and immune cell ratios in multiple sclerosis (MS) patients on ocrelizumab, natalizumab, fingolimod and cladribine to decipher the effect of each disease modifying treatments on counts over time. 

METHOD

Our study included MS patients from Royal Melbourne Hospital and Box Hill Hospital. Peripheral immune cell counts done as part of routine clinical care was retrospectively collected, and the data was entered into iMed (MSBASE) patient register. Immune cell counts of patients treated with natalizumab (n=268 patients), ocrelizumab (n=185), fingolimod (n=265) and cladribine (n=48) were extracted. Data was subsequently analysed using piecewise segmented regression models for pre- and post-treatment. 

RESULTS

After 2 years, peripheral lymphocyte counts were shown to significantly increase with natalizumab (p=8×10−32) and decrease with fingolimod (p=2×10−30) or ocrelizumab therapy (p=0.02). Natalizumab and fingolimod also significantly reduced neutrophil (p=0.006; p=0.00002) and eosinophil counts (p=2×10−9; p=0.00004). Cladribine was shown to cause a reduction in peripheral monocyte count (p=0.00009). Effect on neutrophil-to-lymphocyte ratio (NLR) and the monocyte-to-lymphocyte ratio (MLR) biomarkers of each treatment was shown.

CONCLUSION

ocrelizumab, fingolimod, natalizumab and cladribine affect different peripheral immune cell types in varying degrees, with possible implications on efficacy and safety monitoring of each drug.

Low intensity repetitive transcranial magnetic stimulation does not alter OL survival or demyelination during cuprizone feeding.

Ideally, brain repair strategies for people with multiple sclerosis (MS) will involve the replacement of lost OLs as well as the protection of existing OLs.  Our team previously reported that low intensity repetitive transcranial magnetic stimulation (LI-rTMS) can increase the survival of new oligodendrocytes (OLs) in the adult mouse cortex. However, the ability of LI-rTMS to promote the survival of existing OLs has not been explored.  In this study, OLs were fluorescently labelled with yellow fluorescent protein (YFP) in P60 Plp-CreERT2 :: Rosa26-YFP transgenic mice, and their fate followed over time.  These mice then received 0.2% (w/w) cuprizone for 4 weeks to induce demyelination.  From week 3-4 of cuprizone delivery, mice also received sham-stimulation or LI-rTMS (120 mT) daily.  LI-rTMS was administered as an intermittent theta burst stimulation (iTBS; 600 pulses).  Brain cryosections containing the motor or visual cortices and the underlying corpus callosum were immunolabeled to detect YFP+ mature OLs (YFP+ ASPA+ or YFP+ OLIG2+ PDGFRα-negative).  We found that cuprizone resulted in a significant reduction in the density of YFP+ mature OLs in the visual cortex, motor cortex and the corpus callosum of sham-stimulated and LI-rTMS mice, relative to age-matched controls.  However, the density of mature OLs was equivalent in sham and LI-rTMS mice, in each region examined.  Consistent with this finding, black-gold labelling revealed significant myelin loss in sham and LI-rTMS mice relative to healthy age-matched controls; however, LI-rTMS treatment did not significantly alter the level of demyelination.  Our results suggested that LI-rTMS does not prevent OL loss or alter the level of demyelination produced by cuprizone feeding.  

Keywords: mature oligodendrocyte, demyelination, remyelination, transcranial magnetic stimulation

FUNDING

This study is funded by the MPST, MRFF, NHMRC, MS Research Australia.

Age-related decline in oligodendrogenesis is mediated by neuronal TrkB. 

BACKGROUND

Oligodendrocytes and myelin are generated lifelong, although limited by an age-related decline in efficiency. The spatiotemporal course of decline and the underlying molecular regulation remain unknown. A complete cellular and molecular understanding of lifelong oligodendrogenesis could inform therapeutic strategies to promote remyelination in the CNS. 

OBJECTIVE

We aimed to quantify rates of oligodendrogenesis at distinct stages across adulthood, within disparately myelinated regions, to classify the spatiotemporal course of decline. Given known importance of BDNF-TrkB signalling in developmental myelination, we aimed to identify the role of neuronal TrkB in mediating lifelong oligodendrogenesis. 

METHOD

To cumulatively label adult-born oligodendrocytes we administered thymidine analogue EdU for 6-weeks to C57Bl/6 mice beginning at 2-months (young-adulthood), 12-months (early aging) and 18-months (aging). Immunohistochemistry and confocal microscopy identified oligodendroglial lineage progression in; the optic nerve (almost fully-myelinated tract), corpus callosum (partially-myelinated tract) and somatosensory cortex (sparsely-myelinated grey matter). An inducible, conditional knock-out mouse line was used to delete neuronal TrkB at 12-months and cellular analysis performed 5-months later. 

RESULTS

We found a spatiotemporal decline in oligodendrogenesis with the contribution of new post-mitotic oligodendrocytes at 11% in the callosum, 6% in the cortex and 0.33% in the optic nerve at 2-months, and a brain-wide plateau of <1% by 12-months. Accordingly, the proportion of divided OPCs fell from ~80% to ~60% by 12-months, suggesting age-related quiescence. Loss of neuronal TrkB impaired the survival and differentiation of oligodendrocytes in the cortex only. 

CONCLUSION

These data highlight the inherent spatiotemporal regulation of oligodendrogenesis across the lifespan. Fascinatingly, they identify regional heterogeneity in the underlying molecular program driving lifelong myelination. Together, they comprise an important resource to inform future experimental design and targeted oligodendroglial reparative strategies.

Multiple sclerosis patients positive for HLA-DR15 have fewer CD14+ MERTK+ monocytes at remission, which is further reduced with disease activity  

BACKGROUND

The genotype of MERTK at rs7422195 influences disease course in established multiple sclerosis (MS) and correlates with either an increase or decrease in the risk and severity of disease depending on HLA-DR15 status. This finding provides further evidence for disease stratification based on HLA-DR15 status.

OBJECTIVES

We assessed the influence of HLA-DR15—the major genetic risk factor for MS—on innate immune cell profile during relapse and remission. We aimed to gain mechanistic insight on how MS susceptibility and severity are modulated in a stratified manner based on HLA-DR15 status 

METHOD

CD14+ monocytes were isolated from blood collected from people with MS at relapse (n=40) and three months post-relapse (n=23). Healthy controls underwent two blood collections three months apart (n=18 first time-point, n=16 second time-point). Immunophenotypic profiling of CD14+ monocytes from patients and controls was performed by flow cytometry while MERTK genotype at rs7422195 and HLA-DR15 status were determined by polymerase chain reaction.

RESULTS

HLA-DR15 positive patients had significantly lower proportions of CD14+ MERTK+ monocytes than HLA-DR15 negative patients, independent of rs7422195 genotype, with a similar trend observed in healthy controls. In HLA-DR15-positive patients experiencing relapse, a further contraction in proportions of CD14+ MERTK+ monocytes was observed. Additionally, patients homozygous for the major G allele at rs7422195 exhibited significantly higher frequency of CD14+ MERTK+ monocytes at relapse and remission compared to controls.

CONCLUSION

HLA-DR15 and MERTK genotype influence MERTK expression profile in different ways in MS patients and controls. In HLA-DR15+ MS patients, relapse-associated contraction of CD14+ monocytes expressing MERTK, a receptor involved in resolving inflammation, provides a potential mechanism by which risk genes interact to influence pathogenesis.

Intertwined like a double helix: a meta-synthesis of qualitative literature examining the experiences of living with someone with multiple sclerosis.

BACKGROUND

Carer-related research commonly reduces caring for another to a set of tasks and is often viewed negatively rather than recognising that caregiving is embedded within a relationship. This fails to realise the multidimensionality of the role and the lived experience of the caregiver. 

OBJECTIVE

To synthesise current research about experiences of people living with a person with MS to understand their perspectives and inform healthcare professionals, researchers, and policymakers about how best to incorporate their needs when treating and engaging with a person with MS and their significant others. 

METHOD

We followed scoping review methodology identifying the research question and relevant studies, selecting studies, charting the data, collating, summarising and reporting results. Thematic analysis was then used to conduct a meta-synthesis of the included studies. In the spirit of patient and public involvement, our research team included four members with MS and two members with lived experience of living or working with people with MS. 

RESULTS

People living with a person with MS included partners, spouses, next of kin, support persons, children of a parent with MS, and parents of a child with MS. Five themes were identified that described their experiences: (1) Seeking information and support; (2) Caring is hard work; (3) A changing identity; (4) Living with uncertainty and constant worrying; and, (5) Adapting to life with a person with MS. 

CONCLUSION

Support services and healthcare professionals are currently very much patient centred. They need to look beyond the person with MS and recognise the relational network of people who surround them and broaden their focus to encompass this network. 

FUNDING

This research was funded by and has been delivered in partnership with Our Health in Our Hands (OHIOH), a strategic initiative of the Australian National University, which aims to transform health care by developing new personalized health technologies and solutions in collaboration with patients, clinicians and health-care providers.

Defining how vitamin D promotes tolerogenic dendritic cells to enable its use in combined therapy. 

BACKGROUND

There is compelling genetic, laboratory and environmental evidence that vitamin D contributes to MS pathogenesis, but the inactive forms currently used in clinical trials have failed to provide benefit. This is because of genetic and epigenetic bottlenecks to vitamin D response. 

OBJECTIVE

An improved approach is needed to obtain benefit from the vitamin D pathway. The objective of this study was to define how vitamin D promotes a tolerogenic phenotype in the immunological context most likely to underpin its benefit: control of dendritic cells. 

METHOD

We identified how the chromatin is remodelled using ATACseq (Assay for Transposase-Accessible Chromatin using sequencing) and global gene expression changes using RNAseq in response to treatment with calcipotriol (active vitamin D analogue) for monocyte-derived dendritic cells with a tolerogenic or inflammatory phenotype. We also characterised the effect of co-treatment with vitamin D and a novel bromodomain inhibitor, i-BRD9. 

RESULTS

ATACseq revealed distinct chromatin accessibility changes in response to calcipotriol with over 4,000 genomic locations affected, accounting for approximately 8% of the variation in the ATACseq dataset. Transcriptomic analysis also showed a clear vitamin D response signature, with a far larger number of genes dysregulated in response to calcipotriol the inflammatory cells compared to the tolerogenic cells. Pathway analysis indicated a reduced inflammatory state in cells treated with calcipotriol. 

CONCLUSION

Calcipotriol treatment revealed a distinct a shift away from the inflammatory phenotype to a more immunosuppressive/tolerogenic phenotype. Work is ongoing to assess alternative strategies, including co-treatment with i-BRD9, to upregulate the vitamin D response and bypass the body’s homeostatic setpoint. 

FUNDING

Multiple Sclerosis Research Australia Project Grant
Trish MS Research Foundation Project Grant

Towards evidence-based nutrition: Developing and interpreting targeted nutrition message among Australians with Multiple Sclerosis. 

BACKGROUND

Growing evidence suggests that nutrition may affect multiple sclerosis (MS) progression. However, the communication of consistent, accurate and actionable nutrition information among people with MS (PwMS) is an ongoing challenge. Nutrition messages require attention to encourage dietary change in what are often entrenched health attitudes and behaviours. 

OBJECTIVE

The present study applied the Elaboration Likelihood Model (ELM) of persuasion to develop targeted nutrition messages for PwMS, with the aim to understand the interpretation and acceptance of the nutrition messages. 

METHOD

The ELM theoretical framework was used to translate the literature of diet-MS relationships into the targeted nutritional messages for PwMS. The ‘think-aloud’ methodology was employed to examine the thinking processes that occurred when PwMS and health care professionals (HCPs) were exposed to the messages, with subsequent semi-structured interviews to explore MS-specific needs for nutrition messages. The transcripts were analysed inductively using the principles of grounded theory. 

RESULTS

A total of 15 PwMS, seven allied health professionals and seven HCPs with MS were recruited. PwMS interpreted the messages in terms of food, psychosocial outcomes, personal goals, and specific life experiences (e.g., disease onset). The interpretations of messages by HCPs reflected that training on MS was needed to integrate the messages into their practices. All participants suggested that supportive tools were required to help action the nutrition messages. 

CONCLUSION

Targeted nutrition messages for PwMS were interpreted in complex and diverse ways that reflect personal and social experiences, as well as their environment. The findings will help to think beyond the researcher experience and an offer an audiences’perspective for those who seek nutrition messaging for PwMS.

The relationship between the tryptophan-kynurenine pathway and cognitive fatigue and impairment in multiple sclerosis: An exploratory study. 

BACKGROUND

Cognitive fatigue and impairment are common symptoms of multiple sclerosis, which have broad implications for employment, everyday functioning, and quality of life. Recent, albeit limited, literature has implicated dysregulation of the kynurenine pathway of tryptophan metabolism in multiple sclerosis pathophysiology, including neuronal damage and death. 

OBJECTIVE

This study aimed to explore whether and how nicotinamide adenine dinucleotide and other metabolites along the kynurenine pathway may be associated with cognitive fatigue and impairment in people with multiple sclerosis, relative to healthy controls. 

METHOD

31 people with multiple sclerosis and 26 healthy control participants completed self-report questionnaires assessing cognitive deficits and fatigue. A comprehensive neuropsychological test battery was undertaken by each participant within 7 days of questionnaire completion. All participants provided a blood sample immediately following neuropsychological testing, for profiling of serum kynurenine metabolite concentrations. 

RESULTS

The results provide preliminary evidence that nicotinamide adenine dinucleotide and sixteen other kynurenine metabolites and/or metabolite ratios are differentially related to self-reported cognitive fatigue and several domains of cognitive functioning in people with multiple sclerosis, compared to healthy controls. 

CONCLUSION

The observed results are somewhat consistent with the role of nicotinamide adenine dinucleotide in cellular energy production and repair and the neurotoxic and neuroprotective capacity of several other kynurenine metabolites. Future studies which further elucidate these preliminary findings are required.

FUNDING

This research was generously supported by Incubator Grant funding from MS Research Australia (#15-214)

GluK4-containing kainite receptors impair action potential propagation, synaptic communication and motor function in mice.

Multiple Sclerosis (MS) is an autoimmune and neurodegenerative disease with no clear cause and no known cure.  It is estimated that, depending on the population studied, genetics account for 15–72% of MS risk.  Genome-wide association studies have identified >200 non-MHC loci that are each associated with a very small increase in MS risk (odds ratios ≤1.3) and account for <20% of MS heritability.  We instead study families with an unusually high incidence of MS in which MS-associated genetic signals are amplified.  In one specific family four of seven siblings developed MS and we recently identified a variant in the GRIK4 gene carried by the affected but not the unaffected family members and that is extremely rare in the population.  GRIK4 encodes the Gluk4 high-affinity subunit of ionotropic kainite (glutamate) receptors.  To determine how dysregulated kainite receptor signalling might contribute to MS pathophysiology, we examined the cognitive and motor function of Grik4 knockout (Grik4-/-) mice across adulthood.  Grik4-/- mice were hyperactive and displayed anxiolytic and anti-depressant behaviours, but also developed a progressive motor impairment relative to wildtype mice.  Neuron, oligodendrocyte progenitor cell and oligodendrocyte number were normal.  However, by P180 electron micrographs revealed that the size of the periaxonal space was increased and the callosal axons showed other signs of degeneration.  Whole cell patch clamp recordings from pyramidal neurons in the motor cortex of acute brain slices also revealed a ~50% reduction in the number of excitatory synaptic inputs received, which could readily account for the impaired motor function.  Additionally, the amplitude of the myelinated callosal compound action potential was reduced by ~30% in Grik4-/- mice relative to wildtype mice.  Additional pharmacological studies revealed that this was the result of impaired potassium channel function in Grik4-/- mice. These data suggest that Gluk4-containing kainite receptors are critical for action potential maintenance and synaptic health in the motor cortex indicating that dysfunctional glutamate signalling can directly impair axon health.

High and low efficacy therapy in secondary progressive multiple sclerosis.

BACKGROUND

In secondary progressive multiple sclerosis (SPMS), reduction in the rates of disability accrual after starting treatment has largely been limited to patients with ongoing inflammatory activity. A delayed treatment effect, termed therapeutic lag, may obscure therapeutic benefits in SPMS. 

OBECTIVES

To compare the effect of high- and low-efficacy treatments on disability outcomes in patients with recently active and inactive SPMS. 

METHOD

Patients treated with high (natalizumab, alemtuzumab, mitoxantrone, ocrelizumab, rituximab, fingolimod) or low-efficacy (interferon-β, glatiramer-acetate, teriflunomide) therapies after SPMS onset were selected from MSBase and OFSEP, two large observational cohorts. Personalised estimates of therapeutic lag were calculated. Only events that occurred after the estimated lag period were included. Propensity score matching was used to select groups with comparable baseline characteristics. Disability and relapse outcomes were compared in paired, pairwise-censored analyses. 

RESULTS

Of 7359 patients with SPMS, 1000 patients fulfilled the criteria for study inclusion. For the relapse outcomes, patients with active SPMS treated with high-efficacy therapy experienced lower probabilities of relapses than low-efficacy therapy (HR 0.7 [95%CI 0.5-0.9]).  Patients with inactive SPMS had similar probabilities of relapses in the high and low efficacy DMT groups (0.8 [0.6-1.2]). No difference in the risk of disability accumulation, or proportion of patients reaching EDSS>=7, was observed between groups. 

CONCLUSION

The risk of disability accumulation in SPMS seems to be comparable in patients treated with high- and low- efficacy therapy. High efficacy therapy is superior to low efficacy therapy in reducing relapse activity in patients with active SPMS, but not those with inactive SPMS.

Disease reactivation after cessation of disease-modifying therapy in relapsing-remitting multiple sclerosis. 

BACKGROUND

Multiple sclerosis treatment interruptions are common and leave patients vulnerable to breakthrough disease activity.  The safe duration of the ‘switchover time’ for different therapies is not known. 

OBJECTIVES

To evaluate the rate of return of disease activity after cessation of multiple sclerosis (MS) disease-modifying therapy.  

METHOD

This was a retrospective cohort study from two large observational MS registries: MSBase and OFSEP.  Patients with relapsing-remitting MS who ceased a disease-modifying therapy and were followed up for the subsequent 12-months were included.  The primary study outcome was annualised relapse rate in the 12 months after disease-modifying therapy discontinuation stratified by patients who did, and did not, commence a subsequent therapy.  Predictors of relapse and disability accumulation after treatment discontinuation were a secondary outcome. 

RESULTS

18 029 eligible treatment discontinuation epochs were identified.  Rates of relapse started to increase 2-months after natalizumab cessation.  Commencement of a subsequent therapy within 2-4 months reduced the magnitude of disease reactivation.  After discontinuation of fingolimod, rates of relapse increased overall, and stabilised faster in patients who started a new therapy within 1-2 months.  Predictors of relapse were higher relapse rate in the year before cessation, female sex, younger age and higher EDSS.  

CONCLUSION

Understanding the rate of disease reactivation after discontinuing different MS immunotherapies will help guide optimal wash-out times for therapeutic agents during treatment sequencing. 

Pro-inflammatory diet is associated with worse long-term depression and anxiety levels but not fatigue in MS.

BACKGROUND

MS is an inflammatory disease and diet is a determining factor of chronic inflammation. We examined whether an index of the overall dietary inflammatory potential, residual energy adjusted, Diet Inflammatory Index (E-DII), was associated with depression, anxiety, and fatigue using data from a prospective cohort.

OBJECTIVE

To examine the association between E-DII and depression, anxiety and fatigue in people with MS.

METHOD

AusLong Study participants were followed annually for 10 years (n=223)). Depression and anxiety (Hospital Anxiety & Depression Scale) and fatigue (Fatigue Severity Scale) were assessed at the 5 and 10 year reviews. Dietary intake (preceding 12 months) using the Food Frequency Questionnaire and was assessed at baseline,5 and 10 year reviews. Transformed linear mixed models  were used and we adjusted for age, sex and dietary misreporting to limit measurement error.

RESULTS

When E-DII and the outcomes were examined concurrently, we found no associations. However, when we assessed whether E-DII at baseline and the 5th year review was associated with outcomes five years later, we found that a higher E-DII score (pro-inflammatory) was associated with higher levels of depression and anxiety but not with fatigue (depression: highest vs lowest E-DII quartile, β=2.23, 95%CI=0.98,3.48; anxiety: highest vs lowest E-DII quartile, β=1.90,95%CI=0.59,3.21 ).

CONCLUSION

A more pro-inflammatory diet seemed to have a long-term detrimental effect on depression and anxiety, but not an immediate effect.

A collaborative approach to designing an online nutrition education program for people with multiple sclerosis.

BACKGROUND
People recently diagnosed with multiple sclerosis (MS) want MS-specific dietary advice to reduce the confusion around diet. To address their needs, we used co-design principles and collaborated with people with MS (pwMS) to develop an online nutrition education program.

Objective: To explore the acceptability and ease of comprehension of an online nutrition education program for pwMS.

METHOD
The six-week program was developed using findings from focus groups (six groups, total n=34) and a survey (n=137) of pwMS. The content was refined during two workshops with six pwMS. Cognitive interviews were conducted with eight pwMS (diagnosed within the previous five years). Participants used a ‘talk aloud’ technique and responded to interviewer probing while working through Module One. Audio-recordings were transcribed and analysed using content analysis.

RESULTS
Participants stated that, overall, the module was acceptable, and the content was easy to understand. Person-centred language and information about dietary changes that could potentially improve their symptoms, “things we can do”, were described as empowering and motivating. In contrast, scientific writing about evidence, too much text, and language that was not persuasive, were described as demotivating and disheartening, and the context of the content was misunderstood.

CONCLUSION
Positive language can increase the motivation to make desirable dietary changes and improve willingness to engage with nutrition resources. Nutritionists and other healthcare professionals could apply co-design principles when developing resources for pwMS, and use positive, empowering language.

FUNDING
This research is supported by an MS Australia Incubator Grant (#21-1-072). RDR is supported by an Australian Government Research Training Program Scholarship, a Multiple Sclerosis (MS) Western Australia (MSWA) PhD Top-Up Scholarship, and a Graduate Women (WA) Inc. Open Scholarship. LJB is supported by MS Western Australia (MSWA), an MS Australia Postdoctoral Fellowship, and a Curtin University Research Fellowship.

Predicting Infection Risk in Multiple Sclerosis Patients Treated with Ocrelizumab: A Retrospective Cohort Study. 

BACKGROUND

Ocrelizumab safety outcomes have been well evaluated in clinical trials. However, risk factors for infection in patients with multiple sclerosis (MS) receiving ocrelizumab have not been extensively studied in the real-world setting. 

OBJECTIVE

To examine factors determining risk of self-reported infections and antimicrobial use in patients receiving ocrelizumab for MS. 

METHODS

Retrospective, observational cohort study involving patients receiving ocrelizumab at the Royal Melbourne Hospital. The associations of survey-recorded infections and antimicrobial use with potential clinical and laboratory risk factors for infection were estimated using univariate and multivariable logistic regression models. 

RESULTS

A total of 185 patients were included in the study, and 176 infections were reported in 89 patients (46.1%), with antimicrobial use identified in 47 patients (25.3%). Univariate analyses identified higher serum IgA in associated with reduced odds of infection (OR 0.44, 95% CI 0.25 - 0.76). In multivariable analyses, older age (OR 0.94, 95% CI 0.88 - 0.99), higher serum IgA (OR 0.37, 95% CI 0.17 - 0.80) and higher serum IgG (OR 0.81, 95% CI 0.67 - 0.99) were associated with reduced odds of infection. Older age (OR 0.85, 95% CI 0.75 - 0.96) and higher serum IgA (OR 0.23, 95% CI 0.07 - 0.79) were associated with reduced odds of antimicrobial use, whilst longer MS disease duration (OR 1.22, 95% CI 1.06 - 1.41) and higher EDSS (OR 1.99, 95% CI 1.02 - 3.86) were associated with increased odds of antimicrobial use. 

CONCLUSIONS

Higher serum IgA and IgG and older age were associated with reduced odds of infection. Our findings highlight the non-uniformity of infection risk of patients with MS receiving ocrelizumab, substantiating the need to monitor immunoglobulin levels pre-treatment and whilst on therapy.

Robust label-free imaging to assess demyelination.

BACKGROUND

Demyelination is a distinctive feature and contributes to the dysfunction of numerous conditions affecting the central nervous system (CNS), including Multiple Sclerosis (MS). Therefore, establishing non-destructive and label-free methods to detect demyelination which can be implemented with other techniques, would greatly aid research in this area.   

OBJECTIVE

To establish easily implemented and robust label-free imaging modalities to assess demyelination in a mouse model of MS, neurodegeneration, and human neuropathology. 

METHOD

We chose polarization and confocal reflectance microscopy as two label-free imaging methods to assess demyelination due to their ease of incorporation into existing microscopy pipelines. In order to achieve the objective, this study assessed central nervous system (CNS) samples from a cuprizone (CPZ) mouse model of demyelination, a transgenic mouse model of neurodegeneration (5XFAD), and human post-mortem cases. Samples were imaged with label-free methods combined with established methods such as immunohistochemistry and histological myelin staining methods.  

RESULTS

Polarization microscopy detected demyelination in the white matter tracts in a CPZ mouse model, which correlated with glial activation. It is a sensitive technique that relies on the intact molecular orientation of myelin to generate a signal. Additionally, quantitative imaging is achieved by comparisons with established reference standards. Confocal reflectance microscopy is also a sensitive 3-D imaging technique that relies upon the refractive index difference between lipid-rich myelin and surrounding CNS structures and can detect defective myelination.  

CONCLUSION

Polarization and confocal reflectance microscopy are powerful label-free imaging methods for assessing the molecular orientation of specific structures in the CNS, such as myelin. Myelination is easily assessed and compared to established reference standards to create quantitative imaging and is compatible with techniques including histochemistry.   

FUNDING

MS research at WSU is funded by the Rotary Club of Narellan.

Research at the Centre for Motor Neuron Disease Research, Macquarie University, was funded by the Motor Neuron Disease Institute of Australia and FightMND.

Histological and proteomic investigations of the visual pathway using the cuprizone demyelination model. 

BACKGROUND

A change in visual perception is frequently used as an early indicator of Multiple Sclerosis (MS), the pathoetiology of which remains unclear. 

OBJECTIVE

To investigate histological and proteomics changes in the visual pathway following 12 weeks of feeding 0.1% cuprizone to young adult mice, as a demyelinating model of MS. 

METHOD

Histological analyses of demyelination and gliosis in the optic tracts, pretectal nuclei, superior colliculi, lateral geniculate nuclei and visual cortices were performed. Top-down proteomic assessment followed by liquid chromatography-tandem mass spectrometry (LC-TMS) analysis was used to identify the proteoforms from optic nerve/tract. Literature mining and bioinformatic analyses were used to investigate the relevance of identified proteoforms with previously published literature and molecular functions, respectively.

RESULTS

Histological investigation did not reveal any significant demyelination or gliosis in the visual pathway at this time point. However, proteomic assessment revealed a significant change in the abundance of 34 spots and LC-TMS analysis identified 75 proteoforms. At least 24 proteoforms not previously described in any animal models of MS or MS itself. Bioinformatic analysis indicated involvement of these 75 proteoforms in cytoskeleton organization, metabolic dysregulation, protein aggregation, and axonal support. 

CONCLUSION

These results indicate that continuous cuprizone-ingestion, which evokes a slow, progressive demyelination, results in proteomic changes that preceded any clear histological changes in the visual pathway and that some of these proteoforms may be potential early markers of degenerative demyelinating conditions. 

FUNDING

This study was funded by the Rotary Club of Narellan.

Impact of high fibre diet and its combination with intermittent fasting in CNS autoimmunity. 

BACKGROUND
Dietary fibre is known to benefit health through promoting healthy gut microbiota and boosting the production of bacteria-derived metabolites, particularly short chain fatty acids. High fibre intake confers protection against various preclinical models of immune-mediated disorders including allergy, lupus and colitis, but its impact in multiple sclerosis (MS) remains elusive. Intermittent fasting (IF) attenuates disease in animal models of MS, including experimental autoimmune encephalomyelitis (EAE). Thus, the effects of high-fibre (HF) diet and its combination with IF in MS is of great therapeutic interest.

METHOD
C57Bl/6 mice were fed on either a HF or zero-fibre (ZF) content diet and were either given ad libitum (AL) food access or subjected to 24-hour fasting-feeding cycles. Immune profiles were analysed by flow cytometry – (1) before EAE induction after 5 weeks of feeding and (2) on day 40 post EAE induction. Mice were culled both after feeding and fasting to assess the temporal effects of IF. 

RESULTS
Splenic Th17 cells and blood Ly6Chi monocytes were increased in the HFAL group compared to the ZFAL group under basal conditions before EAE induction. Acute fasting decreased Th17 cells in HFIF mice and decreased Ly6Chi monocytes in both HFIF and ZFIF groups compared to corresponding AL groups, whilst refeeding restored Th17 and monocyte levels to that of AL groups. Consistent with these results, the HFAL group had the highest EAE incidence, whilst IF reduced disease incidence, delayed disease onset and reduced clinical severity compared to AL feeding of the same diet, with the HFIF intervention being the most protective against EAE. In accordance, ex vivo antigen-specific Th17 response was greatest in the HFAL group and was greatly dampened in the HFIF group.

CONCLUSION
Altogether, HF feeding was not protective against EAE development, whilst IF in combination with HF feeding rescued clinical disease parameters, indicating complex interactions between the two interventions.  

Natalizumab versus fingolimod in patients with relapsing-remitting multiple sclerosis: a subgroup analysis from three international cohorts. 

BACKGROUND

Natalizumab has proved to be more effective than fingolimod in reducing disease activity in relapsing-remitting multiple sclerosis (RRMS). Whether this association is universal for all patient groups remains to be determined. 

OBJECTIVE

To compare the relative treatment effects of natalizumab and fingolimod in subgroups of patients with RRMS from three international registries. 

METHOD

RRMS patients from the MSBase, French and Danish Multiple Sclerosis Registries who were given natalizumab or fingolimod were stratified based on sex, age, disease duration, expanded disability status scale score, presence of relapses or disability worsening, and MRI activity 12 months prior to treatment initiation. The study outcomes were number of relapses and cumulative hazards of first relapse, 6-month confirmed disability worsening and improvement events, recorded during study therapy. 

RESULTS

Natalizumab was superior to fingolimod in reducing relapse risk mainly among females, patients of younger age, shorter disease duration, and with pre-treatment relapses. Disability improvement was more commonly associated with natalizumab than fingolimod among females, patients of age˃38 years, patients with longer disease duration, and without brain or spinal cord MRI activity preceding baseline. The effectiveness of natalizumab and fingolimod on disability worsening was, however, comparable across the studied subgroups. 

CONCLUSION

Demographic and clinical characteristics should be considered when choosing between natalizumab and fingolimod for RRMS patients.

Confirmed disability progression as a marker of permanent disability in multiple sclerosis.

BACKGROUND

Prevention of disability over the long-term is the main treatment goal in multiple sclerosis (MS), however, randomized clinical trials provide only short-term (3-6-month) treatment effect on disability. 

OBJECTIVE

This study aimed to define criteria for 6-month confirmed disability progression events of MS with a high probability of resulting in sustained long-term disability worsening. 

METHOD

Prospectively collected demographic and clinical data of 8,741 patients (14,802 6-month confirmed disability progression events) from the MSBase registry were used in the analyses. For each 6-month confirmed progression event, a sustained progression score was calculated based on the demographic and clinical characteristics at the time of progression that were predictive of long-term disability worsening. The score was externally validated in the CLARITY (Cladribine Tablets Treating Multiple Sclerosis Orally) trial. 

RESULTS

The score (discriminatory index:0.89) was based on age, sex, MS phenotype, disability score and its change from baseline, recent relapse activity, and affected functional system domains. The proportions of progression events sustained at 5 years stratified by the score were 1:72%, 2:88%, 3:94%, 4:100% with a positive predictive value of 0.86. Results of the CLARITY trial were confirmed for reduction of disability progression that was >88% likely to be sustained. 

CONCLUSION

Clinical characteristics of 6-month confirmed disability progression events identify those at high risk of sustained long-term disability. This knowledge will allow future trials to better assess the effect of therapy on long-term disability accrual.

Prospective associations of better diet quality with improved quality of life over 7.5 years in people with multiple sclerosis. 

BACKGROUND

There is increasing interest in diet’s role in MS progression and whether a healthier diet may lead to improved health and wellbeing in MS patients. 

OBJECTIVE

To assess the prospective relationship of diet quality with quality of life (QoL) in an international cohort of people with MS followed over 7.5 years. 

METHOD

Data from the Health Outcomes and Lifestyle In a Sample of people with Multiple sclerosis (HOLISM) cohort from baseline to 7.5-year review were utilised. Diet quality was measured by Diet Habits Questionnaire (DHQ). QoL was measured by the MSQOL-54, including physical and mental health composite scores and various subdomains. Linear regression was used to assess prospective relationships with annualised change in QoL, adjusted for clinical and demographic covariates. 

RESULTS

Amongst 948 participants, mean baseline physical- and mental-health QoL scores were 64.4(SD=20.4) and 71.3(SD=19.8). Baseline total DHQ showed positive associations with subsequent change in physical-QoL, such that those in the top two quartiles of baseline DHQ had 0.65 & 0.64 higher physical-QoL per year. Baseline meat consumption was associated with 0.38 lower physical-QoL, while dairy consumption was associated with 0.50 and 0.41 lower physical- and mental-QoL per year, respectively. 

CONCLUSION

These results suggest that efforts to improve diet quality could be beneficial to wellbeing amongst people with MS. Subject to replication, this aspect of lifestyle could be an adjunct to existing care.

Higher quality diet and non-consumption of meat are associated with less disability progression in people with multiple sclerosis: a longitudinal cohort study. 

BACKGROUND

Modifiable lifestyle factors, including diet, have been shown to affect clinical outcomes in multiple sclerosis (MS). 

OBJECTIVE

This study assessed the prospective relationships between diet, and disability, fatigue, and depression-risk in people with MS. 

METHOD

Participants from the HOLISM international cohort were assessed over 2.5-years’ follow-up. Diet was assessed using the modified Diet Habits Questionnaire (DHQ). Disability was assessed by the Patient-determined MS Severity Score, fatigue by the Fatigue Severity Scale, and depression-risk by the Patient Health Questionnaire-2. Cross-sectional and prospective relationships of diet and disease outcomes were assessed, adjusted for relevant confounders. 

RESULTS

Among 1,346 participants, higher DHQ scores showed significant dose-dependent associations with lower frequencies of severe disability, fatigue, and depression-risk, cross-sectionally. Prospectively, higher baseline DHQ scores was associated with 0.30 EDSS points less disability progression, but was not associated with fatigue or depression-risk. Baseline meat consumption was associated with 76% higher risk of increasing disability and 0.18 EDSS points higher disability progression, but dairy consumption showed no consistent associations. 

CONCLUSION

We show that a better quality-of-diet, as well as not consuming meat, were associated with reduced progression of disability in people with MS. Substantiation of these findings in other settings may inform opportunities to slow disability progression in people with MS.

Prospective associations of better diet quality with health state utilities over 5 years in people with multiple sclerosis.

OBJECTIVE
To investigate the prospective associations between diet quality and Health State utility values in an international cohort of people with MS followed over 5 years.  

METHODS
Cohort data was extracted from the Health Outcomes and Lifestyle In a Sample of people with Multiple sclerosis (HOLISM) study. Participant information from baseline to 5-year review was included in analysis. HSU values were estimated by the SF-6D. Diet quality was measured by Diet-Habits Questionnaire (DHQ). Cross-sectional and prospective associations between HSU and diet quality were evaluated via linear regression, adjusted for relevant demographic/clinical covariates.  

RESULTS
Among a sample of 839 participants, better DHQ score showed a dose-dependent positive relationship with HSU at 5-year-review. Participants in the top two quartiles of DHQ had 2.8% (96%CI: 1.1%,4.6%) and 4.5% (95%CI: 1.3%,7.8%) higher HSU. Participants reporting dairy-and-meat consumption had 2.5% (95%CI: 1.1,3.9) and 2.0% (95%CI: 0.1,3.4) lower HSU. Prospective relationships between baseline diet quality, as well as DHQ subdomains, with subsequent change in HSU will also be described.       

CONCLUSIONS
Results suggest higher quality diet, low in meat and dairy has the potential to improve quality of life in pwMS. Should these findings be replicated in other cohorts and substantiated by clinical trial, diet could be a valuable adjunct to care in people with MS.

The UPR preserves mature oligodendrocyte viability and function in adults by regulating autophagy of PLP. 

BACKGROUND

Oligodendrocytes, the myelinating cells of the central nervous system, are highly sensitive to perturbations in endoplasmic reticulum (ER) homeostasis. The unfolded protein response (UPR) aids cell survival under conditions of ER stress. The UPR is important in the pathogenesis of demyelinating disorders including multiple sclerosis. 

OBJECTIVE

The UPR is vital for maintaining cellular proteostasis. The aim of this project was to determine the function of two of the UPR pathways, PERK and ATF6, in oligodendrocytes in physiological conditions to assess the importance of these pathways during developmental myelination and myelin maintenance. 

METHOD

Global ATF6 knock out (KO) mice were crossed with oligodendrocyte specific PERK KO mice to delete both ATF6 and PERK in oligodendrocytes. Motor phenotype was assessed using the rotor rod. Oligodendrocyte number and myelination was assessed by immunohistochemistry (IHC) and electron microscopy at postnatal day (P)21 and 45. IHC was used to assess protein accumulation in oligodendrocytes. RNA-Seq and RT-qPCR were used to investigate perturbations in mRNA expression. 

RESULTS

Interestingly, deletion of PERK and ATF6 in oligodendrocytes did not impair developmental myelination, however, after P21 mice developed a progressive tremoring phenotype that coincided with oligodendrocyte and myelin loss which was extensive by P45. IHC showed accumulation of PLP, p62, and ubiquitin in oligodendrocyte cell bodies, suggesting perturbations in protein degradation pathways. RNA-Seq and RT-qPCR revealed that this was associated with loss of cathepsin D transport proteins. KO of PLP partially rescued the tremoring phenotype. 

CONCLUSION

Surprisingly, PERK and ATF6 are not essential for developmental myelination, however, these pathways are involved in myelin maintenance. These results indicate that loss of the PERK and ATF6 pathways leads to toxic accumulation of PLP in adult oligodendrocytes due to impaired autophagy pathways. 

FUNDING

This study was supported by grants from the NIH (NS094151 and NS105689) to WL.

Gray matter atrophy and axonal damage is more pronounced in males compared to females in mice with experimental autoimmune encephalomyelitis

BACKGROUND
Gray matter atrophy is a hallmark of multiple sclerosis (MS) and correlates with degree of disability. Women are more susceptible to MS due to increased inflammation, men display more severe disease progression and neurodegeneration, suggesting a differential effect of sex on inflammation and neurodegeneration.

OBJECTIVE
The objective of this study was to compare the extent of gray matter atrophy and neurodegeneration in male versus female mice, subject to experimental autoimmune encephalomyelitis (EAE), to elucidate the underlying mechanisms of this sexual dimorphism in MS.

METHOD
Thy1-YFP-H C57BL/6J mice were subjected to the chronic EAE model of MS. Clinical scoring was performed to assess degree of disability. Magnetic resonance imaging was performed, and voxel-based morphometry was used to compare sex differences in gray matter atrophy. Following image acquisition, mice were sacrificed and Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) and immunohistochemistry were used to identify sex differences in neurodegeneration, immune cell infiltration and demyelination.

RESULTS
We reveal gray matter atrophy was worse in males compared to female mice during EAE. Of note, more severe gray matter atrophy in the somatosensory cortex was associated with worse disability in EAE males but not females. This correlated with a greater degree of neurodegeneration in the cerebral cortex of male mice compared to females. Surprisingly, male and female mice displayed similar immune cell infiltration and demyelination during EAE.  

CONCLUSION
These findings demonstrate an increase in gray matter atrophy, neurodegeneration and disability in male mice compared to females subjected to EAE. This supports evidence suggesting male sex is a risk factor for more severe disease progression and neuronal loss in MS.

The kynurenine pathway affects social cognition in people with multiple sclerosis and healthy individuals: implications for neuroprotective and neurotoxic biomarkers.

BACKGROUND

There is a growing understanding that people with multiple sclerosis (pwMS) experience difficulties in social cognitive functioning. MS is also associated with increased dysfunction in the brain’s kynurenine pathway (KP). However, whether there exists a relationship between social-cognitive functioning and the KP, is not known.

OBJECTIVE

In this original study, we examined the relationship between social cognitive functioning and metabolites ratios of the KP of tryptophan metabolism in pwMS and matched healthy individuals. 

METHOD

Participants comprised 31 pwMS (28 relapsing-remitting, 1 secondary-progressive, 2 primary-progressive) and 26 healthy control individuals without MS, demographically matched for sex, age, and education. Social cognitive functioning was objectively assessed using The Awareness of Social Inference Test – Short (TASIT-S) and subjectively assessed using self-reports of abilities in emotion recognition, emotional empathy, and perspective taking (otherwise known as cognitive theory of mind).  

RESULTS

In MS, higher serum concentrations of AA and NAD+ were associated with better social-cognitive ability (i.e., self-reported emotion recognition and TASIT-S sarcasm detection performance). Further, both higher serum concentrations of 3-HK and the NAD+/QA ratio were related to poorer social-cognitive functioning. Collectively, the upstream (i.e., TRP-KYN) and midstream pathways (i.e., KYN-3-HAA) were predominately associated with poorer social-cognitive functioning. However, for the downstream pathway (i.e., QA-NAD+), the findings were mixed. 

CONCLUSION

Overall, our results indicate a relationship between the KP and social-cognitive abilities. Future research may expand the current findings by examining the combined role of the KP and other processes involved in KP regulation (e.g., biologically-derived inflammatory dysregulation) in social cognitive functioning.

FUNDING

The authors acknowledge the importance of funding from a Multiple Sclerosis Research Australia (MSRA) Incubator Grant (2016-17) awarded to Cynthia A. Honan, Chai K. Lim, and Skye McDonald.

MS Specialist Nurse Care: Patterns of access and impact on health outcomes.

BACKGROUND

MS specialist nurses are an integral part of the multidisciplinary health care team of people with MS. However, changes in traditional funding models have led to a reduction in the number of MS nurses, and concern about inequity of access.

OBJECTIVE

To describe the patterns of access to MS nurses, to examine whether people with MS without access had worse disease outcomes, and to describe the overall value of MS nurses and the perceived benefits of MS nurse care.

METHOD

1,417 participants of the Australian MS Longitudinal Study completed the 2020 MS Nurses Survey and 2019 Disease Course Survey. Participants were asked about MS nurse care access, utilisation (frequency, duration, satisfaction, types of support, consultation type), perceived effects and overall value. Regression models were used to assess associations with the severity of 13 MS symptoms, disability, self-reported progression in the past 12 months, and health-related quality of life. 

RESULTS

We found that 33% of people with MS did not have access to MS nurse care, and that people without access had consistently worse health outcomes compared to those with access, even after adjustment for education, age, MS duration, onset type and remoteness. MS nurses reduced the need for other health professionals and prevented hospital admissions. There were high levels of satisfaction with the frequency and types of services provided.

CONCLUSION

MS nurses seem to offer a service that benefits people with MS, and people who are not able to access this service were adversely affected in terms of health outcomes. It calls for policies and practices to improve the access to MS nurse services.

FUNDING

The Australian MS Longitudinal Study is supported by MS Research Australia.
The AMSLS MS Nurses Study was supported by funding from MS Australia.

A systematic review of the effects of lifestyle self-management regimes that improve the life quality of persons living with multiple sclerosis.

BACKGROUND
The evidence exploring the benefits of self-management for people living with multiple sclerosis (MS) is inconsistent. Previous reviews have limited their reporting outcomes highlighting a need to systematically identify elements of self-management regimens that influence the health of people with MS, using a common outcome measure.

OBJECTIVE
To assess the effectiveness of lifestyle self-management strategies and interventions aimed at improving the quality of life (QOL) of people living with MS.

METHOD
A systematic literature search was conducted using Cochrane Library, CINHAIL, MEDLINE, PubMed and Scopus databases. A three-phased screening process was undertaken, according to the 2020 PRISMA statement. Data was qualitatively synthesised under six self-management dimensions: physical activity, fatigue, dietary, stress, coping and emotional; symptom and medical management; and lifestyle and wellbeing programs. The risk of bias of each study was appraised. PROSPERO (Ref: CRD42021235982).

RESULT
A total of 57 research studies (n=40 experimental, n=17 observational) of 5,830 individuals diagnosed with MS were included in this review. Studies that explored lifestyle and wellbeing programs (n=8); dietary interventions (n=2) and coping, depression, stress and/or emotional management (n=10) consistently reported significant improvements in QOL subscales including mental, physical, pain, energy, social and/or fatigue. The remaining dimensions inconsistently improved QOL.

CONCLUSION
Given the complexity of self-management, heterogeneity continues to limit a meta-analysis of the studies from being conducted. As the evidence continues to grow, a future quantitative analysis is warranted

Investigating the shared genetic architecture between multiple sclerosis and inflammatory bowel diseases. 

BACKGROUND

An epidemiological association between multiple sclerosis (MS) and inflammatory bowel disease (IBD) is well-established. 

OBJECTIVE

To determine if the epidemiological association between MS and IBD reflects a shared genetic aetiology and if consistent genetic relationships exist between MS and the two predominant subtypes of IBD, ulcerative colitis (UC) and Crohn’s disease (CD). 

METHOD

We used genome-wide association study (GWAS) summary data to estimate genetic correlations between MS and IBD overall and UC and CD independently. We then used multiple Mendelian randomization methods to investigate causal relationships between these diseases. We also investigated tissue- and cell-type-specific heritability enrichment for each disease using stratified linkage disequilibrium score regression, and identified putative functional genes shared by MS and IBD using Summary-data-based Mendelian Randomisation.

RESULTS

We identified a significantly higher genetic correlation between MS and UC than that between MS and CD. Mendelian randomization methods showed suggestive evidence for a causal effect of MS on UC and IBD, but not for causality in bidirectional analyses of MS and CD. At the tissue level, we observed consistent patterns of enrichment for MS and IBDs in lung, spleen, whole blood, and small intestine. At the cell-type level, we identified significant enrichment for MS and IBDs in CD4+ T cells in lung and CD8+ cytotoxic T cells in both lung and spleen. Finally, we identified three putative functional genes (GPR25, AL031282.2 and AL109917.1) shared by MS and IBD.

CONCLUSION

Our findings progress the understanding of shared genetic mechanisms underlying MS and IBD, and potentially provide points of intervention that may allow the development of new therapies for these common immune disorders.

Neuronal TrkB mediates differentiation and survival of oligodendroglial lineage cells in a spatiotemporal manner during remyelination. 

BACKGROUND

Neuronal activation is crucial for central nervous system(CNS) myelination. Yet, whether neuronal signal also influences remyelination remains unclear. BDNF (Brain-derived neurotrophic factor) acting through oligodendroglial TrkB (tyrosine receptor kinase B) has a promyelinating effect. However, the contribution of neuronal TrkB signaling in remyelination remains unknown. 

OBJECTIVES

The aim of this study was to investigate whether BDNF signaling through neuronally expressed TrkB influences myelin repair following cuprizone-mediated CNS demyelination. 

METHOD

Inducible, neuron-specific TrkB conditional knockout mice (TrkB icKO) and control mice were subjected to a cuprizone diet. Thymidine analogue EdU (5-Ethynyl-2’-deoxyuridine) was given to cumulatively label cells that are newly generated during de- and remyelination. Immunohistochemistry was used to track the proliferation and incorporation of the newly generated cells. Spectral confocal reflectance microscopy and electron microscopy were used to assess myelin. 

RESULTS

In the corpus callosum, TrkBicKO showed a significant increase in newly generated oligodendrocyte precursor cells(OPCs) and a trend reduction in new mature oligodendrocytes(OLs) following 4-week demyelination and 1-week recovery. In the somatosensory cortex, TrkB deletion led to a significant reduction of mature OLs following 3-week remyelination, attributable to a decrease in pre-existing mature OLs. However, analysis of myelin did not reveal significant changes in the extent of remyelination in TrkBicKO.

CONCLUSION

During remyelination, neuronal TrkB promotes differentiation of new OPCs into mature OLs in the corpus callosum, while it makes pre-existing mature OLs less susceptible to death in the somatosensory cortex. This study highlights the role of neuronal signal in the regulation of oligodendroglial lineage cells during remyelination.

Immune cell transcriptomics reveals different pathways regulated by vitamin D in multiple sclerosis.

BACKGROUND

Vitamin D deficiency is a risk factor for MS. Predominantly in vitro studies have demonstrated immunoregulatory effects of vitamin D, but it is not known how vitamin D regulates immune cells in an in vivo setting.

OBJECTIVE

Using transcriptomic datasets from immune cell subsets, we aimed to identify pathways regulated by vitamin D in vivo, and whether there are differences in pathway regulation between people with MS and healthy controls.

METHOD

Microarray transcriptomic datasets available for at least one immune subset (monocyte, B, CD4, CD8, NK cell) were available for 73 MS cases and 102 healthy controls. An independent cohort of 35 MS cases and 33 healthy controls also had RNAseq datasets (monocyte, B cell). Correlation of gene expression with serum 25(OH)D level (LIAISON assay) was determined using Limma or edgeR. Gene set enrichment analysis was performed using ClusterProfiler and MSigDB.

RESULTS

Vitamin D-related pathways across multiple cell types included RNA processing and splicing, mitochondrial function and oxidative phosphorylation, and immune signalling (FDR<0.05). In monocytes, the GO term “vitamin D metabolic process” was enriched by genes downregulated with increasing 25(OH)D level in MS cases. “TNF-alpha signalling via NFKB” was enriched by upregulated genes. Whilst in CD4 cells, downregulated genes led to enrichment of interleukin and TNF-alpha signalling gene sets.

CONCLUSION

We identified signals for vitamin D regulation of immune function in vivo. Inferred differences in pathway regulation by vitamin D in MS cases and controls suggest differences in response to endogenous vitamin D.

Continued adherence to specific diets is associated with lower depression and fatigue in people with multiple sclerosis. 

BACKGROUND
Increasingly, healthy diets are being shown to have a positive role in symptom management in people with multiple sclerosis (pwMS). However, evidence supporting the use of a specific MS-diet is limited, especially, whether continued adherence is necessary in maintaining improved health outcomes, is unknown. 

OBJECTIVE
To assess whether long-term persistency in diet adherence is better than non-persistent adherence (ceased, commenced, and no diet) on fatigue, depression, and disability. 

METHOD
Data from a longitudinal study of 671 pwMS were analysed. Adherence to six MS- diets (Ashton Embry Best Bet, McDougall, Overcoming MS (OMS), Paleolithic (Paleo), Swank, and Wahls Elimination) were queried at 5- and 7.5-year follow-up. Persistence of adherence was defined as continued (both timepoints), ceased (5-year only), commenced (7.5-year only) and non-adherent (neither timepoint). Associations between adherence and fatigue, depression, and disability, was assessed by adjusted log-binomial regression models. 

RESULTS
Forty-two percent of pwMS self-reported ≥12-month continued adherence to any MS-diet, 33% to OMS, 4% to Swank, and < 1.5% to other queried MS-specific diets. Continued adherence to MS-diet was associated with lower depression than other groups, and lower fatigue than partial adherents; whereas continued adherence to OMS-diet was associated with lower fatigue and depression than ceased adherents.  No associations were observed for Swank or other diets. 

CONCLUSION
Current result supports continued adherence to MS- and OMS diet for management of fatigue and depression. More support for pwMS to maintain diet modification in routine care may be beneficial. Future prospective studies with larger populations adhering to specific MS-diets are needed to validate findings.  

FUNDING
Philanthropic gifts to the Neuroepidemiology Unit from Mr Wal Pisciotta and anonymous donors.

The design, development and feasibility on an online lifestyle program for people with MS. 

BACKGROUND
Digital health interventions (DHI) have arisen as an effective means to address complex needs of people with MS (PwMS) and as an aid to manage the disease.  As PwMS are increasingly seeking adjuvant treatment options such as lifestyle management, the potential for DHIs to revolutionise this area is great.

OBJECTIVE

1. To design and develop online lifestyle program for PwMS based upon best-practice in the field. 

2. Test the feasibility of both the data collection and intervention in an international sample of PwMS.

METHOD
The design and development of this lifestyle program was undertaken in collaboration between researchers, members from the MS community and industry. It followed a development plan modelled on the CeHReS roadmap and involved the digital transformation of an existing face-to-face course. This program was then tested for feasibility in an international sample of PwMS. 

RESULTS
This program was developed over two years and adhered to the methodology according to the CeHReS roadmap. Content was developed and implemented by the research team while the development team were responsible for building the platform to host the content. The feasibility study was undertaken over 6 weeks with modules being released twice per week. A total of 34 participants (17 intervention vs 17 standard care intervention) participated from 5 different countries. The study period ends June 7th where feasibility will be analysed. 

CONCLUSION
DHIs may play an important role in self-management for PwMS. This study described a thorough design and development process to ensure the fidelity of the intervention. Further, a feasibility assessment is underway to determine the number of completers of both the assessment protocol and the intervention itself.

Time to relapse after switching to cladribine is predicted by relapse during the washout period.

BACKGROUND

Patients with relapsing-remitting multiple sclerosis (RRMS) commonly switch between disease-modifying therapies (DMTs). Identifying predictors of relapse outcomes when switching to newer therapies such as cladribine could improve patient outcomes.

OBJECTIVE

To characterise relapse activity experienced by patients who switch to cladribine from other DMTs, and in particular to determine predictors of relapse occurrence on cladribine.

METHOD

Data of patients who switched to cladribine from a prior DMT group (pDMT; interferon-β/glatiramer acetate, dimethyl fumarate, teriflunomide, fingolimod or natalizumab) were extracted from the MSBase Registry. Relapse outcomes were compared between pDMT groups and washout duration groups (<1 month, 1-2 months, or 2-6 months). Time to first relapse in the first year of cladribine therapy was analysed using survival curves and Cox proportional hazard regression models.

RESULTS

Of 333 patients, 5.1% relapsed during washout and 7.2% relapsed on cladribine within one year. In the adjusted survival model, relapse on cladribine was predicted by washout relapse (hazard ratio [HR]=7.18, 95% confidence interval [CI] = 1.48-34.88, p=0.015) and younger age (HR=0.96, 95% CI 0.93-0.99, p=0.038). Washout periods under 2m reduced washout relapse risk, but not relapse risk on cladribine. There were no differences in relapse outcomes between pDMTs.

CONCLUSION

Washout relapse and younger age are of prognostic relevance when switching to cladribine. Washout durations shorter than two months were associated with fewer washout relapses, although prospective studies are required to assess if washout period reduction can modify relapse outcomes when switching to cladribine.

Disability and pregnancy in multiple sclerosis: relationships with leukocyte telomere length.

BACKGROUND
Ageing-related processes contribute to neurodegeneration and disability in MS. Biomarkers of senescence such as leukocyte telomere length (LTL) could help personalise prognosis. A history of pregnancy is protective against disability accumulation in women with MS: it is unclear if this could be partly mediated by LTL.

OBJECTIVE
We aimed to cross-sectionally characterise LTL in an MS cohort, and to correlate LTL with disability and pregnancy history.

METHOD
We extracted DNA from the whole blood of 301 patients attending an MS clinic (Alfred Health, Melbourne). Expanded Disability Status Scale (EDSS) score and pregnancy history (for 191 females) were obtained at sample collection. Additional data were extracted from the MSBase Registry. LTL was determined in base pairs (bp) using real-time polymerase chain reaction.

RESULTS
A relationship between EDSS score and shorter LTL was robust to multivariable adjustment for demographic and clinical factors including chronological age (adjusted LTL reduction per 1.0 increase in EDSS = 92.8bp, 95% CI = 5.4-180.2bp). In females with pregnancy data, there were no significant relationships between adjusted LTL and history of pregnancy (LTL increase of 41.8bp, 95% CI = -457.7–541.3bp) or number of pregnancies (LTL increase of 10.5bp per pregnancy, 95% CI = -175.8–196.8bp).

CONCLUSION
LTL was independently associated with cross-sectional disability, which likely reflects the relationship between neurological reserve and biological ageing in MS. Although adjusted LTL did not significantly differ by pregnancy history, longitudinal analyses to assess relationships with disability progression are needed.

Effect of priming on the cargo and function of human amniotic epithelial cell (hAEC)-derived Extracellular Vesicles (EVs)

BACKGROUND

Human amniotic epithelial cells (hAECs) exhibit immunomodulatory and neuroprotective properties in experimental models of stroke, brain injury and multiple sclerosis. Extracellular vesicles (EVs) released from hAECs show the same therapeutic effects as their parent cells, thus providing a safer cell-free based treatment approach. 

OBJECTIVES

To investigate whether hAEC-derived EVs can prevent demyelination and/or enhance remyelination and determine whether priming hAECs with inflammatory cytokines or hypoxia can regulate EV cargo to enhance their therapeutic, using ex vivo organotypic cerebellar slice cultures and the Cuprizone-induced model of demyelination. 

METHODS

EVs were isolated from hAECs cultured under standard or priming conditions (Hypoxia or Inflammatory stimuli), characterised and subjected to proteomics analysis to identify their cargo. Their ability to promote myelin regeneration was examined in brain slices after lysolecithin-induced demyelination. To assess their effect in vivo, C57BL/6 mice were fed a diet of 0.2% cuprizone for 5 weeks to induce demyelination and EVs or vehicle were administered intranasally once per week. 

RESULTS

Proteomic analysis revealed an increase in the protein cargo of EVs primed with inflammatory cytokines and a decrease in the protein cargo of hypoxia primed EVs compared to unprimed EVs. In brain slices, hypoxia conditioned EVs were less effective at promoting remyelination compared to other conditions. Intranasal administration of unprimed hAEC-EVs prevented weight loss associated with cuprizone feeding, and significantly reduced demyelination in the corpus callosum compared with the vehicle-treated group. 

CONCLUSION

These findings suggest that EVs have potential as a regenerative treatment for MS. Future work will focus on combining RNA-seq and proteomic analysis of EV cargo with characterisation of glial cell responses after EV treatment to identify potential mechanisms of action.

Assessment of the breath of spike antibody mediated protection after CVID-19 vaccinations in patients with MS.

BACKGROUND
Current SARS-CoV-2 vaccines rely on protective immunity against early clade SARS-CoV-2 and have resulted in seroconversion in a subset of MS patients receiving immunosuppressive DMTs. However, it is unknown if SARS-CoV-2 spike antibodies afford immunity against emerging variants of concern, such as Delta and Omicron.   

OBJECTIVE
To determine the binding of spike antibodies to early clade, Delta, and Omicron SARS-CoV-2 in patients with MS receiving different DMTs. 

METHOD
Spike antibody binding to early clade SARS-CoV-2, Delta and Omicron was assessed by flow cytometry using sera collected from 58 patients one month post second dose and one patient 1 month post third dose. All patients were previously determined to be seropositive against Wuhan (early clade spike). Clinical and demographic information including DMT treatment and vaccination timing was collected.

RESULTS
53 patients were seropositive for Wuhan, Delta and Omicron spike antibodies. Wuhan Spike antibody titres were high at one month post second vaccination, whereas Delta and Omicron Spike antibody titres were significantly decreased. We observed a 70% and 93% decrease of in immunoreactivity to Delta and Omicron, respectively. Although ocrelizumab and fingolimod decrease Spike antibody titres after vaccination, they did not affect immunoreactivity to variants, in comparison to other DMTS.

CONCLUSION
All 53 patients were able to generate a positive antibody response following vaccination. However, there was an observable decrease in the antibody titres and immunoreactivity to the Delta and Omicron variants, in comparison to Wuhan.

Transcriptomic analyses of normal-appearing CNS white matter from multiple sclerosis donors reveal subtype-specific molecular signatures of disease.  

BACKGROUND
Multiple sclerosis (MS) is a disabling disease of the CNS, whose pattern of progression distinguishes three main subtypes: relapse-remitting, primary- and secondary-progressive forms. Normal-appearing white matter (NAWM) shows pathological changes during MS but has never been employed to assess for transcriptomic differences across MS subtypes.

OBJECTIVE
This study aimed at analysing the transcriptome of NAWM across different MS subtypes, with the goal of identifying distinctive molecular signatures of disease subtype. Additionally, bioinformatics approach was employed to interrogate which MS subtype-specific biological pathways are altered.

METHOD
NAWM tissues from relapsing-remitting (3), primary (4) and secondary progressive MS cases (4) and non-MS aged-matched donors (7) were obtained from the NSW MS and Victorian brain banks, respectively. Total RNA-seq (80M reads/sample) was performed in a NovaSeq 6000 sequencing platform using Illumina Stranded Total RNA Ribo-Zero Plus to identify differentially expressed genes (DEGs), followed functional gene enrichment and pathway analyses with Gene Ontology, KEGG and other databases.

RESULTS
Analyses of DEGs identified only one gene (TTR) commonly dysregulated across the three MS subtypes. 934 genes were uniquely altered in primary and 7950 in secondary progressive MS but not in the relapsing-remitting form. Enrichment studies demonstrated that the NAWM from primary-progressive MS exhibits alterations related to immune cell functions, whereas secondary-progressive MS data analyses revealed changes in pathways associated with brain development and synaptic vesicle trafficking.  

CONCLUSION
Taken together, our findings delineate distinct transcriptomic signatures of NAWM across MS subtypes, which are associated with alterations of diverse of biological pathways. The findings corroborate the use of transcriptomic analyses to characterise MS subtypes and aid in the identification of novel targeted treatment options.

Spike Ab seroconversion following SARS-CoV-2 vaccination in Australian people with Multiple Sclerosis. 

BACKGROUND
SARS-CoV-2 antibody responses after COVID-19 vaccination are attenuated in people living with MS on high efficacy DMTs such as Fingolimod and Ocrelizumab. Uncertainties on how to manage vaccination schedule and DMT administration persist. Furthermore, data on effects of newer related DMTs like Siponimod and Ofatumumab are limited.  

OBJECTIVE
We aimed to determine the seroprevalance of Spike antibody and the longevity of antibody mediatedimmune protection after COVID-19 vaccination in MS patients. 

METHOD
Spike IgG antibodies against Wuhan SARS CoV-2 were assessed in sera (n=520) of MS patients collected pre-vaccination (baseline n= 304), 1 month post second dose (n=172), 6 months post second dose (n=23) and 1 month post third dose (n=21). Demographic and clinical information including age, gender, DMT treatment and timing of COVID-19 vaccination were collected from 160 of these MS patients. 

RESULTS
151/172 sera at 1 month post second dose, 20/23 sera at 6 months post second dose, and 15/21 sera at 1 month post third dose were positive for Spike antibodies. Seropositive patients were treated on Alemtuzumab; n=3, Cladribine; n=32, Dimethyl fumarate; n=8, Fingolimod; n=16, IFN; n =4, Ocrelizumab; n=14, Ofatumumab; n=4, Natalizumab; n=11, Siponimod; n=1 and Teriflunomide; n=1. Out of the 21 patients who did not seroconvert at a month post second dose, treatment information was available in 12 patients. n=9 were treated by Ocrelizumab and n=2 were treated by Cladribine and n=1 was treated by Fingolimod, confirming certain DMTs prevent seroconversion. Out of the 151 patients that did seroconvert, 120  had titers that were comparable to controls (healthy general population) and 31 had reduced titres. Treatment information was available in 21/31 of these patients.  Interestingly, 9/21 were on Ocrelizumab, 8/21 were on Fingolimod, 2/21 were on Ofatumumab, 1/21 was on Natalizumab and 1/21 was on Siponimod. This finding confirmed that certain DMTs such as Fingolimod, Ocrelizumab, Ofatumumab and Siponimod cause a reduction in post-vaccination Spike antibody titers in MS patients in comparison to general population. Expectedly, 304/304 sera were negative at baseline.

CONCLUSION 
Some High efficacy DMTs reduce Spike Ab titers or even prevent seroconversion. To maximize vaccine-mediated immune protection against COVID-19, timing of DMT administration and vaccine schedule may need intricate co-ordination in people living with MS.

Pro-inflammatory effects of the L-proline analogue L-azetidine-2-carboxlyic acid in microglial BV2 cells – implications for multiple sclerosis. 

BACKGROUND
L-Azetidine-2-carboxylic acid (AZE) is a toxic non-protein coding amino acid (npAA) that is highly abundant in sugar and table beets. Due to its high structural similarity with the amino acid L-proline, it can evade the editing process during protein assembly and be misincorporated into proline-rich proteins, potentially triggering a phenomenon known as unfolded protein response (uPR). 

OBJECTIVE
In this study we sought to determine if AZE treatment of BV2 microglial cells at varying times and concentrations could trigger a pro-inflammatory and apoptotic response coherent with uPR. 

METHOD 
Murine BV2 microglial cells were grown assayed for viability (MTT) and nitric oxide release (Griess assay) after exposure to AZE at increasing concentrations (0-2000µM) at 0, 3, 6, 12 and 24h. Annexin V/PI staining was used to assess apoptosis. Real-time qPCR, Western blot and immunocytochemistry were used to interrogate relevant pro- and anti-inflammatory and cell survival molecular targets. 

RESULTS
Treatment of BV2 microglia with AZE (at concentrations > 1000µM) significantly reduced cell viability, increased BAX/Bcl2 ratio and caused cell death. These results were mirrored by a robust increase in nitric oxide release, percentage of activated/polarised cells and expression of pro-inflammatory markers (IL-1β, IL-6, NOS2, CD68 and MHC-2). Additionally, we found that AZE induced the expression of the extracellular matrix degrading enzyme matrix metalloproteinase 9-(MMP-9) and of brain derived neurotrophic factor (BDNF), a trophic factor involved in regulating microglial structural plasticity.

CONCLUSION
Collectively, these data indicates that AZE-induced toxicity is associated with a pro-inflammatory response in BV2 microglia that is coherent with the activation of the uPR. This evidence may prompt for an increased monitoring of AZE consumption by humans.

Time & motion market research survey of three treatments for relapsing remitting multiple sclerosis (RRMS) in Australia. 

BACKGROUND

Relapsing Remitting Multiple Sclerosis (RRMS) is the most common form of multiple sclerosis and is characterised by periods of worsening neurologic symptoms followed by remission. There is currently no cure for multiple sclerosis, however, immunotherapies can reduce symptoms and slow progression of the disease. 

OBJECTIVE

The aim of this market research was to gather healthcare provider (HCP) and patient opinion on the time burden associated with immunotherapies; Mavenclad, Ocrevus and Gilenya. Understanding the time burden will enable HCP’s and patients to make more informed decisions about treatment and clinical management.       

METHODS

Surveys were developed with input from treating HCP’s, reference to the current Approved Product Information of Mavenclad, Ocrevus and Gilenya, and contained questions relating to treatment administration, monitoring requirements and other related activities. Surveys were administered to HCP’s (neurologists and MS nurses) and MS patients via an online platform. Time information was collected for each phase of treatment and extrapolated over four years. Averaged time for each phase of treatment was combined to compare the total mean time burden between the three treatments.

RESULTS

5 Neurologists, 19 nurses and 43 patients completed the survey. Of the patients who agreed to participate in the survey, 6 had experience with Ocrevus and 21 had experience with Gilenya. All 43 patients had experience with Mavenclad. The total mean time burden per patient for nurses, neurologists and patients combined over four years of treatment was less for Mavenclad (103.5 hrs) than for Ocrevus (244.6 hrs) or Gilenya (294.4 hrs). Whilst neurologist time was similar, nurse and patient time was lower for Mavenclad (8.9 hrs and 88.4 hrs) compared to Ocrevus (66 hrs and 173.5 hrs) or Gilenya (29.7 hrs and 252.7 hrs). 

CONCLUSION

Mavenclad has a lower administration and monitoring time burden in treating RRMS over four years when compared to Ocrevus and Gilenya.  The reduction in patient and nurse time could represent an improvement in patient quality of life and a reduced resource burden on the healthcare system.

Clinical and radiological features of atypical initial presentations of multiple sclerosis - a case control analysis.

BACKGROUND

A minority of multiple sclerosis presentations initially resemble other central nervous system pathologies such as acute disseminated encephalomyelitis or CNS tumours. It is unknown whether atypical MS confers a different long-term prognosis compared to typical MS. 

OBJECTIVE

With the aim of better defining the long-term outcomes of this group we have performed a retrospective cohort comparison of the clinical and radiological outcomes in atypical (ADEM-like and tumefactive) versus ‘typical’ MS presentations. 

METHODS

Twenty-eight atypical cases were identified and compared with 76 typical MS cases. Disease features analysed included relapse rates, disability severity, whole brain and lesion volumes, lesion number and distribution. 

RESULTS

Atypical cases demonstrated more severe disability severity during their initial illness and a shorter time to their first relapse. Despite this, there was no difference in long-term clinical outcomes including disability severity, annualised relapse rates (ARR), brain volume, lesion numbers, lesion distributions or cerebrospinal fluid analysis. Atypical MS cases were more likely to receive high potency disease modifying therapy early in the course of illness. 

CONCLUSION

Despite the severity of initial illness, our cohort analysis suggests that atypical MS presentations do not confer a higher risk of long-term adverse outcomes.

Low vitamin D levels do not predict risk of autoimmune disease following alemtuzumab treatment for multiple sclerosis. 

BACKGROUND

Widespread use of alemtuzumab has been tempered by treatment emergent autoimmunity, which develops in approximately one-third of patients within 5 years of treatment. It has been postulated that relative vitamin D deficiency may be a causative factor in this relationship. 

OBJECTIVE

We have undertaken a retrospective, case-control study of vitamin D levels in people with MS treated with alemtuzumab, with the aim of identifying factors conferring an increased risk of autoimmune disease, particularly vitamin D deficiency in the peri- and post-treatment phase.

METHODS

We have conducted a retrospective case-control study looking at the association of vitamin D and autoimmune disease following alemtuzumab. Occurrence of autoimmunity was monitored for clinically and through the Bloodwatch® monitoring program. Clinical data and vitamin D levels obtained as part of routine clinical practice were recorded. Vitamin D levels were seasonally adjusted. Univariable and multivariable Cox proportional hazards analyses were performed. 

RESULTS

There were 113 patients treated with alemtuzumab for whom there was complete data. Median follow up was 4.4 years. Risk of autoimmune disease was not associated with lower vitamin D levels. Risk of autoimmune disease was associated with female sex (HR 3.5) and with higher EDSS score at treatment, but not for four or more years of follow-up. 

CONCLUSION

These data do not support a role for vitamin D supplementation in the prevention of autoimmune disease following alemtuzumab. Males have a lower risk of autoimmunity following alemtuzumab.

Enhancing cardiorespiratory exercise response in people with advanced multiple sclerosis with hybrid functional electrical stimulation cycling.  

BACKGROUND
People with advanced multiple sclerosis (MS) find it difficult to exercise aerobically. Hybrid functional electrical stimulation (FES) cycling, which is a combination of arm cranking ergometry (ACE) and FES cycling, elicits greater aerobic response than ACE or FES cycling in people with spinal injury. 

OBJECTIVE
To determine if Hybrid FES cycling elicits a greater cardiorespiratory response than FES cycling or ACE alone in people with advanced MS to provide preliminary guidance for effective aerobic exercise prescription.  

METHOD
Cardiorespiratory responses were measured in nine participants [7 female, age 54.0 (46-64) years, EDSS 7.0 (6.5-7.5)] during ACE, FES cycling, and Hybrid FES cycling at 40%, 60%, 80% and 100% of mode-specific peak workload. Oxygen consumption (VO2) and heart rate (HR) were analyzed at each workload. Participants completed FES cycling familiarization sessions to assess maximal stimulation tolerance and an ACE peak workload test before the exercise trials.  

RESULTS
Hybrid FES cycling evoked significantly higher VO2relative and HRs at most workloads. At 100% workload, Hybrid FES cycling HR was 125 (113-148) bpm and was higher than ACE at 99 (95-119) bpm (p=0.008) and FES cycling at 94 (79-100) bpm (p=0.008). At 100% workload, Hybrid FES cycling VO2relative was 11.8 (7.6-17.6) mL∙kg−1∙min−1 and was higher than ACE at 8.9 (5.3-12.5) mL∙kg−1∙min−1 (p=0.012) and FES cycling at 6.8 (4.1-9.2) mL∙kg−1∙min−1 (p=0.012). 

CONCLUSION
This study showed that Hybrid FES cycling elicited a greater cardiorespiratory response than ACE or FES cycling in people with advanced MS. Therefore, compared to available modes, Hybrid FES cycling has the  greatest potential to improve aerobic fitness and associated health outcomes in advanced MS.

Correlation of Diffusion Tensor Imaging with Clinical Parameters of Early RRMS Compared to Healthy Controls 

BACKGROUND

MRI is the most common modality to monitor MS patients, however T2-FLAIR intensities are non-specific and only poorly explain some of the clinical symptoms observed. Advanced MRI techniques like diffusion tensor imaging (DTI) could potentially better describe the deficits by studying water diffusion in disrupted tracts. 

OBJECTIVE

To compare DTI parameters (Fractional Anisotropy (FA), Mean, Axial, Radial Diffusivities (MD, AD, 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

D T1-MPRAGE, 3D T2-FLAIR and diffusion-weighted data were acquired 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 statistically significant increases in MD (+3.6%), RD (+4.5%) and AD (+2.7%) and decrease in FA (-4.3%) for total brain-WM in RRMS patients compared to HCs (p<0.01). There was also greater increase in MD, RD and AD by 6.4%, 7.4% and 7.5% respectively for the WM-lesions (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

DTI parameters correlated moderately with disability and mental health of RRMS patients. DTI is a sensitive tool in evaluation of subtle and inconspicuous disease processes within total-brain white matter that is otherwise undetectable with structural MRI.