Advances in Multiple Sclerosis Research Series I Printed Edition of the Special Issue Published in Brain Sciences www.mdpi.com/journal/brainsci John Matsoukas and Vasso Apostolopoulos Edited by Advances in Multiple Sclerosis Research—Series I Advances in Multiple Sclerosis Research—Series I Editors John Matsoukas Vasso Apostolopoulos MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editors John Matsoukas NewDrug, Patras Science Park Greece Vasso Apostolopoulos Victoria University Australia Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Brain Sciences (ISSN 2076-3425) (available at: https://www.mdpi.com/journal/brainsci/special issues/Advance MS Research). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Volume Number , Page Range. ISBN 978-3-03943-947-8 (Hbk) ISBN 978-3-03943-948-5 (PDF) c © Cover image courtesy of Amanda Habib and Rhiannon Filippone, PhD students, Victoria University, Melbourne Australia (Supervisors, Professor Vasso Apostolopoulos andAssociate Professor Kulmira Nurgali). Immunohistochemical image of oligodendrocytes within theprefrontal cortex of the brain. Image showing oligodendrocytes stained for myelin basic protein. Inflammation of the brain results in down regulation of myelin which is associated with variousneurological disorders such as multiple sclerosis. 202 1 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Vasso Apostolopoulos and John Matsoukas Advances in Multiple Sclerosis Research–Series I Reprinted from: Brain Sci. 2020 , 10 , 795, doi:10.3390/brainsci10110795 . . . . . . . . . . . . . . . . 1 Marcello Moccia, Antonio Capacchione, Roberta Lanzillo, Fortunata Carbone, Teresa Micillo, Giuseppe Matarese, Raffaele Palladino and Vincenzo Brescia Morra Sample Size for Oxidative Stress and Inflammation When Treating Multiple Sclerosis with Interferon- β 1a and Coenzyme Q10 Reprinted from: Brain Sci. 2019 , 9 , 259, doi:10.3390/brainsci9100259 . . . . . . . . . . . . . . . . . 9 Tobias Moser, Gayane Harutyunyan, Anush Karamyan, Ferdinand Otto, Carola Bacher, Vaclav Chroust, Markus Leitinger, Helmut F. Novak, Eugen Trinka and Johann Sellner Therapeutic Plasma Exchange in Multiple Sclerosis and Autoimmune Encephalitis: A Comparative Study of Indication, Efficacy, and Safety Reprinted from: Brain Sci. 2019 , 9 , 267, doi:10.3390/brainsci9100267 . . . . . . . . . . . . . . . . . 21 Navzer D. Sachinvala, Angeline Stergiou, Duane E. Haines, Armen Kocharian and Andrew Lawton Post-Craniopharyngioma and Cranial Nerve-VI Palsy Update on a MS Patient with Major Depression and Concurrent Neuroimmune Conditions Reprinted from: Brain Sci. 2019 , 9 , 281, doi:10.3390/brainsci9100281 . . . . . . . . . . . . . . . . . 33 Maria Chiara Buscarinu, Arianna Fornasiero, Giulia Pellicciari, Roberta Reni` e, Anna Chiara Landi, Alessandro Bozzao, Cristina Cappelletti, Pia Bernasconi, Giovanni Ristori and Marco Salvetti Autoimmune Encephalitis and CSF Anti-GluR3 Antibodies in an MS Patient after Alemtuzumab Treatment Reprinted from: Brain Sci. 2019 , 9 , 299, doi:10.3390/brainsci9110299 . . . . . . . . . . . . . . . . . 43 Marco Pitteri, Stefano Ziccardi, Caterina Dapor, Maddalena Guandalini and Massimiliano Calabrese Lost in Classification: Lower Cognitive Functioning in Apparently Cognitive Normal Newly Diagnosed RRMS Patients Reprinted from: Brain Sci. 2019 , 9 , 321, doi:10.3390/brainsci9110321 . . . . . . . . . . . . . . . . . 49 Rechdi Ahdab, Madiha M. Shatila, Abed Rahman Shatila, George Khazen, Joumana Freiha, Maher Salem, Karim Makhoul, Rody El Nawar, Shaza El Nemr, Samar S. Ayache and Naji Riachi Cortical Excitability Measures May Predict Clinical Response to Fampridine in Patients with Multiple Sclerosis and Gait Impairment Reprinted from: Brain Sci. 2019 , 9 , 357, doi:10.3390/brainsci9120357 . . . . . . . . . . . . . . . . . 59 Maria Sofia Basile, Emanuela Mazzon, Katia Mangano, Manuela Pennisi, Maria Cristina Petralia, Salvo Danilo Lombardo, Ferdinando Nicoletti, Paolo Fagone and Eugenio Cavalli Impaired Expression of Tetraspanin 32 (TSPAN32) in Memory T Cells of Patients with Multiple Sclerosis Reprinted from: Brain Sci. 2020 , 10 , 52, doi:10.3390/brainsci10010052 . . . . . . . . . . . . . . . . 69 v Craig D. Workman, Laura L. Boles Ponto, John Kamholz and Thorsten Rudroff No Immediate Effects of Transcranial Direct Current Stimulation at Various Intensities on Cerebral Blood Flow in People with Multiple Sclerosis Reprinted from: Brain Sci. 2020 , 10 , 82, doi:10.3390/brainsci10020082 . . . . . . . . . . . . . . . . 83 Narges Dargahi, John Matsoukas and Vasso Apostolopoulos Streptococcus thermophilus ST285 Alters Pro-Inflammatory to Anti-Inflammatory Cytokine Secretion against Multiple Sclerosis Peptide in Mice Reprinted from: Brain Sci. 2020 , 10 , 126, doi:10.3390/brainsci10020126 . . . . . . . . . . . . . . . . 89 Marina Kleopatra Boziki, Evangelia Kesidou, Paschalis Theotokis, Alexios-Fotios A. Mentis, Eleni Karafoulidou, Mikhail Melnikov, Anastasia Sviridova, Vladimir Rogovski, Alexey Boyko and Nikolaos Grigoriadis Microbiome in Multiple Sclerosis: Where Are We, What We Know and Do Not Know Reprinted from: Brain Sci. 2020 , 10 , 234, doi:10.3390/brainsci10040234 . . . . . . . . . . . . . . . . 103 Vasso Apostolopoulos, Abdolmohamad Rostami and John Matsoukas The Long Road of Immunotherapeutics against Multiple Sclerosis Reprinted from: Brain Sci. 2020 , 10 , 288, doi:10.3390/brainsci10050288 . . . . . . . . . . . . . . . . 121 Athanasios Metaxakis, Dionysia Petratou and Nektarios Tavernarakis Molecular Interventions towards Multiple Sclerosis Treatment Reprinted from: Brain Sci. 2020 , 10 , 299, doi:10.3390/brainsci10050299 . . . . . . . . . . . . . . . . 129 Monika Gudowska-Sawczuk, Joanna Tarasiuk, Alina Kułakowska, Jan Kochanowicz and Barbara Mroczko Kappa Free Light Chains and IgG Combined in a Novel Algorithm for the Detection of Multiple Sclerosis Reprinted from: Brain Sci. 2020 , 10 , 324, doi:10.3390/brainsci10060324 . . . . . . . . . . . . . . . . 149 Olga Kammona and Costas Kiparissides Recent Advances in Antigen-Specific Immunotherapies for the Treatment of Multiple Sclerosis Reprinted from: Brain Sci. 2020 , 10 , 333, doi:10.3390/brainsci10060333 . . . . . . . . . . . . . . . . 159 Maria Chountoulesi and Costas Demetzos Promising Nanotechnology Approaches in Treatment of Autoimmune Diseases of Central Nervous System Reprinted from: Brain Sci. 2020 , 10 , 338, doi:10.3390/brainsci10060338 . . . . . . . . . . . . . . . . 225 Rodolfo Thome, Alexandra Boehm, Larissa Lumi Watanabe Ishikawa, Giacomo Casella, Jaqueline Munhoz, Bogoljub Ciric, Guang-Xian Zhang and Abdolmohamad Rostami Comprehensive Analysis of the Immune and Stromal Compartments of the CNS in EAE Mice Reveal Pathways by Which Chloroquine Suppresses Neuroinflammation Reprinted from: Brain Sci. 2020 , 10 , 348, doi:10.3390/brainsci10060348 . . . . . . . . . . . . . . . . 249 Catherine Koukoulitsa, Eleni Chontzopoulou, Sofia Kiriakidi, Andreas G. Tzakos and Thomas Mavromoustakos A Journey to the Conformational Analysis of T-Cell Epitope Peptides Involved in Multiple Sclerosis Reprinted from: Brain Sci. 2020 , 10 , 356, doi:10.3390/brainsci10060356 . . . . . . . . . . . . . . . . 263 vi Maria Anagnostouli, Artemios Artemiadis, Maria Gontika, Charalampos Skarlis, Nikolaos Markoglou, Serafeim Katsavos, Konstantinos Kilindireas, Ilias Doxiadis and Leonidas Stefanis HLA-DPB1*03 as Risk Allele and HLA-DPB1*04 as Protective Allele for Both Early- and Adult-Onset Multiple Sclerosis in a Hellenic Cohort Reprinted from: Brain Sci. 2020 , 10 , 374, doi:10.3390/brainsci10060374 . . . . . . . . . . . . . . . . 279 Francesca Nuti, Feliciana Real Fernandez, Giuseppina Sabatino, Elisa Peroni, Barbara Mulinacci, Ilaria Paolini, Margherita Di Pisa, Caterina Tiberi, Francesco Lolli, Martina Petruzzo, Roberta Lanzillo, Vincenzo Brescia Morra, Paolo Rovero and Anna Maria Papini A Multiple N -Glucosylated Peptide Epitope Efficiently Detecting Antibodies in Multiple Sclerosis Reprinted from: Brain Sci. 2020 , 10 , 453, doi:10.3390/brainsci10070453 . . . . . . . . . . . . . . . . 293 Efstathios Deskoulidis, Souzana Petrouli, Vasso Apostolopoulos, John Matsoukas and Emmanuel Topoglidis The Use of Electrochemical Voltammetric Techniques and High-Pressure Liquid Chromatography to Evaluate Conjugation Efficiency of Multiple Sclerosis Peptide-Carrier Conjugates Reprinted from: Brain Sci. 2020 , 10 , 577, doi:10.3390/brainsci10090577 . . . . . . . . . . . . . . . . 309 vii About the Editors John Matsoukas has over 30 years of experience in research in the field of organic and peptide chemistry, nuclear magnetic resonance (NMR) and the chemistry of natural products. He has an extensive research background in NMR-based drug discovery, design and development. Professor Matsoukas has studied Chemistry at the University of Patras. He graduated from the University of New Brunswick in Canada with a MSc Degree in Chemistry. His dissertation was on the Total Synthesis of Natural Products and Nuclear Magnetic Resonance. He carried out his Ph.D. studies in Chemistry at the University of Patras, Greece in the peptide field. He joined the University of Calgary, Alberta, Canada, and the group of Professor Graham Moore, studying peptide hormones and peptide mimetics. He was the founder, director, and head of the successful Graduate Program “Medicinal Chemistry: Drug Discovery, Design and Development” of the University of Patras (1997–2013). Since 2000, he has been collaborating with Professor Vasso Apostolopoulos of Victoria University on multiple sclerosis peptides and the development of vaccines. He has published more than 500 articles in peer-reviewed journals, book chapters and conference proceedings. He has been granted many patents, awards and honors for his research and scientific activities. Vasso Apostolopoulos ’s expertise is in the areas of immunology, crystallography, cellular biology, translational research, and the development of drugs and vaccines. Vasso has led/directed several research programs at various research centers and universities around the world. She is currently the Associate Provost, Research Partnership at Victoria University Australia. She has received more than 100 awards, published over 400 research papers, invented 18 patents and her current interests are treating chronic diseases with an immunological focus. ix brain sciences Editorial Advances in Multiple Sclerosis Research–Series I Vasso Apostolopoulos 1, * and John Matsoukas 1,2,3 1 Institute for Health and Sport, Victoria University, Melbourne 8001, Australia; imats1953@gmail.com 2 NewDrug, Patras Science Park, 26500 Patras, Greece 3 Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada * Correspondence: vasso.apostolopoulos@vu.edu.au; Tel.: + 613-9919-2025 Received: 15 October 2020; Accepted: 25 October 2020; Published: 29 October 2020 Abstract: Designing immunotherapeutics, drugs, and anti-inflammatory reagents has been at the forefront of autoimmune research, in particular, multiple sclerosis, for over 20 years. Delivery methods that are used to modulate e ff ective and long-lasting immune responses have been the major focus. This Special Issue, “Advances in Multiple Sclerosis Research—Series I”, focused on delivery methods used for immunotherapeutic approaches, drug design, anti-inflammatories, identification of markers, methods for detection and monitoring MS and treatment modalities. The issue gained much attention with 20 publications, and, as a result, we launched Series II with the deadline for submission being 30 April 2021. Keywords: multiple sclerosis; MS; vaccine; immunomodulation; carriers; MS drugs 1. Multiple Sclerosis The World Health Organization estimates that globally, more than 2.5 million people are a ff ected by multiple sclerosis (MS). With the global population growing to an unparalleled height of 7.0 billion in 2011 and recently reaching 7.8 billion (10 October 2020)—it is estimated to reach 8.5 billion by 2030 and 9.7 billion by 2050—the incidence and onset of MS in young adults is expected to rise exponentially, with an estimate of 2.3 million people living with MS globally. Clinical isolated syndrome is a type of MS which may or may not progress. As such, a person will experience a neurological episode lasting at least 24 h and resulting in damage to the central nervous system (CNS). There are three main subtypes of MS, (i) relapse / remitting MS (RRMS) accounting for 85% of MS cases, with 50% progressing to (ii) secondary progressive MS (SPMS), with (iii) 15% of those diagnosed at onset of primary progressive MS (PPMS) type. It is possible that RRMS patients can remain in that state for up to 30 years, whilst 8% develop a more aggressive disease, named highly active RRMS (HARRMS). In rare occasions, up to 5% are progressive relapsing MS type (PRMS), which is characterized by progressive worsening of the condition from the onset, similar to PPMS. MS is characterized as a chronic demyelinating disorder of the CNS with inflammatory cells infiltrating around the nerve, leading to demyelination of the myelin sheath and immune attack to myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). Inflammatory cells which have been found to be involved in MS include macrophages, T helper type 1 (Th1) cells, Th17 cells, CD8 + T cells and B cells secreting auto-antibodies [ 1 – 3 ]. More recently, it has been shown that tetraspanin-32 is significantly downregulated in Th cells. Tetraspanin-32 controls the development of autoimmune responses, and in EAE models in mice, tetraspanin-32 is significantly expressed at lower levels on activated or encephalitogenic T cells compared to naïve Th cells. In the study by Basile and Cavalli et al., it was noted that tetraspanin-32 was downregulated in memory T cells and was further decreased upon ex vivo restimulation (Figure 1) [ 4 ]. Likewise, myelin-specific memory T cells and peripheral blood mononuclear cells (PBMC) from patients with MS also expressed Brain Sci. 2020 , 10 , 795; doi:10.3390 / brainsci10110795 www.mdpi.com / journal / brainsci 1 Brain Sci. 2020 , 10 , 795 lower levels of tetraspanin-32 compared to memory T cells from healthy subjects. In addition, MS patients with early relapses compared to those with a longer, stable disease expressed lower levels of tetraspanin-32 on their PBMC [ 4 ]. Hence, tetraspanin-32 is involved in immune responses underlying the pathophysiology of MS, and could be a viable diagnostic marker or therapeutic target against MS. Figure 1. Summary of new advances in Multiple Sclerosis Research—Series I, papers in the Special Issue. Created with biorender.com. A number of factors contribute to MS development, including genetic predisposition, especially those who are HLA-DR2 (HLA-DRB1*15, HLA-DRB1*16)- or HLA-DR4 (HLA-DRB1*04)-positive, environmental factors such as Epstein–Barr virus and human herpesvirus 6 exposure, and diet, such as low levels of vitamins B and D [ 1 , 5 , 6 ]. A number of health conditions are related to HLA phenotype, such as type-1 diabetes (HLA-DRB1*03 or HLA-DR3, HLA-DQB1*03 or HLA-DQ8), rheumatoid arthritis (HLA-DRB1*04), juvenile idiopathic arthritis (HLA-DRB1*08), celiac disease (HLA-DQ2, HLA-DQ8) and Graves’ disease (HLA-DRB1*03, HLA-DQA1*0501). The paper by Maria Anagnostouli et al. studied the prevalence of HLA-DPB1 allele in MS patients from a Greek cohort and its association with HLA-DRB1 risk allele [ 7 ]. No significant di ff erences were noted between early onset MS compared to adult onset MS for 23 distinct HLA-DPB1 and 12 HLA-DRB1 alleles. However, the frequency of HLA-DPB1*03 allele was significantly increased, and the frequency of HLA-DPB1*02 allele was significantly decreased, in AOMS patients compared to controls. Interestingly, the frequency of HLA-DPB1*04 allele was significantly decreased in both patients, with early onset and adult onset MS compared to controls, suggesting a protective role of this allele amongst Greek cohort patients (Figure 1) [ 7 ]. Koukoulitsa and colleagues present a nice review articulating the journey of the conformational complex between HLA-peptide with the T cell receptor of agonist peptides and their altered peptide ligands from MBP, MOG and PLP [8]. 2 Brain Sci. 2020 , 10 , 795 2. Detection and Monitoring of Patients with MS Magnetic resonance imaging (MRI) has been the gold standard of diagnosing and monitoring disease by detecting brain lesions and the type of brain lesion which aids treatment decisions. In addition, other detection methods are used in combination with MRI, such as the Kurtzke Expanded Disability Status Scale (EDSS) which measures the body’s function and how well it can move, as well as analysis of cerebrospinal fluid for free light chains and IgG. Together, these increase the accuracy of diagnosis of MS and are used to monitor disease progression. However, there are few simple assays available to follow up disease activity. As such, the detection of auto-antibodies from sera is a method to detect relevant biomarkers. The team by Nuti and Papini et al., developed a method to detect anti-N-glycosylated (N-Glc) peptide antibodies, using a four-branched dendrimeric lysine sca ff old, linked to a polyethylene glycol-based spacer containing 19-amino acids. This e ffi cient multivalent probe has specificity and high a ffi nity for anti-N-Glc antibodies in patients with MS [ 9 ]. In addition, Gudowska-Sawczuk evaluated cerebrospinal fluid and sera from patients with either MS ( n = 34) or other neurological disorders ( n = 42) [ 10 ]. The concentrations of cerebrospinal fluid κ free light chains ( κ FLC) and λ FLC, and sera κ FLC, as well as κ FLC, λ FLC, and κ IgG index, were significantly higher in patients with MS compared to those with other neurological disorders. The κ IgG index showed the highest diagnostic power in the detection of MS with both κ FLC index and κ IgG indexes showing the highest diagnostic sensitivity. This study provides novel information about the diagnostic significance of four markers combined in the κ IgG index [ 10 ] and shows that κ FLC and κ IgG combined in a novel algorithm may improve the detection and disease activity of MS (Figure 1). Cognitive function refers to a range of high-level brain functions, such as the ability to learn and remember information, solve problems, focus, concentration, attention, and verbal fluency. Change in cognitive function is common in patients with advanced MS. However, Pitteri et al, showed that newly diagnosed RRMS patients ( n = 50) performed worse than healthy controls ( n = 36), in particular, in the domains of memory and executive functioning [ 11 ]. These data demonstrate that reduced cognitive functioning can be present early on during the course of disease, even in patients without evidence of cognitive impairment. As such, the cognitive impairment criteria for patients with MS should be re-evaluated and be monitored closely throughout the course of disease (Figure 1). 3. Treatments for MS Treatments for MS include, interferon (IFN) beta-1a, IFN beta-1b (cytokines), fingolimod, ozanimod, siponimod (sphingosine-1-phosphate-receptor modulators), natalizumab (a monoclonal antibody against alpha4-integrin), dimethyl fumarate, glatiramer acetate, teriflunomide, cladribine, ocrelizumab (a humanized anti-CD20 monoclonal antibody) and, alemtuzumab (a humanized anti-CD52 monoclonal antibody) [ 1 , 2 ]. These drugs are focused on speeding recovery from relapse, slowing the progression of disease and managing MS symptoms, and in most cases, there are side e ff ects and patients need to stop treatment due to non-tolerance of the treatment. In rare cases, more severe adverse events occur. In fact, Buscarinu et al., presented a case report of a 45 year old Italian woman with RRMS on alemtuzumab treatment who showed immune thrombocytopenic purpura after the second injection of alemtuzumab. Three months following treatment, the patient presented with transient aphasia, cognitive deficits, and focal epilepsy, consistent encephalitis [ 12 ]. Autoimmune complications following alemtuzumab treatment are generally rare, with only one previous case being reported. Furthermore, Sachinvala et al. reported a male patient with MS, and co-morbid type-2 diabetes, major depression, asthma, developed post craniopharyngioma and cranial nerve-VI palsy. Magnetic resonance imaging, Humphrey’s visual filed and retinal nerve fiber thickening were used to determine changes to help the patient maintain productivity and mental state and mood (Figure 1) [ 13 ]. There is a need for the development of new treatment options which would stop progression and have little to no side e ff ects. Immune therapies have come a long way in recent years, with a number of methods being tested in pre-clinical and clinical settings, such as peptide / protein / DNA based vaccines, tolerogenic dendritic cells, T cell receptor peptide immunotherapy, monoclonal antibody 3 Brain Sci. 2020 , 10 , 795 therapies (anti-integrin a-4, anti-leucine rich repeat and immunoglobin-like domain-containing protein 1 (LINGO-1), anti-CD52), HLA antagonistic co-polymer therapies, cell specific immunotherapies, peptide-carrier conjugates, all of which are extensively reviewed by Kammona and Kiparissides [ 14 ] and Metaxakis et al. [ 15 ] (Figure 1). An editorial entitled, the long road of immunotherapeutics against MS [ 16 ], highlighted 20 years of MS research of an international multi-disciplinary consortia including peptide chemistry, medicinal chemistry, protein synthesis, protein–peptide interactions, nuclear magnetic imaging, molecular modeling, molecular dynamics, molecular biology, immunology, cell biochemistry, animal research and clinical research. This multi-disciplinary consortia led to at least 10 immunotherapeutic peptide-carrier candidates to be tested in human clinical trials. In preclinical studies, these peptide-based immune modulating conjugates showed a safety profile whilst switching immune responses from pro-inflammatory to anti-inflammatory and protection against experimental autoimmune encephalomyelitis (EAE) in mouse models [ 3 , 17 – 24 ]. Characterization of peptide-carrier conjugates was demonstrated using electrochemical voltametric techniques and high-pressure liquid chromatography [ 25 ]. In addition, nanoparticles have been used to deliver MS antigens to the immune system to tolerize T cells or stimulate an anti-inflammatory responses, reviewed by Chountoulesi and Demetzos [ 26 ]. More recently, chloroquine, an anti-malarial drug, was shown to suppress EAE in mice by modulating dendritic cells, Th17 cells, astrocytes, oligodendrocytes and microglia. Microglia cells were also shown to secrete IL-10 and IL-12p70. These data provide evidence that drug repurposing of chloroquine may be useful to patients with MS (Figure 1) [27]. In the last ten years, the incidence of MS has increased considerably, with lifestyle and environmental factors being one of the main contributors. An informative review by Boziki and Grigoriadis et al., provide the current advances in the gut-microbiome-immune–brain axis in patients with MS with altered microbiome, and present the e ff ects of MS treatments on gut microbiome (Figure 1) [ 28 ]. Thus, modification of gut microbiota by either dietary (such as, probiotics) or medicinal approaches is a promising approach for the management of MS. In fact, probiotics have been shown to have beneficial e ff ects not only in the gut flora but also in modulating and maintaining a healthy immune system. Certain probiotics have been shown to have anti-inflammatory e ff ects on immune cells (i.e., monocytes) and in disease settings, such as asthma and allergies [ 29 , 30 ]. The paper by Dargahi et al. showed that the probiotic Streptococcus thermophilus was able alter pro-inflammatory T cells responses against an agonist MBP 83–99 peptide to an anti-inflammatory profile (Figure 1) [ 31 ]. This study suggests that the consumption of Streptococcus thermophilus may be beneficial in the management and treatment of autoimmune diseases such as MS, and further research in this area is warranted. In addition to intravenous or oral steroids that are used as the first line of therapy for MS relapse, therapeutic plasma exchange, or plasmapheresis, is another method used to treat patients with neuromyelitis optica spectrum disorders, autoimmune encephalitis and MS, especially those with sudden, severe attacks or relapse / flare-ups. It is used in MS patients to manage disease by exchanging their plasma with ‘fresh’ plasma to remove pro-inflammatory cytokines and other proteins involved in auto-immune attack. In a study published by Moser et al., in this Special Issue, they compare the indications, e ffi cacy and safety of therapeutic plasma exchange treatment in MS, autoimmune encephalitis and other immune-mediated CNS disorders and noted consistent e ffi cacy and safety [ 32 ]. Measuring biomarkers of inflammation and oxidative stress is important to understand the e ffi cacy of treatments. As such, Moccia et al. studied 60 patients with RRMS who were treated with IFN beta-1a or Coenzyme Q10 and monitored patients for IL-1b, IL-2R, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-13, RANTES, tumor necrosis factor and uric acid (Figure 1) [ 33 ]. These serum biomarkers could be used to determine the e ffi cacy of treatments as well as their mechanisms of action. It is believed that transcranial magnetic stimulation motors with direct current stimulation (tDCS) intensities induce physiological changes to the brain, although the mechanism of action, as well as its validity and e ffi cacy, are not clear. In a pilot study by Workman and colleagues, they noted that there were no immediate changes in cerebral blood flow following direct current stimulation. Hence, further work is required to enable su ffi cient magnitudes of intracranial electrical fields to induce 4 Brain Sci. 2020 , 10 , 795 physiological changes in the brain to patients with MS (Figure 1) [ 34 ]. During disease progression, patients with MS develop walking limitations, and fampridine is usually recommended to improve gait. In the study by Ahdab et al., fampridine was evaluated for cortical excitability e ff ects and whether changes could predict therapeutic responses in 20 patients with MS and gait impairment [ 35 ]. It was noted that fampridine increased the excitatory intracortical processes, as shown by paired-pulse transcranial magnetic stimulation, suggesting that this could be used to select patients with MS who would be likely to experience a favorable response to fampridine (Figure 1) [35]. 4. Conclusions The development of drugs, immunotherapeutics and vaccines against diseases is a long process often taking researchers a lifetime. In this Special Issue, “Advances in Multiple Sclerosis Research—Series I”, a range of papers were published, including MS markers, treatments, detection, monitoring and the role of the microbiome in MS. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http: // creativecommons.org / licenses / by / 4.0 / ). 7 brain sciences Article Sample Size for Oxidative Stress and Inflammation When Treating Multiple Sclerosis with Interferon- β 1a and Coenzyme Q10 Marcello Moccia 1, *, Antonio Capacchione 2 , Roberta Lanzillo 1 , Fortunata Carbone 3,4 , Teresa Micillo 5 , Giuseppe Matarese 4,6 , Ra ff aele Palladino 7,8 and Vincenzo Brescia Morra 1 1 Multiple Sclerosis Clinical Care and Research Centre, Department of Neuroscience, Reproductive Science and Odontostomatology, Federico II University, 80131 Naples, Italy; robertalanzillo@libero.it (R.L.); vincenzo.bresciamorra2@unina.it (V.B.M.) 2 Medical A ff airs Department, Merck, 00176 Rome, Italy; antonio.capacchione@merckgroup.com 3 Neuroimmunology Unit, IRCCS Fondazione Santa Lucia, 00142 Rome, Italy; fortunata.carbone@alice.it 4 Laboratory of Immunology, Institute of Experimental Endocrinology and Oncology, National Research Council (IEOS-CNR), 80131 Naples, Italy; giuseppe.matarese@unina.it 5 Department of Biology, Federico II University, 80131 Naples, Italy; teresa.micillo2@unina.it 6 Treg Cell Lab, Department of Molecular Medicine and Medical Biotechnologies, Federico II University, 80131 Naples, Italy 7 Department of Primary Care and Public Health, Imperial College, London W68RP, UK; palladino.ra ff aele@gmail.com 8 Department of Public Health, Federico II University, 80131 Naples, Italy * Correspondence: moccia.marcello@gmail.com; Tel. / Fax: + 39-0817462670 Received: 23 August 2019; Accepted: 25 September 2019; Published: 27 September 2019 Abstract: Studying multiple sclerosis (MS) and its treatments requires the use of biomarkers for underlying pathological mechanisms. We aim to estimate the required sample size for detecting variations of biomarkers of inflammation and oxidative stress. This is a post-hoc analysis on 60 relapsing-remitting MS patients treated with Interferon- β 1a and Coenzyme Q10 for 3 months in an open-label crossover design over 6 months. At baseline and at the 3 and 6-month visits, we measured markers of scavenging activity, oxidative damage, and inflammation in the periphe