Identification of disease phenotypes in acetylcholine receptor-antibody myasthenia gravis using proteomics-based consensus clustering.

BACKGROUND: The clinical heterogeneity of myasthenia gravis (MG), an autoimmune disease defined by antibodies (Ab) directed against the postsynaptic membrane, constitutes a challenge for patient stratification and treatment decision making. Novel strategies are needed to classify patients based on their biological phenotypes aiming to improve patient selection and treatment outcomes. METHODS: For this purpose, we assessed the serum proteome of a cohort of 140 patients with anti-acetylcholine receptor-Ab-positive MG and utilised consensus clustering as an unsupervised tool to assign patients to biological profiles. For in-depth analysis, we used immunogenomic sequencing to study the B cell repertoire of a subgroup of patients and an in vitro assay using primary human muscle cells to interrogate serum-induced complement formation. FINDINGS: This strategy identified four distinct patient phenotypes based on their proteomic patterns in their serum. Notably, one patient phenotype, here named PS3, was characterised by high disease severity and complement activation as defining features. Assessing a subgroup of patients, hyperexpanded antibody clones were present in the B cell repertoire of the PS3 group and effectively activated complement as compared to other patients. In line with their disease phenotype, PS3 patients were more likely to benefit from complement-inhibiting therapies. These findings were validated in a prospective cohort of 18 patients using a cell-based assay. INTERPRETATION: Collectively, this study suggests proteomics-based clustering as a gateway to assign patients to a biological signature likely to benefit from complement inhibition and provides a stratification strategy for clinical practice. FUNDING: CN and CBS were supported by the Forschungskommission of the Medical Faculty of the Heinrich Heine University Düsseldorf. CN was supported by the Else Kröner-Fresenius-Stiftung (EKEA.38). CBS was supported by the Deutsche Forschungsgemeinschaft (DFG-German Research Foundation) with a Walter Benjamin fellowship (project 539363086). The project was supported by the Ministry of Culture and Science of North Rhine-Westphalia (MODS, "Profilbildung 2020" [grant no. PROFILNRW-2020-107-A]).

Inter-alpha-trypsin inhibitor heavy chain H3 is a potential biomarker for disease activity in myasthenia gravis.

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.

Triage Performance Across Large Language Models, ChatGPT, and Untrained Doctors in Emergency Medicine: Comparative Study.

BACKGROUND: Large language models (LLMs) have demonstrated impressive performances in various medical domains, prompting an exploration of their potential utility within the high-demand setting of emergency department (ED) triage. This study evaluated the triage proficiency of different LLMs and ChatGPT, an LLM-based chatbot, compared to professionally trained ED staff and untrained personnel. We further explored whether LLM responses could guide untrained staff in effective triage. OBJECTIVE: This study aimed to assess the efficacy of LLMs and the associated product ChatGPT in ED triage compared to personnel of varying training status and to investigate if the models' responses can enhance the triage proficiency of untrained personnel. METHODS: A total of 124 anonymized case vignettes were triaged by untrained doctors; different versions of currently available LLMs; ChatGPT; and professionally trained raters, who subsequently agreed on a consensus set according to the Manchester Triage System (MTS). The prototypical vignettes were adapted from cases at a tertiary ED in Germany. The main outcome was the level of agreement between raters' MTS level assignments, measured via quadratic-weighted Cohen κ. The extent of over- and undertriage was also determined. Notably, instances of ChatGPT were prompted using zero-shot approaches without extensive background information on the MTS. The tested LLMs included raw GPT-4, Llama 3 70B, Gemini 1.5, and Mixtral 8x7b. RESULTS: GPT-4-based ChatGPT and untrained doctors showed substantial agreement with the consensus triage of professional raters (κ=mean 0.67, SD 0.037 and κ=mean 0.68, SD 0.056, respectively), significantly exceeding the performance of GPT-3.5-based ChatGPT (κ=mean 0.54, SD 0.024; P<.001). When untrained doctors used this LLM for second-opinion triage, there was a slight but statistically insignificant performance increase (κ=mean 0.70, SD 0.047; P=.97). Other tested LLMs performed similar to or worse than GPT-4-based ChatGPT or showed odd triaging behavior with the used parameters. LLMs and ChatGPT models tended toward overtriage, whereas untrained doctors undertriaged. CONCLUSIONS: While LLMs and the LLM-based product ChatGPT do not yet match professionally trained raters, their best models' triage proficiency equals that of untrained ED doctors. In its current form, LLMs or ChatGPT thus did not demonstrate gold-standard performance in ED triage and, in the setting of this study, failed to significantly improve untrained doctors' triage when used as decision support. Notable performance enhancements in newer LLM versions over older ones hint at future improvements with further technological development and specific training.

Recombinant Acetylcholine Receptor Immunization Induces a Robust Model of Experimental Autoimmune Myasthenia Gravis in Mice.

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.

The Use of Nitrosative Stress Molecules as Potential Diagnostic Biomarkers in Multiple Sclerosis.

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.

K2P2.1 is a regulator of inflammatory cell responses in idiopathic inflammatory myopathies.

K2P2.1 (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 K2P2.1 in the autoimmune response of IIMs. We detected K2P2.1 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 K2P2.1 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 K2P2.1 function also demonstrated a strong pro-inflammatory response. Conversely, these features were abrogated by activation of K2P2.1 and improved the disease course of a myositis mouse model. In humans, K2P2.1 expression was diminished in IIM patients compared to non-diseased controls arguing for the translatability of our findings. In summary, K2P2.1 may regulate the inflammatory response of skeletal muscle. Further research is required to understand whether K2P2.1 could serve as novel therapeutic target.

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice.

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

Introducing electronic monitoring of disease activity in patients with chronic inflammatory demyelinating polyneuropathy (EMDA CIDP): trial protocol of a proof of concept study.

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.

Eculizumab treatment alters the proteometabolome beyond the inhibition of complement.

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.

Validation of TREK1 ion channel activators as an immunomodulatory and neuroprotective strategy in neuroinflammation.

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.

Cladribine treatment improves cortical network functionality in a mouse model of autoimmune encephalomyelitis.

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.

High-Dimensional Cytometry Dissects Immunological Fingerprints of Idiopathic Inflammatory Myopathies.

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.

Immunological consequences of cladribine treatment in multiple sclerosis: A real-world study.

BACKGROUND: Cladribine is a synthetic deoxyadenosine analogue approved for the treatment of highly active relapsing multiple sclerosis (RMS). Cladribine is considered to be a semi-selective immune-reconstitution therapy (IRT) that induces long-term remission following short course of treatment. Here, we evaluated the effect of cladribine on immune cell reduction and reconstitution during the first two years of treatment. METHODS: We analyzed our longitudinal, prospective, real-world cohort of 80 cladribine-treated RMS patients from two tertiary centers in Germany. Laboratory testing was conducted monthly and included evaluation of cellular as well as soluble parameters. Laboratory outcomes were correlated with infectious adverse events (AEs) and clinical or paraclinical disease activity. RESULTS: Selective alterations in immune cell populations occurred following cladribine treatment, with the most marked effects observed in year two of treatment. Specifically, a rapid reduction in CD56+ natural killer cells (nadir: month 1 (year 1) and 14 (year 2); -37 and -41% from baseline) was followed by a greater reduction in CD19+ B cells (nadir: month 2 and 14; -81 and -82%); a moderate effect on CD4+ (nadir: month 3 and 15; -48 and -61%) and CD8+ T cells (nadir: month 5 and 18; -40 and -48%). Despite the marked effect on B cells, immunoglobulin levels were unaffected. There was no or minimal effect on thrombocytes and innate immune cells. Clinical and paraclinical disease activity was unrelated to the observed immune alterations. Lymphopenia was the most commonly observed AE (86.3% of patients; grade III-IV lymphopenia: 38.8%). The cumulative incidence of infections was 55% with cladribine treatment, which were mostly mild or moderate. In total, 19 herpes infections developed in 8 (10%) cladribine-treated patients; all cases were dermatomal and 94.7% of the herpetic infections occurred during a period of lymphopenia. CONCLUSIONS: The immunophenotyping data obtained in our real-world setting are comparable to those demonstrated in pivotal clinical trials and provide further evidence that cladribine may represent a form of IRT. However, regarding the side-effect profile of cladribine, severe lymphopenia (exceeding grade II CTCAE) was more frequent, which may have prompted the development of herpes infections. Of note, lymphocyte dynamics did not correlate with clinical and paraclinical measures of disease activity in the two-year follow-up period.

Independent risk factors for myasthenic crisis and disease exacerbation in a retrospective cohort of myasthenia gravis patients.

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.

Eculizumab versus rituximab in generalised myasthenia gravis.

OBJECTIVE: Myasthenia gravis (MG) is the most common autoimmune disorder affecting the neuromuscular junction. However, evidence shaping treatment decisions, particularly for treatment-refractory cases, is sparse. Both rituximab and eculizumab may be considered as therapeutic options for refractory MG after insufficient symptom control by standard immunosuppressive therapies. METHODS: In this retrospective observational study, we included 57 rituximab-treated and 20 eculizumab-treated patients with MG to compare the efficacy of treatment agents in generalised, therapy-refractory anti-acetylcholine receptor antibody (anti-AChR-ab)-mediated MG with an observation period of 24 months. Change in the quantitative myasthenia gravis (QMG) score was defined as the primary outcome parameter. Differences between groups were determined in an optimal full propensity score matching model. RESULTS: Both groups were comparable in terms of clinical and demographic characteristics. Eculizumab was associated with a better outcome compared with rituximab, as measured by the change of the QMG score at 12 and 24 months of treatment. Minimal manifestation of disease was more frequently achieved in eculizumab-treated patients than rituximab-treated patients at 12 and 24 months after baseline. However, the risk of myasthenic crisis (MC) was not ameliorated in either group. INTERPRETATION: This retrospective, observational study provides the first real-world evidence supporting the use of eculizumab for the treatment of refractory, anti-AChR-ab positive MG. Nonetheless, the risk of MC remained high and prompts the need for intensified monitoring and further research effort aimed at this vulnerable patient cohort.

Immune response to SARS-CoV-2 vaccination in relation to peripheral immune cell profiles among patients with multiple sclerosis receiving ocrelizumab.

BACKGROUND: Vaccination has proven to be effective in preventing SARS-CoV-2 transmission and severe disease courses. However, immunocompromised patients have not been included in clinical trials and real-world clinical data point to an attenuated immune response to SARS-CoV-2 vaccines among patients with multiple sclerosis (MS) receiving immunomodulatory therapies. METHODS: We performed a retrospective study including 59 ocrelizumab (OCR)-treated patients with MS who received SARS-CoV-2 vaccination. Anti-SARS-CoV-2-antibody titres, routine blood parameters and peripheral immune cell profiles were measured prior to the first (baseline) and at a median of 4 weeks after the second vaccine dose (follow-up). Moreover, the SARS-CoV-2-specific T cell response and peripheral B cell subsets were analysed at follow-up. Finally, vaccination-related adverse events were assessed. RESULTS: After vaccination, we found anti-SARS-CoV-2(S) antibodies in 27.1% and a SARS-CoV-2-specific T cell response in 92.7% of MS cases. T cell-mediated interferon (IFN)-γ release was more pronounced in patients without anti-SARS-CoV-2(S) antibodies. Antibody titres positively correlated with peripheral B cell counts, time since last infusion and total IgM levels. They negatively correlated with the number of previous infusion cycles. Peripheral plasma cells were increased in antibody-positive patients. A positive correlation between T cell response and peripheral lymphocyte counts was observed. Moreover, IFN-γ release was negatively correlated with the time since the last infusion. CONCLUSION: In OCR-treated patients with MS, the humoral immune response to SARS-CoV-2 vaccination is attenuated while the T cell response is preserved. However, it is still unclear whether T or B cell-mediated immunity is required for effective clinical protection. Nonetheless, given the long-lasting clinical effects of OCR, monitoring of peripheral B cell counts could facilitate individualised treatment regimens and might be used to identify the optimal time to vaccinate.

Mini-Review: Two Brothers in Crime - The Interplay of TRESK and TREK in Human Diseases.

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.

An optimized and validated protocol for inducing chronic experimental autoimmune encephalomyelitis in C57BL/6J mice.

BACKGROUND: 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. OPTIMIZED METHOD: 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, MOG35-55-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. RESULTS: 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. COMPARISON WITH EXISTING METHODS: 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. CONCLUSION: 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.

Vaccine-based clinical protection against SARS-CoV-2 infection and the humoral immune response: A 1-year follow-up study of patients with multiple sclerosis receiving ocrelizumab.

Introduction: Given 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. Methods: We 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. Results: We 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. Discussion: Additional 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.

Crosstalk of Microorganisms and Immune Responses in Autoimmune Neuroinflammation: A Focus on Regulatory T Cells.

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.