Dimethyl fumarate for treatment of multiple sclerosis : clinical effects and mechanisms

Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system (CNS). Dimethyl fumarate (DMF) is one of the more recent additions to a rapidly expanding treatment repertoire for MS. While DMF has proven beneficial for relapsing-remitting MS (RRMS) patients, its clinical profile in relation to current alternatives as well as its immunological effects are less known. The overarching aim of the thesis was to assess the clinical effects of DMF for MS patients and investigate the underlying immunological mechanisms. Since both DMF and physical exercise is known to elicit an antioxidative response through the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), we further explored their immunological commonalities. In Paper I, we showed that treatment discontinuations with DMF were lower than with interferons, the main existing initial drug choice, among newly diagnosed MS patients in Stockholm and Västerbotten Counties. Risks of having persistent disease activity, as shown by relapses and/or magnetic resonance imaging (MRI), were similar to fingolimod and natalizumab; two more recent disease modulatory therapies (DMTs). The main finding of the article, however, was that the comparator treatment, rituximab (Mabthera®; RTX), had a superior clinical effect compared to all other DMTs in terms of both clinical effect and treatment discontinuation. In Paper II, we used Swedish nationwide data to compare DMF to interferons and glatiramer acetate, two common initial DMT choices, and fingolimod, which mainly is used as an escalation treatment. DMF proved more effective and had better drug survival in the first line comparison with interferons and glatiramer acetate but was less well tolerated than fingolimod when used second line. In Paper III, we explored the immunological mechanisms of DMF treatment in humans underlying the clinical effects we observed in Paper I and II. We observed that DMF increased production of reactive oxygen species (ROS) in monocytes and that methylation changes occurred earlier in monocytes than in T cells. In addition, monocyte counts and levels of oxidized fat in blood were higher among treatment responders compared to non-responders, supporting the notion that DMF act by increasing oxidative burst in myeloid cells. In Paper IV, we investigated the effects of aerobic exercise of moderate and high intensity on immune protein markers and kynurenine pathway (KP) metabolites in cerebrospinal fluid (CSF) and plasma of healthy participants. Participants in the high intensity group displayed changes in concentration of several immune markers and KP metabolites in both CSF and plasma, whereas participants in the moderate intensity group displayed few changes, suggesting a dose-response relationship. A separate comparison with DMF treated MS patients revealed few overlapping immune markers despite indications of overlapping mechanisms. In conclusion, by affecting Nrf2 and oxidative burst, DMF has a unique mode of action among existing DMT options for RRMS, however, with limited overlap to effects mediated by physical exercise. Its clinical effectiveness is superior to traditional DMTs for newly diagnosed patients, but inferior to RTX. As an escalation DMT, it is less well tolerated than existing alternatives

Multiple sclerosis -the impact of environmental- and lifestyle factors

Background: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), likely caused by an interaction of genetic and environmental factors. Epstein-Barr virus infection, low serum vitamin D levels, smoking and obesity increase the risk of MS. However, knowledge of their effect on disease activity and progression have been limited. Objective: The main objective was to explore the role of different environmental and lifestyle factors for MS disease activity. In more detail, we sought to evaluate whether there is an association between tobacco use or body mass index (BMI) and MS disease activity. We also explored the potential of two adipokines, leptin and adiponectin as biomarkers for disease course or interferon-beta (IFNβ) treatment response in MS. Methods: All data in our studies were based on the OFAMS study, a randomized placebo-controlled multicenter study of 92 patients with relapsing-remitting MS (RRMS) that was conducted between 2004 -2008. The patients were followed for 24 months with repeated magnetic resonance imaging (MRI) of the brain, blood tests and clinical evaluations, 6 months prior to and 18 months during IFNβ-treatment. For the current thesis, we analyzed serum samples for cotinine, a biomarker for tobacco use, and the adipokines leptin and adiponectin. For the first study, the patients were categorized as tobacco-users and non-tobacco-users according to their serum cotinine level. For the second and third study, patients were categorized based on the World Health Organisation (WHO) classification of BMI into three groups; normal weight patients (BMI30 kg/m²). All analyses were adjusted for age, gender and BMI. Results: We did not find any association between tobacco use and MRI activity (paper I). Further, there was no difference between tobacco users and non-tobacco users regarding baseline Expanded Disability Status Scale (EDSS) score, EDSS-progression or relapse-rate. For tobacco users, there was no correlation between serum cotinine levels and disease activity. There was no difference in clinical and MRI activity between patients stratified by BMI prior to IFNβ-treatment. During IFNβ-treatment, 80 % of overweight or obese patients had MRI activity compared to 48 % in the group of normal weight patients (p=0.001). The number of patients obtaining NEDA (no evidence of disease activity)-status differed according to BMI; 26 % in the normal weight group compared to only 13 % in the group of overweight and obese patients (p=0.05) (paper II). There was no association between serum levels of leptin or adiponectin and MRI disease activity (paper III). The serum levels of leptin were lower and the levels of adiponectin higher during IFNβ-treatment compared to the treatment-naïve period, reflecting the anti-inflammatory effect of the drug. Conclusion: In our studies, we found no direct association between tobacco use or BMI and MS disease activity. During IFNβ-treatment fewer of the overweight and obese patients obtained NEDA-status compared to patients with normal weight, indicating that BMI could affect IFNβ-treatment response. Serum levels of leptin and adiponectin seem not to be suited as biomarkers for disease activity or IFNβ-treatment response in MS.Doktorgradsavhandlin

Imaging Neuroinflammation in Progressive Multiple Sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system CNS), where inflammation and neurodegeneration lead to irreversible neuronal damage. In MS, a dysfunctional immune system causes auto‐reactive lymphocytes to migrate into CNS where they initiate an inflammatory cascade leading to focal demyelination, axonal degeneration and neuronal loss. One of the hallmarks of neuronal injury and neuroinflammation is the activation of microglia. Activated microglia are found not only in the focal inflammatory lesions, but also diffusely in the normal‐appearing white matter (NAWM), especially in progressive MS. The purine base, adenosine is a ubiquitous neuromodulator in the CNS and also participates in the regulation of inflammation. The effect of adenosine mediated via adenosine A2A receptors has been linked to microglial activation, whereas modulating A2A receptors may exert neuroprotective effects. In the majority of patients, MS presents with a relapsing disease course, later advancing to a progressive phase characterised by a worsening, irreversible disability. Disease modifying treatments can reduce the severity and progression in relapsing MS, but no efficient treatment exists for progressive MS. The aim of this research was to investigate the prevalence of adenosine A2A receptors and activated microglia in progressive MS by using in vivo positron emission tomography (PET) imaging and [11C]TMSX and [11C](R)‐PK11195 radioligands. Magnetic resonance imaging (MRI) with diffusion tensor imaging (DTI) was performed to evaluate structural brain damage. Non‐invasive input function methods were also developed for the analyses of [11C]TMSX PET data. Finally, histopathological correlates of [11C](R)‐PK11195 radioligand binding related to chronic MS lesions were investigated in post‐mortem samples of progressive MS brain using autoradiography and immunohistochemistry. [11C]TMSX binding to A2A receptors was increased in NAWM of secondary progressive MS (SPMS) patients when compared to healthy controls, and this correlated to more severe atrophy in MRI and white matter disintegration (reduced fractional anisotropy, FA) in DTI. The non‐invasive input function methods appeared as feasible options for brain [11C]TMSX images obviating arterial blood sampling. [11C](R)‐PK11195 uptake was increased in the NAWM of SPMS patients when compared to patients with relapsing MS and healthy controls. Higher [11C](R)‐PK11195 binding in NAWM and total perilesional area of T1 hypointense lesions was associated with more severe clinical disability, increased brain atrophy, higher lesion load and reduced FA in NAWM in the MS patients. In autoradiography, increased perilesional [11C](R)‐PK11195 uptake was associated with increased microglial activation identified using immunohistochemistry. In conclusion, brain [11C]TMSX PET imaging holds promise in the evaluation of diffuse neuroinflammation in progressive MS. Being a marker of microglial activation, [11C](R)‐ PK11195 PET imaging could possibly be used as a surrogate biomarker in the evaluation of the neuroinflammatory burden and clinical disease severity in progressive MS.Siirretty Doriast

Multiple Sclerosis

Multiple sclerosis is among the most frequent neurological diseases, which affect seriously the quality of life of a constantly increasing number of patients, inducing physical and mental invalidism with indefinite perspectives. The ongoing investigation of the pathogenetic background and the inconclusive analysis of many pathophysiological mechanisms and serious neuropathological alterations of the disease are dominant crucial topics in the field of neurosciences, aimed at tracing a definite way to a therapeutic approach. The authors of this volume attempt to throw light on the labyrinth of multiple sclerosis, approaching the disease from the viewpoint of epidemiology, autoimmune reactions, symptomatology, mental and physical decline, diagnostic procedures, prognosis, and treatment. The authors submit herein, along with scientific data, their hope of contributing effectively to ameliorating the quality of life of those suffering from multiple sclerosis who wait patiently for the day of recovery

Current developments in MRI for assessing rodent models of multiple sclerosis

MRI is a key radiological imaging technique that plays an important role in the diagnosis and characterization of heterogeneous multiple sclerosis (MS) lesions. Various MRI methodologies such as conventional T 1/T 2 contrast, contrast agent enhancement, diffusion-weighted imaging, magnetization transfer imaging and susceptibility weighted imaging have been developed to determine the severity of MS pathology, including demyelination/remyelination and brain connectivity impairment from axonal loss. The broad spectrum of MS pathology manifests in diverse patient MRI presentations and affects the accuracy of patient diagnosis. To study specific pathological aspects of the disease, rodent models such as experimental autoimmune encephalomyelitis, virus-induced and toxin-induced demyelination have been developed. This review aims to present key developments in MRI methodology for better characterization of rodent models of MS

Imaging Neuroinflammation – from Bench to Bedside

Neuroinflammation plays a central role in a variety of neurological diseases, including stroke, multiple sclerosis, Alzheimer’s disease, and malignant CNS neoplasms, among many other. Different cell types and molecular mediators participate in a cascade of events in the brain that is ultimately aimed at control, regeneration and repair, but leads to damage of brain tissue under pathological conditions. Non-invasive molecular imaging of key players in the inflammation cascade holds promise for identification and quantification of the disease process before it is too late for effective therapeutic intervention. In this review, we focus on molecular imaging techniques that target inflammatory cells and molecules that are of interest in neuroinflammation, especially those with high translational potential. Over the past decade, a plethora of molecular imaging agents have been developed and tested in animal models of (neuro)inflammation, and a few have been translated from bench to bedside. The most promising imaging techniques to visualize neuroinflammation include MRI, positron emission tomography (PET), single photon emission computed tomography (SPECT), and optical imaging methods. These techniques enable us to image adhesion molecules to visualize endothelial cell activation, assess leukocyte functions such as oxidative stress, granule release, and phagocytosis, and label a variety of inflammatory cells for cell tracking experiments. In addition, several cell types and their activation can be specifically targeted in vivo, and consequences of neuroinflammation such as neuronal death and demyelination can be quantified. As we continue to make progress in utilizing molecular imaging technology to study and understand neuroinflammation, increasing efforts and investment should be made to bring more of these novel imaging agents from the “bench to bedside.

Tertiary lymphoid organs in central nervous system autoimmunity

Multiple sclerosis (MS) is an autoimmune disease characterized by chronic inflammation in the central nervous system (CNS), which results in permanent neuronal damage and substantial disability in patients. Autoreactive T cells are important drivers of the disease; however, the efficacy of B cell depleting therapies uncovered an essential role for B cells in disease pathogenesis. They can contribute to inflammatory processes via presentation of autoantigen, secretion of pro-inflammatory cytokines, and production of pathogenic antibodies. Recently, B cell aggregates reminiscent of tertiary lymphoid organs (TLOs) were discovered in the meninges of MS patients, leading to the hypothesis that differentiation and maturation of autopathogenic B and T cells may partly occur inside the CNS. Since these structures were associated with a more severe disease course, it is extremely important to gain insight into the mechanism of induction, their precise function, and clinical significance. Mechanistic studies in patients are limited. However, a few studies in the MS animal model experimental autoimmune encephalomyelitis (EAE) recapitulate TLO formation in the CNS and provide new insight into CNS TLO features, formation, and function. This review summarizes what we know so far about CNS TLOs in MS and what we have learned about them from EAE models. It also highlights the areas that are in need of further experimental work, as we are just beginning to understand and evaluate the phenomenon of CNS TLOs

Current Evidence for a Role of the Kynurenine Pathway of Tryptophan Metabolism in Multiple Sclerosis

The kynurenine pathway (KP) is the major metabolic pathway of the essential amino acid tryptophan (TRP). Stimulation by inflammatory molecules, such as interferon-γ (IFN-γ), is the trigger for induction of the KP, driving a complex cascade of production of both neuroprotective and neurotoxic metabolites, and in turn, regulation of the immune response and responses of brain cells to the KP metabolites. Consequently, substantial evidence has accumulated over the past couple of decades that dysregulation of the KP and the production of neurotoxic metabolites are associated with many neuroinflammatory and neurodegenerative diseases, including Parkinson's disease, AIDS-related dementia, motor neurone disease, schizophrenia, Huntington's disease, and brain cancers. In the past decade, evidence of the link between the KP and multiple sclerosis (MS) has rapidly grown and has implicated the KP in MS pathogenesis. KP enzymes, indoleamine 2,3-dioxygenase (IDO-1) and tryptophan dioxygenase (highest expression in hepatic cells), are the principal enzymes triggering activation of the KP to produce kynurenine from TRP. This is in preference to other routes such as serotonin and melatonin production. In neurological disease, degradation of the blood-brain barrier, even if transient, allows the entry of blood monocytes into the brain parenchyma. Similar to microglia and macrophages, these cells are highly responsive to IFN-γ, which upregulates the expression of enzymes, including IDO-1, producing neurotoxic KP metabolites such as quinolinic acid. These metabolites circulate systemically or are released locally in the brain and can contribute to the excitotoxic death of oligodendrocytes and neurons in neurological disease principally by virtue of their agonist activity at N-methyl-d-aspartic acid receptors. The latest evidence is presented and discussed. The enzymes that control the checkpoints in the KP represent an attractive therapeutic target, and consequently several KP inhibitors are currently in clinical trials for other neurological diseases, and hence may make suitable candidates for MS patients. Underpinning these drug discovery endeavors, in recent years, several advances have been made in how KP metabolites are assayed in various biological fluids, and tremendous advancements have been made in how specimens are imaged to determine disease progression and involvement of various cell types and molecules in MS.22 page(s

Post mortem and in vivo study of multiple sclerosis pathogenesis

Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system. A number of pathological mechanisms could be responsible for acute demyelination and chronic tissue remodelling in MS, including inflammation, oxidative stress, microglia activation, and astrocyte infiltrates. In the present work, we aim to further explore the heterogeneity of MS pathogenesis on post mortem brains, and to evaluate the possibility to study MS pathogenesis by using magnetic resonance imaging (MRI) and peripheral blood biomarkers. In the first part of the study, we applied a data driven approach to classify MS patients in relation to the variety of pathological changes occurring in lesional and normal-appearing (NA) white matter (WM) and grey matter (GM), with subsequent clinical correlates. Tissue blocks from 16 MS brains were immunostained and quantified for neuro-axonal structures (NF200), myelin (SMI94), macrophages (CD68), B-lymphocytes (CD20), T-lymphocytes (CD3), cytotoxic T-lymphocytes (CD8), microglia (IBA1), astrocytes (GFAP), and mitochondrial damage. After semi-automatic registration of digitized histologic sections, regions-of-interest (ROIs) were manually defined in lesion and NA WM and GM. A latent class analysis was employed to characterize pathology subtypes in MS; different goodness of fit parameters (AIC, BIC, and G2 statistics) were used to identify the number of classes that better characterize the MS sub-populations. Profile 1 (active remodelling) was characterized by normal-appearing neuro-axonal structures and intact energetic metabolism, with high levels of macrophages/microglia and astrocytes. Profile 2 (mitochondrial dysfunction) was characterized by severely impaired mitochondrial function, along with demyelination and neuroaxonal loss, and ongoing inflammatory changes, mainly driven by cytotoxic T-cells (CD8+); patients in profile 2 presented with more severe symptoms at onset and faster disability accrual, when compared with other profiles. Profile 3 (inactive) was characterized by severe demyelination and axonal loss, with similarly reduced mitochondrial function, without any concomitant pathological process contributing to further tissue remodelling and/or damage. The possibility to classify each patient depending on his/her prevalent pathology profile support the concept of MS immunopathological homogeneity within the same patient and heterogeneity between different patients, and could be used to better profile MS patients and individualize their treatment. In the second part of the study, we explored post mortem pathology-MRI correlates and specifically focused on an advanced MRI technique (magnetization transfer ratio -MTR-), ideally detecting myelin content. MTR is widely used in MS observational studies and clinical trials, but its pathological correlates remain unclear. MTR maps were acquired at 3 Tesla from sixteen fixed MS brains and four healthy controls. 101 tissue blocks were immunostained and quantified, as previously described. After semi-automatic registration of digitized histologic sections and MTR maps, ROIs were manually defined. Associations between MTR and each stain were explored using linear mixed regression models (with cassettes nested within patients); differences in the associations between ROIs were explored using interaction terms. Lower MTR was associated with lower levels of NF200, SMI94, CD68, IBA1 and GFAP, with higher levels of CD8 and greater mitochondrial damage; MTR was more strongly associated with SMI94 in GM than WM. In a multivariate linear mixed regression model including all ROIs and brains, SMI94 was the best correlate of MTR. Myelin immunostain intensity is the strongest correlate of MTR, especially when measured in the GM. However, the additional histological correlates of MTR have to be kept in mind when interpreting the results of MTR clinical studies and designing experimental trials in MS. Finally, we evaluated the possibility to study (and to modify) MS pathology in vivo, by using biomarkers in the peripheral blood. Considering that oxidative stress is a driver of MS pathology, we evaluated the effect of coenzyme Q10 (CoQ10) on laboratory markers of oxidative stress and inflammation, and on MS clinical severity, and, then, calculated the sample size needed to detect significant variations to define most promising biomarkers. We included 60 relapsing-remitting MS patients treated with Interferon-Beta1a-44μg with CoQ10 for 3 months, and with Interferon-Beta1a-44μg alone for 3 more months (open-label cross-over design). At baseline, 3- and 6-month visits, we measured markers of scavenging activity, oxidative damage and inflammation in the peripheral blood, and collected data on disease severity. After 3 months, CoQ10 supplementation was associated with improved scavenging activity (as mediated by uric acid), reduced intracellular reactive oxygen species production, reduced oxidative DNA damage, and shift towards a more anti-inflammatory milieu in the peripheral blood (with higher IL-4 and IL-13, and lower Eotaxin, GM-CSF, HGF, IFN-γ, IL-1α, IL-2R, IL-9, IL-17F, MIP-1α, RANTES, TNF-α and VEGF). Also, CoQ10 supplementation was associated with lower expanded disability status scale, fatigue severity scale, Beck's depression inventory, and visual analogic scale for pain. For sample size estimates, we used adjusted-beta-coefficients of observed 3-month variation for each laboratory measure (and respective standard deviation); we assumed that the observed variation was the highest achievable treatment effect (100%), and we estimated sample size for conservative treatment effects (e.g., 70%), smaller than what observed. Setting 5% alpha-error and 80% power, low sample size requirements to detect 70% observed variation from a baseline pre-treatment timepoint to a 3-month follow-up were found for IL-3 (n=1), IL-5 (n=1), IL-7 (n=4), IL-2R (n=4), IL-13 (n=6), IL-6 (n=14), IL-8 (n=22), IL-4 (n=23), RANTES (n=25), TNF-α (n=26), IL-1β (n=27), and uric acid (n=29). CoQ10 supplementation improved scavenging activity, reduced oxidative damage, and induced a shift towards a more anti-inflammatory milieu, in the peripheral blood of relapsing-remitting MS patients treated with Interferon-Beta1a 44μg, along with clinical improvements. Peripheral biomarkers of oxidative stress and inflammation could be used in small proof-of-concept studies to quickly screen the mechanisms of action of new or already-existing medications for MS