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In the recent past, a plethora of drugs have been approved for the treatment of multiple sclerosis (MS). These therapeutics are mainly confined to immunomodulatory or immunosuppressive strategies but do not sufficiently address remyelination and neuroprotection. However, several neuroregenerative agents have shown potential in pre-clinical research and entered Phase I to III clinical trials. Although none of these compounds have yet proceeded to approval, understanding the causes of failure can broaden our knowledge about neuroprotection and neuroregeneration in MS. Moreover, most of the investigated approaches are characterised by consistent mechanisms of action and proved convincing efficacy in animal studies. Therefore, learning from their failure will help us to enforce the translation of findings acquired in pre-clinical studies into clinical application. Here, we summarise trials on MS treatment published since 2015 that have either failed or were interrupted due to a lack of efficacy, adverse events, or for other reasons. We further outline the rationale underlying these drugs and analyse the background of failure to gather new insights into MS pathophysiology and optimise future study designs. For conciseness, this review focuses on agents promoting remyelination and medications with primarily neuroprotective properties or unconventional approaches. Failed clinical trials that pursue immunomodulation are presented in a separate article. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is a prototypical autoimmune disease of the neuromuscular junction (NMJ). The study of the underlying pathophysiology has provided novel insights into the interplay of autoantibodies and complement-mediated tissue damage. Experimental autoimmune myasthenia gravis (EAMG) emerged as a valuable animal model, designed to gain further insight and to test novel therapeutic approaches for MG. However, the availability of native acetylcholine receptor (AChR) protein is limited favouring the use of recombinant proteins. To provide a simplified platform for the study of MG, we established a model of EAMG using a recombinant protein containing the immunogenic sequence of AChR in mice. This model recapitulates key features of EAMG, including fatigable muscle weakness, the presence of anti-AChR-antibodies, and engagement of the NMJ by complement and a reduced NMJ density. Further characterization of this model demonstrated a prominent B cell immunopathology supported by T follicular helper cells. Taken together, the herein-presented EAMG model may be a valuable tool for the study of MG pathophysiology and the pre-clinical testing of therapeutic applications. [ABSTRACT FROM AUTHOR]

Myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model of multiple sclerosis. However, variations in the induction protocol can affect EAE progression, and may reduce the comparability of data. In the present study, we investigated the influence of the different components used for EAE induction in C57BL/6J mice on disease progression. In the present study, MOG 35–55 -induced chronic EAE in C57BL/6J mice has been applied as a model to challenge optimal pertussis toxin (PTx) dosing, while considering variations in batch potency. We demonstrate that the dosage of PTx, adjusted to its potency, influences EAE development in a dose-dependent manner. Our data show that with our protocol, which considers PTx potency, C57BL/6J mice consistently develop symptoms of EAE. The mice show a typical chronic course with symptom onset after 10.5 ± 1.08 days and maximum severity around day 16 postimmunization followed by a mild remission of symptoms. Previously studies reveal that alterations in PTx dosing directly modify EAE progression. Our present study highlights that PTx batches differ in potency, resulting in inconsistent EAE induction. We also provide a clear protocol that allows a reduction in the number of mice used in EAE experiments, while maintaining consistent results. Higher standards for comparability and reproducibility are needed to ensure and maximize the generation of reliable EAE data. Specifically, consideration of PTx potency. With our method of establishing consistent EAE pathogenesis, improved animal welfare standards and a reduction of mice used in experimentation can be achieved. • Pertussis toxin lots may vary in its potency, influencing the pathogenesis of EAE. • We titrated pertussis toxin to an optimal dose for a consistent EAE induction. • We provide a reliable protocol to achieve a higher degree of comparability. • This protocol helps to reduce the number of mice used in preclinical research. [ABSTRACT FROM AUTHOR]

• TREK and TRESK contribute significantly to the pathophysiology of pain symptoms, migraine, and inflammatory diseases. • Interactions of TREK and TRESK might involve heterodimerization and compensatory regulation of their subunit expression. • Insights into this interplay might provide innovative therapeutic targets. TWIK-related spinal cord potassium (TRESK) and TWIK-related potassium (TREK) channels are both subfamilies of the two-pore domain potassium (K2P) channel group. Despite major structural, pharmacological, as well as biophysical differences, emerging data suggest that channels of these two subfamilies are functionally more closely related than previously assumed. Recent studies, for instance, indicate an assembling of TRESK and TREK subunits, leading to the formation of heterodimeric channels with different functional properties compared to homodimeric ones. Formation of tandems consisting of TRESK and TREK subunits might thus multiply the functional diversity of both TRESK and TREK activity. Based on the involvement of these channels in the pathophysiology of migraine, we here highlight the role as well as the impact of the interplay of TRESK and TREK subunits in the context of different disease settings. In this regard, we focus on their involvement in migraine and pain syndromes, as well as on their influence on (neuro-)inflammatory processes. Furthermore, we describe the potential implications for innovative therapeutic strategies that take advantage of TRESK and TREK modulation as well as obstacles encountered in the development of therapies related to the aforementioned diseases. [ABSTRACT FROM AUTHOR]

Myasthenia gravis is a chronic antibody-mediated autoimmune disease disrupting neuromuscular synaptic transmission. Informative biomarkers remain an unmet need to stratify patients with active disease requiring intensified monitoring and therapy; their identification is the primary objective of this study. We applied mass spectrometry-based proteomic serum profiling for biomarker discovery. We studied an exploration and a prospective validation cohort consisting of 114 and 140 anti-acetylcholine receptor antibody (AChR-Ab)-positive myasthenia gravis patients, respectively. For downstream analysis, we applied a machine learning approach. Protein expression levels were confirmed by ELISA and compared to other myasthenic cohorts, in addition to myositis and neuropathy patients. Anti-AChR-Ab levels were determined by a radio receptor assay. Immunohistochemistry and immunofluorescence of intercostal muscle biopsies were employed for validation in addition to interactome studies of inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3). Machine learning identified ITIH3 as potential serum biomarker reflective of disease activity. Serum levels correlated with disease activity scores in the exploration and validation cohort and were confirmed by ELISA. Lack of correlation between anti-AChR-Ab levels and clinical scores underlined the need for biomarkers. In a subgroup analysis, ITIH3 was indicative of treatment responses. Immunostaining of muscle specimens from these patients demonstrated ITIH3 localization at the neuromuscular endplates in myasthenia gravis but not in controls, thus providing a structural equivalent for our serological findings. Immunoprecipitation of ITIH3 and subsequent proteomics lead to identification of its interaction partners playing crucial roles in neuromuscular transmission. This study provides data on ITIH3 as a potential pathophysiological-relevant biomarker of disease activity in myasthenia gravis. Future studies are required to facilitate translation into clinical practice. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) of still unclear etiology. In recent years, the search for biomarkers facilitating its diagnosis, prognosis, therapy response, and other parameters has gained increasing attention. In this regard, in a previous meta-analysis comprising 22 studies, we found that MS is associated with higher nitrite/nitrate (NOx) levels in the cerebrospinal fluid (CSF) compared to patients with non-inflammatory other neurological diseases (NIOND). However, many of the included studies did not distinguish between the different clinical subtypes of MS, included pre-treated patients, and inclusion criteria varied. As a follow-up to our meta-analysis, we therefore aimed to analyze the serum and CSF NOx levels in clinically well-defined cohorts of treatment-naïve MS patients compared to patients with somatic symptom disorder. To this end, we analyzed the serum and CSF levels of NOx in 117 patients (71 relapsing–remitting (RR) MS, 16 primary progressive (PP) MS, and 30 somatic symptom disorder). We found that RRMS and PPMS patients had higher serum NOx levels compared to somatic symptom disorder patients. This difference remained significant in the subgroup of MRZ-negative RRMS patients. In conclusion, the measurement of NOx in the serum might indeed be a valuable tool in supporting MS diagnosis. [ABSTRACT FROM AUTHOR]

Modulation of two-pore domain potassium (K2P) channels has emerged as a novel field of therapeutic strategies as they may regulate immune cell activation and metabolism, inflammatory signals, or barrier integrity. One of these ion channels is the TWIK-related potassium channel 1 (TREK1). In the current study, we report the identification and validation of new TREK1 activators. Firstly, we used a modified potassium ion channel assay to perform high-throughput-screening of new TREK1 activators. Dose-response studies helped to identify compounds with a high separation between effectiveness and toxicity. Inside-out patch-clamp measurements of Xenopus laevis oocytes expressing TREK1 were used for further validation of these activators regarding specificity and activity. These approaches yielded three substances, E1, B3 and A2 that robustly activate TREK1. Functionally, we demonstrated that these compounds reduce levels of adhesion molecules on primary human brain and muscle endothelial cells without affecting cell viability. Finally, we studied compound A2 via voltage-clamp recordings as this activator displayed the strongest effect on adhesion molecules. Interestingly, A2 lacked TREK1 activation in the tested neuronal cell type. Taken together, this study provides data on novel TREK1 activators that might be employed to pharmacologically modulate TREK1 activity. [ABSTRACT FROM AUTHOR]

Background: Cladribine is a synthetic purine analogue that interferes with DNA synthesis and repair next to disrupting cellular proliferation in actively dividing lymphocytes. The compound is approved for the treatment of multiple sclerosis (MS). Cladribine can cross the blood-brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. Here, we explored compartment-specific immunosuppressive as well as potential direct neuroprotective effects of oral cladribine treatment in experimental autoimmune encephalomyelitis (EAE) mice.Methods: In the current study, we compare immune cell frequencies and phenotypes in the periphery and CNS of EAE mice with distinct grey and white matter lesions (combined active and focal EAE) either orally treated with cladribine or vehicle, using flow cytometry. To evaluate potential direct neuroprotective effects, we assessed the integrity of the primary auditory cortex neuronal network by studying neuronal activity and spontaneous synaptic activity with electrophysiological techniques ex vivo.Results: Oral cladribine treatment significantly attenuated clinical deficits in EAE mice. Ex vivo flow cytometry showed that cladribine administration led to peripheral immune cell depletion in a compartment-specific manner and reduced immune cell infiltration into the CNS. Histological evaluations revealed no significant differences for inflammatory lesion load following cladribine treatment compared to vehicle control. Single cell electrophysiology in acute brain slices was performed and showed an impact of cladribine treatment on intrinsic cellular firing patterns and spontaneous synaptic transmission in neurons of the primary auditory cortex. Here, cladribine administration in vivo partially restored cortical neuronal network function, reducing action potential firing. Both, the effect on immune cells and neuronal activity were transient.Conclusions: Our results indicate that cladribine exerts a neuroprotective effect after crossing the blood-brain barrier independently of its peripheral immunosuppressant action. [ABSTRACT FROM AUTHOR]

Chronic inflammation of skeletal muscle is the common feature of idiopathic inflammatory myopathies (IIM). Given the rarity of the disease and potential difficulty of routinely obtaining target tissue, i.e., standardized skeletal muscle, our understanding of immune signatures of the IIM spectrum remains incomplete. Further insight into the immune topography of IIM is needed to determine specific treatment targets according to clinical and immunological phenotypes. Thus, we used high-dimensional flow cytometry to investigate the immune phenotypes of anti-synthetase syndrome (ASyS), dermatomyositis (DM) and inclusion-body myositis (IBM) patients as representative entities of the IIM spectrum and compared them to healthy controls. We studied the CD8, CD4 and B cell compartments in the blood aiming to provide a contemporary overview of the immune topography of the IIM spectrum. ASyS was characterized by altered CD4 composition and expanded T follicular helper cells supporting B cell-mediated autoimmunity. For DM, unsupervised clustering identified expansion of distinct B cell subtypes highly expressing immunoglobulin G4 (IgG4) and CD38. Lastly, terminally differentiated, cytotoxic CD8 T cells distinguish IBM from other IIM. Interestingly, these terminally differentiated CD8 T cells highly expressed the integrin CD18 mediating cellular adhesion and infiltration. The distinct immune cell topography of IIM might provide the framework for targeted treatment approaches potentially improving therapeutic outcomes. [ABSTRACT FROM AUTHOR]

Background: Myasthenic crisis (MC) and disease exacerbation in myasthenia gravis (MG) are associated with significant lethality and continue to impose a high disease burden on affected patients. Therefore, we sought to determine potential predictors for MC and exacerbation as well as to identify factors affecting outcome.Methods: We examined a retrospective, observational cohort study of patients diagnosed with MG between 2000 and 2021 with a mean follow-up of 62.6 months after diagnosis from eight tertiary hospitals in Germany. A multivariate Cox regression model with follow-up duration as the time variable was used to determine independent risk factors for MC and disease exacerbation.Results: 815 patients diagnosed with MG according to national guidelines were included. Disease severity at diagnosis (quantitative MG score or Myasthenia Gravis Foundation of America class), the presence of thymoma and anti-muscle specific tyrosine kinase-antibodies were independent predictors of MC or disease exacerbation. Patients with minimal manifestation status 12 months after diagnosis had a lower risk of MC and disease exacerbation than those without. The timespan between diagnosis and the start of immunosuppressive therapy did not affect risk. Patients with a worse outcome of MC were older, had higher MGFA class before MC and at admission, and had lower vital capacity before and at admission. The number of comorbidities, requirement for intubation, prolonged mechanical ventilation, and MC triggered by infection were associated with worse outcome. No differences between outcomes were observed comparing treatments with IVIG (intravenous immunoglobulin) vs. plasma exchange vs. IVIG together with plasma exchange.Conclusions: MC and disease exacerbations inflict a substantial burden of disease on MG patients. Disease severity at diagnosis and antibody status predicted the occurrence of MC and disease exacerbation. Intensified monitoring with emphasis on the prevention of infectious complications could be of value to prevent uncontrolled disease in MG patients. [ABSTRACT FROM AUTHOR]

Aside from the established immune-mediated etiology of multiple sclerosis (MS), compelling evidence implicates platelets as important players in disease pathogenesis. Specifically, numerous studies have highlighted that activated platelets promote the central nervous system (CNS)-directed adaptive immune response early in the disease course. Platelets, therefore, present a novel opportunity for modulating the neuroinflammatory process that characterizes MS. We hypothesized that the well-known antiplatelet agent acetylsalicylic acid (ASA) could inhibit neuroinflammation by affecting platelets if applied at low-dose and investigated its effect during experimental autoimmune encephalomyelitis (EAE) as a model to study MS. We found that oral administration of low-dose ASA alleviates symptoms of EAE accompanied by reduced inflammatory infiltrates and less extensive demyelination. Remarkably, the percentage of CNS-infiltrated CD4+ T cells, the major drivers of neuroinflammation, was decreased to 40.98 ± 3.28% in ASA-treated mice compared to 56.11 ± 1.46% in control animals at the disease maximum as revealed by flow cytometry. More interestingly, plasma levels of thromboxane A2 were decreased, while concentrations of platelet factor 4 and glycoprotein VI were not affected by low-dose ASA treatment. Overall, we demonstrate that low-dose ASA could ameliorate the platelet-dependent neuroinflammatory response in vivo, thus indicating a potential treatment approach for MS. [ABSTRACT FROM AUTHOR]

K 2P 2.1 (TREK1), a two-pore domain potassium channel, has emerged as regulator of leukocyte transmigration into the central nervous system. In the context of skeletal muscle, immune cell infiltration constitutes the pathogenic hallmark of idiopathic inflammatory myopathies (IIMs). However, the underlying mechanisms remain to be elucidated. In this study, we investigated the role of K 2P 2.1 in the autoimmune response of IIMs. We detected K 2P 2.1 expression in primary skeletal muscle and endothelial cells of murine and human origin. We observed an increased pro-inflammatory cell response, adhesion and transmigration by pharmacological blockade or genetic deletion of K 2P 2.1 in vitro and in in vivo myositis mouse models. Of note, our findings were not restricted to endothelial cells as skeletal muscle cells with impaired K 2P 2.1 function also demonstrated a strong pro-inflammatory response. Conversely, these features were abrogated by activation of K 2P 2.1 and improved the disease course of a myositis mouse model. In humans, K 2P 2.1 expression was diminished in IIM patients compared to non-diseased controls arguing for the translatability of our findings. In summary, K 2P 2.1 may regulate the inflammatory response of skeletal muscle. Further research is required to understand whether K 2P 2.1 could serve as novel therapeutic target. • K 2P 2.1 (TREK1) is a potassium channel with immunoregulatory properties. • Immune cell infiltration is a pathophysiological hallmark of idiopathic inflammatory myopathies. • K 2P 2.1 regulates immune cell migration across the myovascular barrier, providing a potentially novel therapeutic target. [ABSTRACT FROM AUTHOR]

In the past decades, multiple sclerosis (MS) treatment has experienced vast changes resulting from major advances in disease-modifying therapies (DMT). Looking at the overall number of studies, investigations with therapeutic advantages and encouraging results are exceeded by studies of promising compounds that failed due to either negative or inconclusive results or have been interrupted for other reasons. Importantly, these failed clinical trials are informative experiments that can help us to understand the pathophysiological mechanisms underlying MS. In several trials, concepts taken from experimental models were not translatable to humans, although they did not lack a well-considered pathophysiological rationale. The lessons learned from these discrepancies may benefit future studies and reduce the risks for patients. This review summarizes trials on MS since 2015 that have either failed or have been interrupted for various reasons. We identify potential causes of failure or inconclusiveness, looking at the path from basic animal experiments to clinical trials, and discuss the implications for our current view on MS pathogenesis, clinical practice, and future study designs. We focus on anti-inflammatory treatment strategies, without including studies on already approved and effective DMT. Clinical trials addressing neuroprotective and alternative treatment strategies are presented in a separate article. [ABSTRACT FROM AUTHOR]