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Abstract Background Immune dysregulation is a hallmark of autoimmune diseases of the central nervous system (CNS), characterized by an excessive immune response, and primary CNS tumors (pCNS-tumors) showing a highly immunosuppressive parenchymal microenvironment. Methods Aiming to provide novel insights into the pathogenesis of CNS autoimmunity and cerebral tumor immunity, we analyzed the peripheral blood (PB) and cerebrospinal fluid (CSF) of 81 autoimmune limbic encephalitis (ALE), 148 relapsing–remitting multiple sclerosis (RRMS), 33 IDH-wildtype glioma, 9 primary diffuse large B cell lymphoma of the CNS (CNS-DLBCL), and 110 controls by flow cytometry (FC). Additionally, an in-depth immunophenotyping of the PB from an independent cohort of 20 RRMS and 18 IDH-wildtype glioblastoma patients compared to 19 controls was performed by FC combined with unsupervised computational approaches. Results We identified alterations in peripheral and intrathecal adaptive immunity, mainly affecting the T cell (Tc) but also the B cell (Bc) compartment in ALE, RRMS, and pCNS-tumors compared to controls. ALE, RRMS, and pCNS-tumors featured higher expression of the T cell activation marker HLA-DR, which was even more pronounced in pCNS-tumors than in ALE or RRMS. Glioblastoma patients showed signs of T cell exhaustion that were not visible in RRMS patients. In-depth characterization of the PB revealed differences mainly in the T effector and memory compartment between RRMS and glioblastoma patients and similar alterations in the Bc compartment, including atypical Bc, CD19+CD20− double negative Bc, and plasma cells. PB and CSF mFC together with CSF routine parameters could reliably differentiate ALE and RRMS from pCNS-tumors facilitating early diagnosis and treatment. Conclusions ALE, RRMS, and pCNS-tumors show distinct but partially overlapping changes mainly in HLA-DR+ Tc, memory Tc, exhausted Tc, and Bc subsets providing insights into disease pathogenesis. Moreover, mFC shows diagnostic potential facilitating early diagnosis and treatment. Graphical Abstract

Abstract To gain a deeper understanding of skeletal muscle function in younger age and aging in elderly, identification of molecular signatures regulating these functions under physiological conditions is needed. Although molecular studies of healthy muscle have been conducted on adults and older subjects, there is a lack of research on infant muscle in terms of combined morphological, transcriptomic and proteomic profiles. To address this gap of knowledge, we performed RNA sequencing (RNA-seq), tandem mass spectrometry (LC–MS/MS), morphometric analysis and assays for mitochondrial maintenance in skeletal muscle biopsies from both, infants aged 4–28 months and adults aged 19–65 years. We identified differently expressed genes (DEGs) and differentially expressed proteins (DEPs) in adults compared to infants. The down-regulated genes in adults were associated with functional terms primarily related to sarcomeres, cellular maintenance, and metabolic, immunological and developmental processes. Thus, our study indicates age-related differences in the molecular signatures and associated functions of healthy skeletal muscle. Moreover, the findings assert that processes previously associated solely with aging are indeed part of development and healthy aging. Hence, combined findings of this study also indicate that age-dependent controls are crucial in muscle disease studies, as otherwise the comparative results may not be reliable.

Abstract 5q-associated spinal muscular atrophy (SMA) is a motoneuron disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Adaptive immunity may contribute to SMA as described in other motoneuron diseases, yet mechanisms remain elusive. Nusinersen, an antisense treatment, enhances SMN2 expression, benefiting SMA patients. Here we have longitudinally investigated SMA and nusinersen effects on local immune responses in the cerebrospinal fluid (CSF) - a surrogate of central nervous system parenchyma. Single-cell transcriptomics (SMA: N = 9 versus Control: N = 9) reveal NK cell and CD8+ T cell expansions in untreated SMA CSF, exhibiting activation and degranulation markers. Spatial transcriptomics coupled with multiplex immunohistochemistry elucidate cytotoxicity near chromatolytic motoneurons (N = 4). Post-nusinersen treatment, CSF shows unaltered protein/transcriptional profiles. These findings underscore cytotoxicity’s role in SMA pathogenesis and propose it as a therapeutic target. Our study illuminates cell-mediated cytotoxicity as shared features across motoneuron diseases, suggesting broader implications.

Abstract Introduction Chronic inflammatory demyelinating polyneuropathy (CIDP) is one of the most common immune neuropathies leading to severe impairments in daily life. Current treatment options include intravenous immunoglobulins (IVIG), which are administered at intervals of 4–12 weeks. Determination of individual treatment intervals is challenging since existing clinical scores lack sensitivity to objectify small, partially fluctuating deficits in patients. End-of-dose phenomena described by patients, manifested by increased fatigue and worsening of (motor) symptoms, are currently difficult to detect. From a medical and socio-economic point of view, it is necessary to identify and validate new, more sensitive outcome measures for accurate mapping of disease progression and, thus, for interval finding. Digital health technologies such as wearables may be particularly useful for this purpose, as they record real-life data and consequently, in contrast to classic clinical 'snapshots’, can continuously depict the disease course. Methods In this prospective, observational, non-interventional, single-center, investigator-initiated study, CIDP patients treated with IVIG will be continuously monitored over a period of 6 months. Clinical scores and blood analyses will be assessed and collected during three visits (V1, V2, V3). Additionally, activity, sleep, and cardiac parameters will be recorded over the entire period using a wearable device. Further, patients' subjective disease development and quality of life will be recorded at various visits (read-outs). The usability of the smartwatch will be assessed at the end of the study. Perspective The study aims to evaluate different digital measurements obtained with the smartwatch and blood-based analyses for monitoring disease activity and progress in CIDP patients. In conjunction, both means of monitoring might offer detailed insights into behavioral and biological patterns associated with treatment-related fluctuations such as end-of-dose phenomena. Trial registration The study protocol was registered at ClinicalTrials.gov. Identifier: NCT05723848. Initially, the protocol was submitted prospectively on January 10, 2023. The trial was publicly released after formal improvements on February 13, 2023, after first patients were included according to the original protocol.

Abstract The TWIK-related spinal cord K + channel (TRESK, K2P18.1) is a K2P channel contributing to the maintenance of membrane potentials in various cells. Recently, physiological TRESK function was identified as a key player in T-cell differentiation rendering the channel a new pharmacological target for treatment of autoimmune diseases. The channel activator cloxyquin represents a promising lead compound for the development of a new class of immunomodulators. Identification of cloxyquin binding site and characterization of the molecular activation mechanism can foster the future drug development. Here, we identify the cloxyquin binding site at the M2/M4 interface by mutational scan and analyze the molecular mechanism of action by protein modeling as well as in silico and in vitro electrophysiology using different permeating ion species (K+ / Rb+). In combination with kinetic analyses of channel inactivation, our results suggest that cloxyquin allosterically stabilizes the inner selectivity filter facilitating the conduction process subsequently activating hTRESK.

Abstract Background Myasthenia gravis (MG) is a rare neuromuscular disorder. Symptoms can range from ptosis only to life threatening myasthenic crisis. Thymectomy is recommended for anti-acetylcholine receptor-antibody positive patients with early-onset MG. Here, we investigated prognostic factors shaping therapeutic outcomes of thymectomy to improve patient stratification. Methods We retrospectively collected single-center data from a specialized center for MG from all consecutive adult patients that underwent thymectomy from 01/2012 to 12/2020. We selected patients with thymoma-associated and non-thymomatous MG for further investigations. We analyzed the patient collective regarding perioperative parameters in relation to the surgical approach. Furthermore, we investigated the dynamics of the anti-acetylcholine receptor-antibody titers and concurrent immunosuppressive therapies, as well as the therapeutic outcomes in dependence of clinical classifications. Results Of 137 patients 94 were included for further analysis. We used a minimally invasive approach in 73 patients, whereas 21 patients underwent sternotomy. A total of 45 patients were classified as early-onset MG (EOMG), 28 as late-onset MG (LOMG) and 21 as thymoma-associated MG (TAMG). The groups differed in terms of age at diagnosis (EOMG: 31.1 ± 12.2 years; LOMG: 59.8 ± 13.7 years; TAMG: 58.6 ± 16.7 years; p

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 Background The medical care of patients with myositis is a great challenge in clinical practice. This is due to the rarity of these disease, the complexity of diagnosis and management as well as the lack of systematic analyses. Objectives Therefore, the aim of this project was to obtain an overview of the current care of myositis patients in Germany and to evaluate epidemiological trends in recent years. Methods In collaboration with BARMER Insurance, retrospective analysis of outpatient and inpatient data from an average of approximately 8.7 million insured patients between January 2005 and December 2019 was performed using ICD-10 codes for myositis for identification of relevant data. In addition, a comparative analysis was performed between myositis patients and an age-matched comparison group from other populations insured by BARMER. Results 45,800 BARMER-insured individuals received a diagnosis of myositis during the observation period, with a relatively stable prevalence throughout. With regard to comorbidities, a significantly higher rate of cardiovascular disease as well as neoplasm was observed compared to the control group within the BARMER-insured population. In addition, myositis patients suffer more frequently from psychiatric disorders, such as depression and somatoform disorders. However, the ICD-10 catalogue only includes the specific coding of “dermatomyositis” and “polymyositis” and thus does not allow for a sufficient analysis of all idiopathic inflammatory myopathies subtypes. Conclusion The current data provide a comprehensive epidemiological analysis of myositis in Germany, highlighting the multimorbidity of myositis patients. This underlines the need for multidisciplinary management. However, the ICD-10 codes currently still in use do not allow for specific analysis of the subtypes of myositis. The upcoming ICD-11 coding may improve future analyses in this regard.

Abstract Background Therapeutic options targeting inflammation in multiple sclerosis (MS) have evolved rapidly for relapsing–remitting MS, whereas few therapies are available for progressive forms of MS, in particular secondary progressive MS (SPMS). The approval of siponimod for SPMS has allowed for optimism in the otherwise discouraging therapeutic landscape. Methods We conducted a retrospective, multicenter, non-interventional study analyzing the efficacy and safety of siponimod under real-world conditions in 227 SPMS patients. According to the retrospective study framework, data was acquired at prespecified time points. Clinical readouts were assessed every three months. Disease progression was determined as increase in expanded disability status scale (EDSS), radiological progression, or the occurrence of new relapses under treatment. For safety analyses, adverse events (AE) and reasons for discontinuation were documented. The collected data points were analyzed at baseline and after 6, 12 and 18 months. However, data were predominately collected at the 6- and 12-month time points as many patients were lost to follow-up. In a group consisting of 41 patients, a more detailed investigation regarding disease progression was conducted, including data from measurement of cognitive and motoric functions. Results Under siponimod therapy, 64.8% of patients experienced sustained clinical disease stability at 12 months. Out of the stable patients 21.4% of patients improved. Of the remaining patients, 31.5% experienced EDSS progression, 3.7% worsened without meeting the threshold for progression. Relapses occurred in 7.4%. Radiological disease activity was detected in 24.1% of patients after six months of treatment and in 29.6% of patients at 12 months follow-up. The in-depth cohort consisting of 41 patients demonstrated no substantial changes in cognitive abilities measured by Paced Auditory Serial Addition Test and Symbol Digit Modalities Test or motoric functions measured with Timed 25-Foot Walk, 100-m timed test, and 9-Hole Peg Test throughout the 12-month study period. Radiological assessment showed a stable volume of white and grey matter, as well as a stable lesion count at 12 months follow-up. AE were observed in nearly half of the included patients, with lymphopenia being the most common. Due to disease progression or AE, 31.2% of patients discontinued therapy. Conclusion Treatment with siponimod had an overall stabilizing effect regarding clinical and radiological outcome measures. However, there is a need for more intensive treatment management and monitoring to identify disease progression and AE.

Abstract 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 Objective Intravenous methylprednisolone is the standard treatment for a multiple sclerosis relapse; however, this fails to improve symptoms in up to one quarter of patients. Immunoadsorption is an accepted treatment for refractory relapses, but prospective comparator-controlled studies are missing. Methods In this observational study, patients with steroid-refractory acute multiple sclerosis relapses receiving either six courses of tryptophan-immunoadsorption or double-dose methylprednisolone therapy were analysed. Outcomes were evaluated at discharge and three months later. Immune profiling of blood lymphocytes and proteomic analysis were performed by multi-parameter flow cytometry and Olink analysis, respectively (NCT04450030). Results 42 patients were enrolled (methylprednisolone: 26 patients; immunoadsorption: 16 patients). For determination of the primary outcome, treatment response was stratified according to relative function system score changes (“full/best” vs. “average” vs. “worse/none”). Upon discharge, the adjusted odds ratio for any treatment response (“full/best” + ”average” vs. “worse/none”) was 10.697 favouring immunoadsorption (p = 0.005 compared to methylprednisolone). At follow-up, the adjusted odds ratio for the best treatment response (“full/best” vs. “average” + ”worse/none”) was 103.236 favouring IA patients (p = 0.001 compared to methylprednisolone). Similar results were observed regarding evoked potentials and quality of life outcomes, as well as serum neurofilament light-chain levels. Flow cytometry revealed a profound reduction of B cell subsets following immunoadsorption, which was closely correlated to clinical outcomes, whereas methylprednisolone had a minimal effect on B cell populations. Immunoadsorption treatment skewed the blood cytokine network, reduced levels of B cell-related cytokines and reduced immunoglobulin levels as well as levels of certain coagulation factors. Interpretation Immunoadsorption demonstrated favourable outcomes compared to double-dose methylprednisolone. Outcome differences were significant at discharge and follow-up. Further analyses identified modulation of B cell function as a potential mechanism of action for immunoadsorption, as reduction of B cell subsets correlated with clinical improvement.

IntroductionIn multiple sclerosis (MS), chronic disability primarily stems from axonal and neuronal degeneration, a condition resistant to conventional immunosuppressive or immunomodulatory treatments. Recent research has indicated that selective sphingosine-1-phosphate receptor S1PR-1 and -5 modulators yield positive effects in progressive MS and mechanistic models of inflammation-driven neurodegeneration and demyelination. MethodsIn this study, the S1PR-1/-5 modulator RP-101074 was evaluated as a surrogate for ozanimod in the non-inflammatory, primary degenerative animal model of light-induced photoreceptor loss (LI-PRL) in CX3CR1-GFP mice to assess potential neuroprotective effects, independent of its immunomodulatory mechanism of action.ResultsProphylactic administration of RP-101074 demonstrated protective effects in the preclinical, non-inflammatory LI-PRL animal model, following a bell-shaped dose-response curve. RP-101074 treatment also revealed activity-modulating effects on myeloid cells, specifically, CX3CR1+ cells, significantly reducing the marked infiltration occurring one week post-irradiation. Treatment with RP-101074 produced beneficial outcomes on both retinal layer thickness and visual function as evidenced by optical coherence tomography (OCT) and optomotor response (OMR) measurements, respectively. Additionally, the myelination status and the quantity of neural stem cells in the optic nerve suggest that RP-101074 may play a role in the activation and/or recruitment of neural stem cells and oligodendrocyte progenitor cells, respectively.Conclusion/DiscussionThe data from our study suggest that RP-101074 may have a broader role in MS treatment beyond immunomodulation, potentially offering a novel approach to mitigate neurodegeneration, a core contributor to chronic disability in MS.

IntroductionGiven its wide availability and cost-effectiveness, multidimensional flow cytometry (mFC) became a core method in the field of immunology allowing for the analysis of a broad range of individual cells providing insights into cell subset composition, cellular behavior, and cell-to-cell interactions. Formerly, the analysis of mFC data solely relied on manual gating strategies. With the advent of novel computational approaches, (semi-)automated gating strategies and analysis tools complemented manual approaches.MethodsUsing Bayesian network analysis, we developed a mathematical model for the dependencies of different obtained mFC markers. The algorithm creates a Bayesian network that is a HC tree when including raw, ungated mFC data of a randomly selected healthy control cohort (HC). The HC tree is used to classify whether the observed marker distribution (either patients with amyotrophic lateral sclerosis (ALS) or HC) is predicted. The relative number of cells where the probability q is equal to zero is calculated reflecting the similarity in the marker distribution between a randomly chosen mFC file (ALS or HC) and the HC tree.ResultsIncluding peripheral blood mFC data from 68 ALS and 35 HC, the algorithm could correctly identify 64/68 ALS cases. Tuning of parameters revealed that the combination of 7 markers, 200 bins, and 20 patients achieved the highest AUC on a significance level of p < 0.0001. The markers CD4 and CD38 showed the highest zero probability. We successfully validated our approach by including a second, independent ALS and HC cohort (55 ALS and 30 HC). In this case, all ALS were correctly identified and side scatter and CD20 yielded the highest zero probability. Finally, both datasets were analyzed by the commercially available algorithm ‘Citrus’, which indicated superior ability of Bayesian network analysis when including raw, ungated mFC data.DiscussionBayesian network analysis might present a novel approach for classifying mFC data, which does not rely on reduction techniques, thus, allowing to retain information on the entire dataset. Future studies will have to assess the performance when discriminating clinically relevant differential diagnoses to evaluate the complementary diagnostic benefit of Bayesian network analysis to the clinical routine workup.

Therapeutic strategies targeting complement have revolutionized the treatment of myasthenia gravis (MG). However, a deeper understanding of complement modulation in the human system is required to improve treatment responses and identify off-target effects shaping long-term outcomes. For this reason, we studied a cohort of patients with MG treated with either eculizumab or azathioprine as well as treatment-naive patients using a combined proteomics and metabolomics approach. This strategy validated known effects of eculizumab on the terminal complement cascade. Beyond that, eculizumab modulated the serum proteometabolome as distinct pathways were altered in eculizumab-treated patients, including the oxidative stress response, mitogen-activated protein kinase signaling, and lipid metabolism with particular emphasis on arachidonic acid signaling. We detected reduced levels of arachidonate 5-lipoxygenase (ALOX5) and leukotriene A4 in eculizumab-treated patients. Mechanistically, ligation of the C5a receptor (C5aR) is needed for ALOX5 metabolism and generation of downstream leukotrienes. As eculizumab prevents cleavage of C5 into C5a, decreased engagement of C5aR may inhibit ALOX5-mediated synthesis of pro-inflammatory leukotrienes. These findings indicate distinct off-target effects induced by eculizumab, illuminating potential mechanisms of action that may be harnessed to improve treatment outcomes.

Background Typically defined as a thromboinflammatory disease, ischemic stroke features early and delayed inflammatory responses, which determine the extent of ischemia‐related brain damage. T and natural killer cells have been implicated in neuronal cytotoxicity and inflammation, but the precise mechanisms of immune cell‐mediated stroke progression remain poorly understood. The activating immunoreceptor NKG2D is expressed on both natural killer and T cells and may be critically involved. Methods and Results An anti‐NKG2D blocking antibody alleviated stroke outcome in terms of infarct volume and functional deficits, coinciding with reduced immune cell infiltration into the brain and improved survival in the animal model of cerebral ischemia. Using transgenic knockout models devoid of certain immune cell types and immunodeficient mice supplemented with different immune cell subsets, we dissected the functional contribution of NKG2D signaling by different NKG2D‐expressing cells in stroke pathophysiology. The observed effect of NKG2D signaling in stroke progression was shown to be predominantly mediated by natural killer and CD8+ T cells. Transfer of T cells with monovariant T‐cell receptors into immunodeficient mice with and without pharmacological blockade of NKG2D revealed activation of CD8+ T cells irrespective of antigen specificity. Detection of the NKG2D receptor and its ligands in brain samples of patients with stroke strengthens the relevance of preclinical observations in human disease. Conclusions Our findings provide a mechanistic insight into NKG2D‐dependent natural killer– and T‐cell–mediated effects in stroke pathophysiology.

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 Inclusion body myositis (IBM) is the most prevalent idiopathic inflammatory myopathy (IIM) affecting older adults. The pathogenic hallmark of IBM is chronic inflammation of skeletal muscle. At present, we do not classify IBM into different sub-entities, with the exception perhaps being the presence or absence of the anti-cN-1A-antibody. In contrast to other IIM, IBM is characterized by a chronic and progressive disease course. Here, we discuss the pathophysiological framework of IBM and highlight the seemingly prototypical situations where IBM occurs in the context of other diseases. In this context, understanding common immune pathways might provide insight into the pathogenesis of IBM. Indeed, IBM is associated with a distinct set of conditions, such as human immunodeficiency virus (HIV) or hepatitis C—two conditions associated with premature immune cell exhaustion. Further, the pathomorphology of IBM is reminiscent of other muscle diseases, notably HIV-associated myositis or granulomatous myositis. Distinct immune pathways are likely to drive these commonalities and senescence of the CD8+ T cell compartment is discussed as a possible mechanism of pathogenesis. Future effort directed at understanding the co-occurrence of IBM and associated diseases could prove valuable to better understand the enigmatic IBM pathophysiology.

Abstract 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. Graphical Abstract

Inflammatory neuromuscular disorders encompass a diverse group of immune-mediated diseases with varying clinical manifestations and treatment responses. The identification of specific biomarkers has the potential to provide valuable insights into disease pathogenesis, aid in accurate diagnosis, predict disease course, and monitor treatment efficacy. However, the rarity and heterogeneity of these disorders pose significant challenges in the identification and implementation of reliable biomarkers. Here, we aim to provide a comprehensive review of biomarkers currently established in Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), myasthenia gravis (MG), and idiopathic inflammatory myopathy (IIM). It highlights the existing biomarkers in these disorders, including diagnostic, prognostic, predictive and monitoring biomarkers, while emphasizing the unmet need for additional specific biomarkers. The limitations and challenges associated with the current biomarkers are discussed, and the potential implications for disease management and personalized treatment strategies are explored. Collectively, biomarkers have the potential to improve the management of inflammatory neuromuscular disorders. However, novel strategies and further research are needed to establish clinically meaningful biomarkers.

Background & Aims: The 2-pore potassium channel subfamily K member 9 (KCNK9) regulates intracellular calcium concentration and thus modulates cell survival and inflammatory signaling pathways. It also was recognized as a risk allele for inflammatory bowel disease. However, it remains unclear whether KCNK9 modulates inflammatory bowel disease via its impact on immune cell function or whether its influence on calcium homeostasis also is relevant in intestinal epithelial cells. Methods: Kcnk9-/- mice were challenged with 3% dextran sulfate sodium (DSS) to induce experimental acute colitis. Primary cultures of intestinal epithelial cells were generated, and expression of potassium channels as well as cytosolic calcium levels and susceptibility to apoptosis were evaluated. Furthermore, we evaluated whether KCNK9 deficiency was compensated by the closely related 2-pore potassium channel KCNK3 in vivo or in vitro. Results: Compared with controls, KCNK9 deficiency or its pharmacologic blockade were associated with aggravated DSS-induced colitis compared with wild-type animals. In the absence of KCNK9, intestinal epithelial cells showed increased intracellular calcium levels and were more prone to mitochondrial damage and caspase-9–dependent apoptosis. We found that expression of KCNK3 was increased in Kcnk9-/- mice but did not prevent apoptosis after DSS exposure. Conversely, increased levels of KCNK9 in Kcnk3-/- mice were associated with an ameliorated course of DSS-induced colitis. Conclusions: KCNK9 enhances mitochondrial stability, reduces apoptosis, und thus supports epithelial cell survival after DSS exposure in vivo and in vitro. Conversely, its increased expression in Kcnk3-/- resulted in less mitochondrial damage and apoptosis and was associated with beneficial outcomes in DSS-induced colitis.

Objective Due to the growing complexity in monitoring and treatment of many disorders, disease-specific care and research networks offer patients certified healthcare. However, the networks’ ability to provide health services close to patients’ homes usually remains vague. Digital Health Technologies (DHTs) help to provide better care, especially if implemented in a targeted manner in regions undersupplied by specialised networks. Therefore, we used a car travel time-based isochrone approach to identify care gaps using the example of the neuroinflammation-focused German healthcare and research networks for multiple sclerosis (MS), myasthenia gravis (MG), myositis and immune-mediated neuropathy. Methods Excellence centres were mapped, and isochrones for 30, 60, 90 and 120 minutes were calculated. The resulting geometric figures were aggregated and used to mask the global human settlement population grid 2019 to estimate German inhabitants that can reach centres within the given periods. Results While 96.48% of Germans can drive to an MS-focused centre within one hour, coverage is lower for the rare disease networks for MG (48.3%), myositis (43.1%) and immune-mediated neuropathy (56.7%). Within 120 minutes, more than 80% of Germans can reach a centre of any network. Besides the generally worse covered rural regions such as North-Eastern Germany, the rare disease networks also show network-specific regional underrepresentation. Conclusion An isochrone-based approach helps identify regions where specialised care is hard to reach, which might be especially troublesome in the case of an often disabled patient collective. Patient care could be improved by focusing deployments of disease-specific DHTs on these areas.

IntroductionGiven the varying severity of coronavirus disease 2019 (COVID-19) and the rapid spread of Severe-Acute-Respiratory-Syndrome-Corona-Virus-2 (SARS-CoV-2), vaccine-mediated protection of particularly vulnerable individuals has gained increasing attention during the course of the pandemic.MethodsWe performed a 1-year follow-up study of 51 ocrelizumab-treated patients with multiple sclerosis (OCR-pwMS) who received COVID-19 vaccination in 2021. We retrospectively identified 37 additional OCR-pwMS, 42 pwMS receiving natalizumab, 27 pwMS receiving sphingosine 1-phosphate receptor modulators, 59 pwMS without a disease-modifying therapy, and 61 controls without MS (HC). In OCR-pwMS, anti-SARS-CoV-2(S)-antibody titers were measured prior to the first and after the second, third, and fourth vaccine doses (pv2/3/4). The SARS-CoV-2-specific T cell response was analyzed pv2. SARS-CoV-2 infection status, COVID-19 disease severity, and vaccination-related adverse events were assessed in all pwMS and HC.ResultsWe found a pronounced and increasing anti-SARS-CoV-2(S)-antibody response after COVID-19 booster vaccinations in OCR-pwMS (pv2: 30.4%, pv3: 56.5%, and pv4 90.0% were antibody positive). More than one third of OCR-pwMS without detectable antibodies pv2 developed positive antibodies pv3. 23.5% of OCR-pwMS had a confirmed SARS-CoV-2 infection, of which 84.2% were symptomatic. Infection rates were comparable between OCR-pwMS and control groups. None of the pwMS had severe COVID-19. An attenuated humoral immune response was not associated with a higher risk of SARS-CoV-2 infection.DiscussionAdditional COVID-19 vaccinations can boost the humoral immune response in OCR-pwMS and improve clinical protection against COVID-19. Vaccines effectively protect even OCR-pwMS without a detectable COVID-19 specific humoral immune response, indicating compensatory, e.g., T cell-mediated immunological mechanisms.

Vascularization of tissue-engineered constructs remains a key challenge in the field of skeletal muscle tissue engineering. One strategy for vascularizing organoids is in vitro pre-vascularization, relying on de novo assembly of undifferentiated endothelial cells into capillaries, a process termed vasculogenesis. In most endothelial cell research to date, human umbilical vein endothelial cells have been used primarily because of their availability. Nevertheless, this endothelial cell type is naturally not occurring in skeletal muscle tissue. Since endothelial cells display a tissue-specific phenotype, it is of interest to use muscle-specific microvascular endothelial cells to study pre-vascularization in skeletal muscle tissue engineering research. Thus far, tissue biopsies had to be processed in two separate protocols to obtain cells from the myogenic and the endothelial compartment. Here, we describe a novel, detailed protocol for the co-isolation of human skeletal muscle microvascular endothelial cells and satellite cell-derived myoblasts. It incorporates an automated mechanical and enzymatic tissue dissociation followed by magnetically activated cell sorting based on a combination of endothelial and skeletal muscle cell markers. Qualitative, quantitative, and functional characterization of the obtained cells is described and demonstrated by representative results. The simultaneous isolation of both cell types from the same donor is advantageous in terms of time efficiency. In addition, it may be the only possible method to isolate both cell types as the amount of tissue biopsy is often limited. The isolation of the two cell types is crucial for further studies to elucidate cell crosstalk in health and disease. Furthermore, the use of muscle-specific microvascular endothelial cells allows a shift towards engineering more physiologically relevant functional tissue, with downstream applications including drug screening and regenerative medicine.

Background“Presbyphagia” refers to characteristic age-related changes in the complex neuromuscular swallowing mechanism. It has been hypothesized that cumulative impairments in multiple domains affect functional reserve of swallowing with age, but the multifactorial etiology and postulated compensatory strategies of the brain are incompletely understood. This study investigates presbyphagia and its neural correlates, focusing on the clinical determinants associated with adaptive neuroplasticity.Materials and methods64 subjects over 70 years of age free of typical diseases explaining dysphagia received comprehensive workup including flexible endoscopic evaluation of swallowing (FEES), magnetoencephalography (MEG) during swallowing and pharyngeal stimulation, volumetry of swallowing muscles, laboratory analyzes, and assessment of hand-grip-strength, nutritional status, frailty, olfaction, cognition and mental health. Neural MEG activation was compared between participants with and without presbyphagia in FEES, and associated clinical influencing factors were analyzed. Presbyphagia was defined as the presence of oropharyngeal swallowing alterations e.g., penetration, aspiration, pharyngeal residue pooling or premature bolus spillage into the piriform sinus and/or laryngeal vestibule.Results32 of 64 participants showed swallowing alterations, mainly characterized by pharyngeal residue, whereas the airway was rarely compromised. In the MEG analysis, participants with presbyphagia activated an increased cortical sensorimotor network during swallowing. As major clinical determinant, participants with swallowing alterations exhibited reduced pharyngeal sensation. Presbyphagia was an independent predictor of a reduced nutritional status in a linear regression model.ConclusionsSwallowing alterations frequently occur in otherwise healthy older adults and are associated with decreased nutritional status. Increased sensorimotor cortical activation may constitute a compensation attempt to uphold swallowing function due to sensory decline. Further studies are needed to clarify whether the swallowing alterations observed can be considered physiological per se or whether the concept of presbyphagia may need to be extended to a theory with a continuous transition between presbyphagia and dysphagia.

Abstract Background COVID-19 pandemic caused a decline in stroke care in several countries. The objective was to describe lockdown stroke care in a tertiary stroke center in Düsseldorf, Germany near Heinsberg, a German hot spot for COVID-19 in spring 2020. Methods In a retrospective, observational, single-center study, we compared all patients treated in our emergency department (ED), patients seen by a neurologist in the ED, ED patients suffering from ischemic and hemorrhagic strokes and transient ischemic attacks (TIAs) as well as stroke patients admitted to our stroke unit during lockdown in spring 2020 (16 March 2020–12 April 2020) to those cared for during the same period in 2019 and lockdown light in fall 2020 (2 November – 29 November 2020). Results In spring 2020 lockdown the mean number of patients admitted to our ED dropped by 37.4%, seen by a neurologist by 35.6%, ED stroke patients by 19.2% and number of patients admitted to our stroke unit by 10% compared to the same period in 2019. In fall lockdown light 2020 effects were comparable but less pronounced. Thrombolysis rate was stable during spring and fall lockdown, however, endovascular treatment (EVT) rate declined by 58% in spring lockdown and by 51% in fall lockdown compared to the period in 2019. Conclusions Our study indicates a profound reduction of overall ED patients, neurological ED patients and EVT during COVID-19 pandemic caused lockdowns. Planning for pandemic scenarios should include access to effective emergency therapies.

Cladribine has been approved for the treatment of multiple sclerosis (MS) and its administration results in a long-lasting depletion of lymphocytes. As lymphopenia is known to hamper immune responses to vaccination, we evaluated the immunogenicity of the influenza vaccine in patients undergoing cladribine treatment at different stages vs. controls. The antibody response in 90 cladribine-treated MS patients was prospectively compared with 10 control subjects receiving platform immunotherapy (NCT05019248). Serum samples were collected before and six months after vaccination. Response to vaccination was determined by the hemagglutination-inhibition test. Postvaccination seroprotection rates against influenza A were comparable in cladribine-treated patients and controls (H1N1: 94.4% vs. 100%; H3N2: 92.2% vs. 90.0%). Influenza B response was lower in the cladribine cohort (61.1% vs. 80%). The increase in geometric mean titers was lower in the cladribine group vs. controls (H1N1: +98.5 vs. +188.1; H3N2: +225.3 vs. +300.0; influenza B: +40.0 vs. +78.4); however, titers increased in both groups for all strains. Seroprotection was achieved irrespective of vaccination timing and lymphocyte subset counts at the time of vaccination in the cladribine cohort. To conclude, cladribine-treated MS patients can mount an adequate immune response to influenza independently of treatment duration and time interval to the last cladribine administration.

Abstract Background Multiple sclerosis is one of the most prevalent neurological diseases in young adults affecting over 2 million people worldwide. Alemtuzumab is a highly effective therapy in relapsing remitting MS. Alemtuzumab is a monoclonal CD52 antibody that proved its efficacy against an active comparator (interferon [IFN]-β1a) in a phase II trial and two phase III trials regarding clinical and MRI outcomes. Nevertheless, the exact mode of action is still unknown. Alemtuzumab is commonly associated with secondary autoimmune disorders significantly affecting the risk-benefit ratio. Therefore, new biomarkers predicting treatment response and adverse events are urgently needed. This study aims to further elucidate the mechanism of action of the neuroprotective potential of alemtuzumab in relapsing-remitting multiple sclerosis (RRMS). Methods/Design This is a 3-year multicentre, explorative study including overall 150 patients comprising three different groups: (i) de novo patients prior and after alemtuzumab treatment initiation, (ii) patients under alemtuzumab treatment and (iii) patients requiring more than two alemtuzumab infusions. Peripheral blood and serum samples will be collected semi-annually for several in vitro/ex vivo assays to detect and characterize immune cells including their functional activity. Furthermore, data of MRI scans and disease-related impairment (using EDSS and MSFC), as well as the number and time of relapses, will be assessed. The clinical study is registered at clinicaltrials.gov (NCT04082260). Perspective Our study will provide deep insights into the underlying immunological changes in a longitudinal analysis of alemtuzumab treated RRMS patients. By combining clinical, radiological and functional immune-phenotype data, we will be able to identify biomarkers and/or immune signatures predicting treatment response and adverse events. Thereby, the understanding of the mechanisms of action of alemtuzumab will improve its efficacy and safety for present and future patients.

Abstract Allelic variants of genes encoding for the Fc gamma receptors IIIA and IIA have been associated with the clinical response to cell‐depleting antibodies in lymphoma patients. Here, we tested the hypothesis that FCGR3A and FCGR2A high‐affinity polymorphisms predict clinical outcomes to alemtuzumab therapy in 85 patients with relapsing‐remitting multiple sclerosis. No differences in clinical and MRI‐based efficacy parameters, the development of severe infusion‐associated reactions and secondary autoimmune diseases during a 2 year follow‐up was observed based on FCGR3A or FCGR2A polymorphisms. This study does not support the use of FCGR genetic variants to predict clinical outcomes to alemtuzumab.

Regulatory T cells (Tregs) are the major determinant of peripheral immune tolerance. Many Treg subsets have been described, however thymus-derived and peripherally induced Tregs remain the most important subpopulations. In multiple sclerosis, a prototypical autoimmune disorder of the central nervous system, Treg dysfunction is a pathogenic hallmark. In contrast, induction of Treg proliferation and enhancement of their function are central immune evasion mechanisms of infectious pathogens. In accordance, Treg expansion is compartmentalized to tissues with high viral replication and prolonged in chronic infections. In friend retrovirus infection, Treg expansion is mainly based on excessive interleukin-2 production by infected effector T cells. Moreover, pathogens seem also to enhance Treg functions as shown in human immunodeficiency virus infection, where Tregs express higher levels of effector molecules such as cytotoxic T-lymphocyte-associated protein 4, CD39 and cAMP and show increased suppressive capacity. Thus, insights into the molecular mechanisms by which intracellular pathogens alter Treg functions might aid to find new therapeutic approaches to target central nervous system autoimmunity. In this review, we summarize the current knowledge of the role of pathogens for Treg function in the context of autoimmune neuroinflammation. We discuss the mechanistic implications for future therapies and provide an outlook for new research directions.

Background: Easily accessible biomarkers enabling the identification of those patients with multiple sclerosis (MS) who will accumulate irreversible disability in the long term are essential to guide early therapeutic decisions. We here examine the utility of serum neurofilament light chain (sNfL) for forecasting relapse-free disability progression and conversion to secondary progressive MS (SPMS) in the prospective Neurofilament and longterm outcome in MS (NaloMS) cohort. Methods: The predictive ability of sNfL at Baseline and sNfL follow-up (FU)/ Baseline (BL) ratio with regard to disability progression was assessed within a development cohort (NaloMS, n=196 patients with relapsing-remitting MS (RRMS) or clinically isolated syndrome) and validated with an external independent cohort (Düsseldorf, Essen, n=204). Both relapse-free EDSS-progression (RFP: inflammatory-independent EDSS-increase 12 months prior to FU) and SPMS-transition (minimum EDSS-score of 3.0) were investigated. Findings: During the study period, 17% (n=34) of NaloMS patients suffered from RFP and 14% (n=27) converted to SPMS at FU (validation cohort RFP n=42, SPMS-conversion n=24). sNfL at BL was increased in patients with RFP (10.8 pg/ml (interquartile range (IQR) 7.7-15.0) vs. 7.2 pg/ml (4.5-12.5), p

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.

Dominant VCP–mutations cause a variety of neurological manifestations including inclusion body myopathy with early–onset Paget disease and frontotemporal dementia 1 (IBMPFD). VCP encodes a ubiquitously expressed multifunctional protein that is a member of the AAA+ protein family, implicated in multiple cellular functions ranging from organelle biogenesis to ubiquitin–dependent protein degradation. The latter function accords with the presence of protein aggregates in muscle biopsy specimens derived from VCP–patients. Studying the proteomic signature of VCP–mutant fibroblasts, we identified a (pathophysiological) increase of FYCO1, a protein involved in autophagosome transport. We confirmed this finding applying immunostaining also in muscle biopsies derived from VCP–patients. Treatment of fibroblasts with arimoclomol, an orphan drug thought to restore physiologic cellular protein repair pathways, ameliorated cellular cytotoxicity in VCP–patient derived cells. This finding was accompanied by increased abundance of proteins involved in immune response with a direct impact on protein clearaqnce as well as by elevation of pro–survival proteins as unravelled by untargeted proteomic profiling. Hence, the combined results of our study reveal a dysregulation of FYCO1 in the context of VCP–etiopathology, highlight arimoclomol as a potential drug and introduce proteins targeted by the pre–clinical testing of this drug in fibroblasts.

Aims: The flexible endoscopic evaluation of swallowing-tensilon test (FTT) was developed to diagnose myasthenia gravis (MG) in patients with unclear pharyngeal dysphagia. The purpose of this study was to determine sensitivity and specificity of the FTT and compare its diagnostic validity with that of other diagnostic markers. Methods: In this single-centre pragmatic clinical cohort study, a total of 100 patients with unclear pharyngeal dysphagia were eligible to undergo FTT. All patients were subjected to FTT and subsequently followed up clinically. FTT was considered positive if a significant improvement of pharyngeal swallowing function could be objectified endoscopically upon administration of edrophonium chloride. In addition, repetitive nerve stimulation test and serum MG antibody analysis were conducted. Results: All subjects (mean age 62.5 ± 14.1 years, female 33) underwent FTT without any complications. According to the results of the diagnostic procedures and based on long-term clinical follow-up for at least 3 years, 51 patients were finally diagnosed with MG. The sensitivity and specificity for the FTT was 88.2% and 95.9%, respectively. Application of the Cochran’s Q test showed statistically significant heterogeneity among the diagnostic tests, with results indicating FTT performance to be more accurate than the repetitive nerve stimulation results ( p 0.99). Conclusion: FTT has excellent clinical properties to be used routinely in the assessment of dysphagia with isolated or predominant pharyngeal muscle involvement allowing rapid and accurate diagnosis of MG.

The past decades have yielded major therapeutic advances in many autoimmune conditions – such as multiple sclerosis (MS) – and thus ushered in a new era of more targeted and increasingly potent immunotherapies. Yet this growing arsenal of therapeutic immune interventions has also rendered therapy much more challenging for the attending physician, especially when treating patients with more than one autoimmune condition. Importantly, some therapeutic strategies are either approved for several autoimmune disorders or may be repurposed for other conditions, therefore opening new curative possibilities in related fields. In this article, we especially focus on frequent and therapeutically relevant concomitant autoimmune conditions faced by neurologists when treating patients with MS, namely psoriasis, rheumatoid arthritis and inflammatory bowel diseases. We provide an overview of the available disease-modifying therapies, highlight possible contraindications, show pathophysiological overlaps and finally present which therapeutics can be utilized as a combinatory treatment, in order to ‘kill two birds with one stone’.

As our population grows older, age-related pathologies are becoming more prevalent. Deterioration of skeletal muscle and the immune system manifests as sarcopenia and immune senescence respectively. The disease burden of these pathologies emphasizes the need for a better understanding of the underlying mechanisms. Skeletal muscle has emerged as a potent regulator of immune system function. As such, skeletal muscle might be the central integrator between sarcopenia and immune senescence in an aging biological system. Therapeutic approaches targeting skeletal muscle might be able to restore both muscle and immune system function. In this review, we therefore outline the current - however still fragmentary - knowledge about the potential communication pathways of muscle and immune system, how they are affected by aging of skeletal muscle and discuss possible treatment strategies. The review intends to be hypothesis-generating and should thereby stimulate further research in this important scientific field. Keywords: Skeletal muscle, Sarcopenia, Immune senescence, Myokines, IL-6, IL-7, IL-15

Background: Alemtuzumab is approved for the treatment of active relapsing-remitting multiple sclerosis (RRMS). Alemtuzumab-related secondary autoimmune disorders (sAID) are common, with thyroid sAID being the most frequent, and fundamentally affect the risk-benefit ratio. Therefore, biomarkers indicating the development of sAID are urgently needed to instruct clinical decisions. Methods: We evaluated whether the anti-thyroid autoantibodies (ThyAb) anti-thyroglobulin (anti-TG) and anti–thyroid-peroxidase (anti-TPO) detected at baseline by standard testing are able to indicate increased risk for thyroid sAID following alemtuzumab treatment in a multicentre prospective cohort of 106 alemtuzumab-treated RRMS patients. We here present an interim-analysis with a median follow-up of 36 months. Findings: Baseline characteristics demonstrated no significant differences between patients with or without thyroid sAID. 29/106 (27·4%) patients developed thyroid sAID between 5 and 51 months following alemtuzumab treatment initiation. 14/29 patients (48·3%) were positive for ThyAb at baseline and developed thyroid sAID. Hazard ratio for time to thyroid autoimmunity was 12.15 (95% CI 4.73–31.2) indicating a highly increased risk for ThyAb positive patients. Baseline ThyAb were associated with shorter time to sAID, but not with a specific disease entity of thyroid sAID. Hazard ratios for age, sex, previous treatment, disease duration, disability and smoking status demonstrated no significant association with thyroid autoimmunity. Interpretation: Standard ThyAb-testing for anti-TPO and anti-TG antibodies at baseline was able to indicate increased risk for clinically manifest thyroid sAID and should therefore be used in clinical decisions concerning alemtuzumab treatment initiation. Fund: German Ministry of Education, Science, Research and Technology and the German Research foundation. Keywords: Multiple sclerosis, Alemtuzumab, Secondary autoimmune disorders, Thyroid autoimmunity, Risk estimation

Autoimmune diseases of the central nervous system (CNS) like multiple sclerosis (MS) are characterized by inflammation and demyelinated lesions in white and grey matter regions. While inflammation is present at all stages of MS, it is more pronounced in the relapsing forms of the disease, whereas progressive MS (PMS) shows significant neuroaxonal damage and grey and white matter atrophy. Hence, disease-modifying treatments beneficial in patients with relapsing MS have limited success in PMS. BAF312 (siponimod) is a novel sphingosine-1-phosphate receptor modulator shown to delay progression in PMS. Besides reducing inflammation by sequestering lymphocytes in lymphoid tissues, BAF312 crosses the blood-brain barrier and binds its receptors on neurons, astrocytes and oligodendrocytes. To evaluate potential direct neuroprotective effects, BAF312 was systemically or locally administered in the CNS of experimental autoimmune encephalomyelitis mice with distinct grey- and white-matter lesions (focal experimental autoimmune encephalomyelitis using an osmotic mini-pump). Ex-vivo flow cytometry revealed that systemic but not local BAF312 administration lowered immune cell infiltration in animals with both grey and white matter lesions. Ex-vivo voltage-sensitive dye imaging of acute brain slices revealed an altered spatio-temporal pattern of activation in the lesioned cortex compared to controls in response to electrical stimulation of incoming white-matter fiber tracts. Here, BAF312 administration showed partial restore of cortical neuronal circuit function. The data suggest that BAF312 exerts a neuroprotective effect after crossing the blood-brain barrier independently of peripheral effects on immune cells. Experiments were carried out in accordance with German and EU animal protection law and approved by local authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen; 87-51.04.2010.A331) on December 28, 2010.

Background and aims: Various attempts have been made to support recovery following optic neuritis (ON), but the respective trials have mostly been negative. The aim of this study was to determine whether disease-modifying treatment (DMT) following ON as first manifestation of relapsing-remitting multiple sclerosis influences long-term outcomes. Methods: A total of 79 patients with ON were identified and evaluated at relapse, DMT induction, and 12 months following treatment induction with either glatiramer acetate (GLAT), interferon-beta (IFN), or teriflunomide (TRF). Low-contrast letter acuity (LCLA) and full-field visual-evoked potentials (FF-VEP) were compared between treatment groups using multivariable regression models. The impact of TRF treatment induction compared with IFN or GLAT following relapses outside the optic nerves was evaluated in an independent cohort of 122 patients. Magnetic resonance imaging (MRI) outcomes and rates of confirmed improvement of relapse-related disability were evaluated. Results: TRF-treated patients exhibited higher LCLA and lower relative P100 latencies normalized to the fellow-eye. Findings were significant following covariate-adjustment by multivariable analyses. Cranial MRI lesion load as well as disability progression rates were not significantly different between groups. The cohort of patients following relapses other than ON showed no differences in confirmed improvement of disability. Conclusion: TRF treatment is associated with favorable outcomes regarding functional optic nerve recovery following ON in early multiple sclerosis.

Background and purpose: In 2017, eculizumab has been approved for treatment-refractory generalised myasthenia gravis (TRgMG). The German Myasthenia Foundation has published a consensus statement on the use of eculizumab, with a recent update. However, a treatment-refractory state is still ill-defined and the term warrants further clarification. We aimed at developing a sum score to operationalise the definition of a TRgMG status, which is easy- to-handle in clinical decision making. Methods: We established a structured consensus process according to the Delphi consensus methodology, with 12 members of the medical advisory board of the German Myasthenia Foundation. Accordingly, 4 consensus rounds were accomplished. Additionally, a literature survey covering the years 2004-2020 was done and relevant information offered to the consensus group. Consensus criteria were predefined. In the consensus process the relative importance of scoring items were to be consented, with a sum score of 20 and above indicating a TRgMG status. Results: The sum score considers the categories disease severity, inefficiency of antecedent therapies, cessation of therapies due to side effects, and long term stay on the intensive care unit. Categories were specified by a total of 13 scoring items. Eventually, the Delphi process developed an unanimous scoring consensus. Conclusion: We suggest a sum score to define treatment refractory state in generalised myasthenia gravis. Beyond clarifying the indication of eculizumab, this easy-to-handle score facilitates clinical decision making and offers new inclusion criteria for clinical studies that explore new therapeutic perspectives in myasthenia gravis treatment.

To prevent severe and potentially life-threatening consequences of bilateral vocal fold paralysis (BVFP), the identification and management of reversible causes is pivotal. Myasthenia gravis (MG) presenting with BVFP is rarely reported and remains incompletely understood. Although symptom control is achievable for most MG patients with sufficient therapy, atypical clinical presentation such as BVFP might preclude diagnosis and thus effective treatment. Here, we present a case of BVFP as leading manifestation of MG successfully treated with plasmapheresis. Moreover, we performed a literature review of the few existing cases reported between 1980 and 2020 indicating that elderly patients are particularly at risk for MG presenting with severe BVFP and that edrophonium testing with fiber optic endoscopic evaluation of swallowing (FEES) might be valuable for establishing the diagnosis. We conclude that clinicians should consider MG as possible and reversible cause for BVFP.

Immunosuppression and immunomodulation are valuable therapeutic approaches for managing neuroimmunological diseases. In times of the Coronavirus disease 2019 (COVID-19) pandemic, clinicians must deal with the question of whether immunotherapy should currently be initiated or discontinued in neurological patients. Uncertainty exists especially because different national medical associations publish different recommendations on the extent to which immunotherapies must be continued, monitored, or possibly switched during the current pandemic.Based on the most recently available data both about the novel coronavirus and the approved immunotherapies for neurological diseases, we provide an updated overview that includes current treatment strategies and the associated COVID-19 risk, but also the potential of immunotherapies to treat COVID-19.

Background/Aims: Multiple sclerosis (MS) is a prototypical autoimmune central nervous system (CNS) disease. Particularly progressive forms of MS (PMS) show significant neuroaxonal damage as consequence of demyelination and neuronal hyperexcitation. Immuno-modulatory treatment strategies are beneficial in relapsing MS (RMS), but mostly fail in PMS. Pregabalin (Lyrica®) is prescribed to MS patients to treat neuropathic pain. Mechanistically, it targets voltage-dependent Ca2+ channels and reduces harmful neuronal hyperexcitation in mouse epilepsy models. Studies suggest that GABA analogues like pregabalin exert neuroprotective effects in animal models of ischemia and trauma. Methods: We tested the impact of pregabalin in a mouse model of MS (experimental autoimmune encephalomyelitis, EAE) and performed histological and immunological evaluations as well as intravital two-photon-microscopy of brainstem EAE lesions. Results: Both prophylactic and therapeutic treatments ameliorated the clinical symptoms of EAE and reduced immune cell infiltration into the CNS. On neuronal level, pregabalin reduced long-term potentiation in hippocampal brain slices indicating an impact on mechanisms of learning and memory. In contrast, T cells, microglia and brain endothelial cells were unaffected by pregabalin. However, we found a direct impact of pregabalin on neurons during CNS inflammation as it reversed the pathological elevation of neuronal intracellular Ca2+ levels in EAE lesions. Conclusion: The presented data suggest that pregabalin primarily acts on neuronal Ca2+ channel trafficking thereby reducing Ca2+-mediated cytotoxicity and neuronal damage in an animal model of MS. Future clinical trials need to assess the benefit for neuronal survival by expanding the indication for pregabalin administration to MS patients in further disease phases.

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system of unknown etiology. As it is still a diagnosis of exclusion, there is an urgent need for biomarkers supporting its diagnosis. Increasing evidence suggests that nitrosative stress may play a pivotal role in the pathogenesis of MS. However, previous reports supporting the role of nitrosative stress molecules as disease biomarkers are inconsistent overall. We therefore systematically analyzed the existing literature to compare the serum and cerebrospinal fluid (CSF) levels of nitrite/nitrate in MS patients with those in patients with noninflammatory other neurological diseases (NIOND) and healthy controls (HC), respectively. We searched the PubMed database and included original articles investigating nitrite/nitrate levels in MS patients and NIOND patients or HC based on predefined selection criteria. Effect sizes were estimated by the standardized mean difference using a random effects model. Our results suggest that MS is associated with higher nitrite/nitrate levels within the CSF compared with patients with NIOND (SMD of 1.51; 95% CI: 0.72, 2.30; p = 0.0008). Likewise, nitrite/nitrate in the CSF of MS patients trends towards increased levels compared with those of HC but does not reach statistical significance (SMD of 3.35; 95% CI: −0.48, 7.19; p = 0.07). Measurement of nitrite/nitrate in the CSF might be a valuable tool facilitating the differentiation of MS and NIOND. Further studies with more homogeneous study criteria are needed to corroborate this hypothesis.

Vitamin E is often associated with health benefits, such as antioxidant, anti-inflammatory and cholesterol-lowering effects. These properties make its supplementation a suitable therapeutic approach in neurodegenerative disorders, for example, Alzheimer’s or Parkinson’s disease. However, trials evaluating the effects of vitamin E supplementation are inconsistent. In randomized controlled trials, the observed associations often cannot be substantiated. This could be due to the wide variety of study designs regarding the dosage and duration of vitamin E supplementation. Furthermore, genetic variants can influence vitamin E uptake and/or metabolism, thereby distorting its overall effect. Recent studies also show adverse effects of vitamin E supplementation regarding Alzheimer’s disease due to the increased synthesis of amyloid β. These diverse effects may underline the inhomogeneous outcomes associated with its supplementation and argue for a more thoughtful usage of vitamin E. Specifically, the genetic and nutritional profile should be taken into consideration to identify suitable candidates who will benefit from supplementation. In this review, we will provide an overview of the current knowledge of vitamin E supplementation in neurodegenerative disease and give an outlook on individualized, sustainable neuro-nutrition, with a focus on vitamin E supplementation.

Background: Pulmonary affection (PA) is associated with a substantial increase in morbidity and mortality in patients with idiopathic inflammatory myopathies (IIM). However, the underlying immune mechanisms of PA remain enigmatic and prompt deeper immunological analyses. Importantly, the Janus-faced role of natural killer (NK) cells, capable of pro-inflammatory as well as regulatory effects, might be of interest for the pathophysiologic understanding of PA in IIM. Methods: To extend our understanding of immunological alterations in IIM patients with PA, we compared the signatures of NK cells in peripheral blood using multi-color flow cytometry in IIM patients with (n = 12, of which anti-synthetase syndrome = 8 and dermatomyositis = 4) or without PA (n = 12). Results: We did not observe any significant differences for B cells, CD4, and CD8 T cells, while total NK cell numbers in IIM patients with PA were reduced compared to non-PA patients. NK cell alterations were driven by a particular decrease of CD56dim NK cells, while CD56bright NK cells remained unchanged. Comparisons of the cell surface expression of a large panel of NK receptors revealed an increased mean fluorescence intensity of NKG2D+ on NK cells from patients with PA compared with non-PA patients, especially on the CD56dim subset. NKG2D+ and NKp46+ cell surface levels were associated with reduced vital capacity, serving as a surrogate marker for clinical severity of PA. Conclusion: Our data illustrate that PA in IIM is associated with alterations of the NK cell repertoire, suggesting a relevant contribution of NK cells in certain IIMs, which might pave the way for NK cell-targeted therapeutic approaches.

(1) Background: Immune–mediated necrotizing myopathy (IMNM) is a rare form of inflammatory muscle disease which is even more rare in pediatric patients. To increase the knowledge of juvenile IMNM, we here present the clinical findings on long-term follow-up, myopathological changes, and therapeutic strategies in two juvenile patients. (2) Methods: Investigations included phenotyping, determination of antibody status, microscopy on muscle biopsies, MRI, and response to therapeutic interventions. (3) Results: Anti-signal recognition particle (anti-SRP54) and anti- 3-hydroxy-3-methylglutarly coenzyme A reductase (anti-HMGCR) antibodies (Ab) were detected in the patients. Limb girdle presentation, very high CK-levels, and a lack of skin rash at disease-manifestation and an absence of prominent inflammatory signs accompanied by an abnormal distribution of α-dystroglycan in muscle biopsies initially hinted toward a genetically caused muscle dystrophy. Further immunostaining studies revealed an increase of proteins involved in chaperone-assisted autophagy (CASA), a finding already described in adult IMNM-patients. Asymmetrical muscular weakness was present in the anti-SRP54 positive Ab patient. After initial stabilization under therapy with intravenous immunoglobulins and methotrexate, both patients experienced a worsening of their symptoms and despite further therapy escalation, developed a permanent reduction of their muscle strength and muscular atrophy. (4) Conclusions: Diagnosis of juvenile IMNM might be complicated by asymmetric muscle weakness, lack of cutaneous features, absence of prominent inflammatory changes in the biopsy, and altered α-dystroglycan.

Background/Aims: Substance P (SP) is a neuropeptide, likely acting as a neurotransmitter in the pharyngeal mucosa enhancing the swallow and cough reflex. Pharyngeal Electrical Stimulation (PES) induces a temporary increase of salivary SP levels in healthy adults. Previous evidence suggests that post-stroke dysphagia is related to reduced SP levels. Here, we investigated the effects of PES on SP levels in severely dysphagic stroke patients and a possible link between increase of SP and treatment success. Methods: 23 tracheotomized stroke patients who could not be decannulated due to severe and persisting dysphagia according to endoscopic evaluation received PES for 10 minutes a day over three consecutive days in this prospective single-center study. If initial treatment failed, repetitive stimulation cycles were provided. Saliva samples were collected before and directly after each PES. Results: 61% of participants were decannulated after the first treatment cycle. Increase of SP levels post-stimulation was closely related to treatment success, i.e. decannulation with 79% of successfully treated patients showing increase of SP, whereas 89% of unsuccessfully treated patients had stable or decreased SP levels. Applying logistic regression analysis, increase of SP level remained the only independent predictor of decannulation after PES. All 3 repetitively treated patients showed increased SP levels when progressing from the 1st to the 2nd cycle, two of whom were decannulated hereafter. Conclusions: The physiological mechanism of PES may consist in restoration of sensory feedback, which is known to be crucial for the execution of a safe swallow. SP possibly acts as a biomarker for indicating response to PES.