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Background and purpose Although myasthenia gravis (MG) is recognized as an immunoglobulin G autoantibody-mediated disease, the relationship between autoantibody levels and disease activity in MG is unclear. We sought to evaluate this landscape through systematically assessing the evidence, testing the impact of predefined variables on any relationship, and augmenting with expert opinion. Methods In October 2020, a forum of leading clinicians and researchers in neurology from across Europe (Expert Forum for Rare Autoantibodies in Neurology in Myasthenia Gravis) participated in a series of virtual meetings that took place alongside the conduct of a systematic literature review (SLR). Results Forty-two studies were identified meeting inclusion criteria. Of these, 10 reported some correlation between a patient's autoantibody level and disease severity. Generally, decreased autoantibody levels (acetylcholine receptor, muscle-specific kinase, and titin) were positively and significantly correlated with improvements in disease severity (Quantitative Myasthenia Gravis score, Myasthenia Gravis Composite score, Myasthenia Gravis Activities of Daily Living score, Myasthenia Gravis Foundation of America classification). Given the limited evidence, testing the impact of predefined variables was not feasible. Conclusions This first SLR to assess whether a correlation exists between autoantibody levels and disease activity in patients with MG has indicated a potential positive correlation, which could have clinical implications in guiding treatment decisions. However, in light of the limited and variable evidence, we cannot currently recommend routine clinical use of autoantibody level testing in this context. For now, patient's characteristics, clinical disease course, and laboratory data (e.g., autoantibody status, thymus histology) should inform management, alongside patient-reported outcomes. We highlight the need for future studies to reach more definitive conclusions on this relationship.

Pericytes (PCs) are mesenchymal stromal cells (MSCs) that function as support cells and play a role in tissue regeneration and, in particular, vascular homeostasis. PCs promote endothelial cells (ECs) survival which is critical for vessel stabilization, maturation, and remodeling. In this study, PCs were isolated from human micro-fragmented adipose tissue (MFAT) obtained from fat lipoaspirate and were characterized as NG2

Background Mesenchymal stromal cells (MSCs) are able to migrate and engraft at sites of inflammation, injuries, and tumours, but little is known about their fate after local injection. The purpose of this study is to perform MSC tracking, combining in vivo 7-T magnetic resonance imaging (MRI) and histological assessment, following lung injection in a rat model. Methods Five lungs were injected with ferumoxide-labelled MSCs and five with perfluorocarbon-labelled MSCs and underwent 7-T MRI. MRI acquisitions were recorded immediately (T0), at 24 h (T24) and/or 48 h (T48) after injection. For each rat, labelled cells were assessed in the main organs by MRI. Target organs were harvested under sterile conditions from rats sacrificed 0, 24, or 48 h after injection and fixed for histological analysis via confocal and structured illumination microscopy. Results Ferumoxide-labelled MSCs were not detectable in the lungs, whereas they were not visible in the distant sites. Perfluorocarbon-labelled MSCs were seen in 5/5 injected lungs at T0, in 1/2 at T24, and in 1/3 at T48. The fluorine signal in the liver was seen in 3/5 at T0, in 1/2 at T24, and in 2/3 at T48. Post-mortem histology confirmed the presence of MSCs in the injected lung. Conclusions Ferumoxide-labelled cells were not seen at distant sites; a linear decay of injected perfluorocarbon-labelled MSCs was observed at T0, T24, and T48 in the lung. In more than half of the experiments, perfluorocarbon-labelled MSCs scattering to the liver was observed, with a similar decay over time as observed in the lung.

Here, we demonstrate that introduction of halogen atoms at the tyrosine 10 phenol ring of the DSGYEV sequence derived from the flexible amyloid‐β N‐terminus, promotes its self‐assembly in the solid state. In particular, we report the crystal structures of two halogen‐modified sequences, which we found to be stabilized in the solid state by halogen‐mediated interactions. The structural study is corroborated by Non‐Covalent Interaction (NCI) analysis. Our results prove that selective halogenation of an amino acid enhances the supramolecular organization of otherwise unstructured biologically‐relevant sequences. This method may develop as a general strategy for stabilizing highly polymorphic peptide regions., The power of halogens! The high‐resolution single crystal X‐ray structures of two halogenated derivatives of the amyloid‐β sequence 7–12 demonstrate the role of halogen‐mediated intermolecular interactions in stabilizing the solid‐state supramolecular organization of this otherwise unstructured biologically‐relevant sequence.

Background Patients with neuroimmunological conditions such as multiple sclerosis (MS) often receive disease-modifying therapies (DMTs) or immunosuppressants which may reduce the response to vaccines. BNT162b2 (Pfizer-BioNTech) is the first COVID-19 vaccine authorized in Italy. Its clinical efficacy and serological response were not evaluated in MS patients receiving DMTs or immunosuppressants. This early multicenter study evaluated serological response to BNT162b2 and safety in these patients. Methods From February 2021 we enrolled consecutive MS patients, treated with at least one DMT and all healthcare workers (HCWs), having received or being scheduled to receive the first dose of BNT162b2. Blood samples were collected after the second vaccine dose and analyzed to quantitatively detect the presence of anti-Spike antibodies. Serological response was compared to the one from a control population of HCWs, with neither neuroimmunological conditions nor receiving immunosuppressants. Patients receiving treatments associated with a possible reduced response (Under-scrutiny treatment group) were also compared to those undergoing other treatments. Anti-Spike levels were described as median and interquartile range (IQR). Comparisons were performed with Wilcoxon-Mann-Whitney test. Solicited and unsolicited adverse events (AEs) were collected. Results 39 MS patients and a control population of 273 HCWs were included. One patient, under treatment with ocrelizumab, did not respond to BNT162b2, while all the remaining patients and all controls developed a serological response to the vaccine. Median anti-Spike levels were similar between patients (1471.0 BAU/ml; IQR 779.7 to 2357.0) and controls (1479.0 BAU/ml; IQR 813.1 to 2528.0) (p = 0.53). Patients included in the Under-scrutiny treatments group showed reduced anti-Spike levels (156.4 BAU/ml; IQR 33.4 to 559.1) compared to those receiving other treatments (1582.4 BAU/ml; IQR 1296.5 to 2219.0) (p = 0.001). Solicited AEs were all mild to moderate in severity, generally reported in the first days after vaccination, and resolved in the following days. Two MS patients reported a clinical relapse after the second vaccine dose. Conclusion BNT162b2 induced a serological response in MS patients treated with DMTs similar to controls not receiving DMTs or immunosuppressants. Some treatments were associated with reduced levels of anti-Spike antibodies in patients. These observations have relevant implications for treated patients receiving BNT162b2 and the community.

Alzheimer's disease (AD) is a serious neuropathology related to the misfolded assembly state of amyloid-beta (A?40 and A?42) peptides. It has been demonstrated that protein post-translation modifications (PPTMs) of the more hydrophilic N-term moiety of the A? peptide affect its aggregation kinetics and interaction with the environment. Considering that chlorination and bromination are non-canonical PPTMs found in various metabolic pathways and often correlated to inflammatory responses, halogenation of the Y10 of the A? N-term could be a putative in vivo modification with implications in the A? peptide aggregation propensity. In this framework, we chose as a model system, a short peptide sequence, DSGYEV (i.e. residues 7-12 of the A? N-term) and studied its self-assembly behaviour in comparison to its chlorinated and brominated derivatives. Our results show that Y10 halogenation works as a molecular trigger of the peptide self-assembly in solution, promoting the formation of more structured aggregates.

Pathogen infections and dysregulated Toll-like receptor (TLR)-mediated innate immune responses are suspected to play key roles in autoimmunity. Among TLRs, TLR7 and TLR9 have been implicated in several autoimmune conditions, mainly because of their ability to promote abnormal B cell activation and survival. Recently, we provided evidence of Epstein-Barr virus (EBV) persistence and reactivation in the thymus of myasthenia gravis (MG) patients, suggesting an involvement of EBV in the intrathymic pathogenesis of the disease. Considerable data highlight the existence of pathogenic crosstalk among EBV, TLR7, and TLR9: EBV elicits TLR7/9 signaling, which in turn can enhance B cell dysfunction and autoimmunity. In this article, after a brief summary of data demonstrating TLR activation in MG thymus, we provide an overview on the contribution of TLR7 and TLR9 to autoimmune diseases and discuss our recent findings indicating a pivotal role for these two receptors, along with EBV, in driving, perpetuating, and/or amplifying intrathymic B cell dysregulation and autoimmune responses in MG. Development of therapeutic approaches targeting TLR7 and TLR9 signaling could be a novel strategy for treating the chronic inflammatory autoimmune process in myasthenia gravis.

Gut microorganisms (microbiota) live in symbiosis with the host and influence human nutrition, metabolism, physiology, and immune development and function. The microbiota prevents pathogen infection to the host, and in turn the host provides a niche for survival. The alteration of gut bacteria composition (dysbiosis) could contribute to the development of immune-mediated diseases by influencing the immune system activation and driving the pro- and anti-inflammatory responses in order to promote or counteract immune reactions. Probiotics are nonpathogenic microorganisms able to interact with the gut microbiota and provide health benefits; their use has recently been exploited to dampen immunological response in several experimental models of autoimmune diseases. Here, we focus on the relationships among commensal bacteria, probiotics, and the gut, describing the main interactions occurring with the immune system and recent data supporting the clinical efficacy of probiotic administration in rheumatoid arthritis, multiple sclerosis, and myasthenia gravis (MG) animal models. The encouraging results suggest that selected strains of probiotics should be evaluated in clinical trials as adjuvant therapy to restore the disrupted tolerance in myasthenia gravis.

The thymus plays a key role in myasthenia gravis (MG), a B cell-mediated autoimmune disorder affecting neuromuscular junction. Most MG patients have thymic abnormalities, including hyperplasia and thymoma, a neoplasm of thymic epithelial cells. Epstein-Barr virus (EBV) is associated with autoimmune diseases and tumors. Recently, we showed EBV persistence and reactivation in hyperplastic MG thymuses, suggesting that EBV might contribute to intra-thymic B cell dysregulation in MG patients. Here, we investigated EBV involvement in thymoma-associated MG, by searching for EBV markers in MG (n=26) and non-MG (n=14) thymomas. EBV DNA and EBV-encoded small nuclear RNA (EBER) 1 transcript were detected in 14/26 (53.8%) and 22/26 (84.6%) MG thymomas, and only in 3 of 14 (21.4%) non-MG thymomas. Latent EBNA2 and late gp350/220 lytic transcripts were undetectable in all, but one, thymomas, and early lytic BZLF1 transcript was absent in all samples, suggesting that early infection events and EBV reactivation were very rare in thymomas. EBER1 and 2-positive cells were detected in MG, but not in non-MG, thymomas, as well as cells expressing EBV latency proteins (EBNA1, LMP1, LMP2A), that were mainly of B cell phenotype, indicating EBV association with MG rather than with thymoma. Toll-like receptor (TLR) 3 transcriptional levels were higher in MG than non-MG thymomas and positively correlated with EBER1 levels, suggesting a role for EBERs in TLR3 activation. Our findings show that EBV is commonly present in thymoma-infiltrating B cells of myasthenic patients, indicating a contribution of EBV to B cell-mediated autoreactivity in MG associated with thymic tumor.

The purpose of this study is to report on the validity, reliability, and sensitivity of the myasthenia gravis activities of daily living profile (MG-ADL) in a sample of Italian patients. Patients with myasthenia gravis (MG) completed a protocol that included the MG-ADL, the WHO Disability Assessment Schedule (WHODAS 2.0), the Besta Neurological Institute rating scale for myasthenia gravis, and the MG-composite. Cronbach’s alpha was used to test reliability, Spearman’s correlation and intra-class correlation coefficient (ICC) to test short-term test-retest, Kruskal-Wallis test to assess differences in MG-ADL between patients with different disease severity, and Wilcoxon signed-rank test to assess sensitivity to change. In total, 58 patients were enrolled: 44 were females, mean MG duration 10.5 ± 10.4 years, mean MG-ADL 3.98 ± 3.07. The MG-ADL showed good internal consistency (alpha = .774), stability (test-retest correlation = .98, ICC = .97). It was superior to the WHODAS 2.0 in differentiating patients with different MG type and severity (P

Objective:We performed propensity score (PS) models to compare the outcome of patients with myasthenia gravis (MG) submitted to 2 different surgical approaches: extended transsternal (T-3b) or thoracoscopic extended thymectomy (VATET).Methods:Patients' clinical data were retrieved from the MG database of the C. Besta Neurologic Institute Foundation. In the PS analysis, a matching ratio of 1:1 of the main clinical variables was obtained for the 2 groups of patients and treatment effect was estimated by comparing their outcome.Results:A total of 210 patients met the inclusion criteria, by having a complete set of clinical data, and were included in the PS model; a matched dataset of 122 participants (61 per group) showed an adequate balance of all the covariates. Our analysis demonstrated that 68.9% of patients who had thymectomy by the VATET technique reached the pharmacologic remission/remission status at 2 years from thymectomy compared to 34.4% of those operated on by the T-3b technique (p < 0.001), had a lower INCB-MG score (p < 0.001), and had less muscle fatigability (p = 0.004). Similar results were found considering only nonthymomatous patients with MG. Results were also confirmed by paired statistical tests.Conclusions:Our PS matching analysis showed that VATET is a reliable and effective surgical approach alternative to T-3b in patients with MG who are candidates for thymectomy.Classification of evidence:This study provides Class IV evidence that for patients with MG, VATET is more effective than T-3b thymectomy.

Introduction In this study we assess the Italian version of the 15-item Myasthenia Gravis Quality-of-Life questionnaire (MG-QOL15). Methods The validation protocol included the MG-QOL15, the 36-item Short Form (SF-36), the Besta Neurological Institute Rating Scale for Myasthenia Gravis, and the MG-Composite. We used the Cronbach α to test reliability, the Spearman correlation to test short-term test–retest, the Kruskal–Wallis test to assess differences in MG-QOL15 between patients with different disease severity, and the Wilcoxon signed-rank test to assess sensitivity to change. Results Seventy-two patients were enrolled in the study. The mean MG-QOL15 score was 15.2 ± 12.2, with α = 0.93 and test–retest correlation = 0.93. Compared with the SF-36, the MG-QOL15 was superior in differentiating patients with different MG types (P = 0.041) and severity (P = 0.004), showed higher sensitivity to change (P = 0.003 for improved and P = 0.024 for worsened patients), and had higher correlations with the MG-Composite (rho = 0.367 vs. –0.213 and –0.154). Conclusion The Italian version of the MG-QOL15 is valid, reliable, stable, and sensitive to changes. Muscle Nerve 56: 716–720, 2017

LYVE-1 is 'on stage' now: an emerging player in dendritic cell docking to lymphatic endothelial cells

Considerable data implicate the thymus as the main site of autosensitization to the acetylcholine receptor in myasthenia gravis (MG), a B-cell-mediated autoimmune disease affecting the neuromuscular junction. We recently demonstrated an active Epstein-Barr virus (EBV) infection in the thymus of MG patients, suggesting that EBV might contribute to the onset or maintenance of the autoimmune response within MG thymus, because of its ability to activate and immortalize autoreactive B cells. EBV has been reported to elicit and modulate Toll-like receptor (TLR) 7- and TLR9-mediated innate immune responses, which are known to favor B-cell dysfunction and autoimmunity. Aim of this study was to investigate whether EBV infection is associated with altered expression of TLR7 and TLR9 in MG thymus. By real-time PCR, we found that TLR7 and TLR9 mRNA levels were significantly higher in EBV-positive MG compared to EBV-negative normal thymuses. By confocal microscopy, high expression levels of TLR7 and TLR9 proteins were observed in B cells and plasma cells of MG thymic germinal centers (GCs) and lymphoid infiltrates, where the two receptors co-localized with EBV antigens. An increased frequency of Ki67-positive proliferating B cells was found in MG thymuses, where we also detected proliferating cells expressing TLR7, TLR9 and EBV antigens, thus supporting the idea that EBV-associated TLR7/9 signaling may promote abnormal B-cell activation and proliferation. Along with B cells and plasma cells, thymic epithelium, plasmacytoid dendritic cells and macrophages exhibited enhanced TLR7 and TLR9 expression in MG thymus; TLR7 was also increased in thymic myeloid dendritic cells and its transcriptional levels positively correlated with those of interferon (IFN)-β. We suggested that TLR7/9 signaling may be involved in antiviral type I IFN production and long-term inflammation in EBV-infected MG thymuses. Our overall findings indicate that EBV-driven TLR7- and TLR9-mediated innate immune responses may participate in the intra-thymic pathogenesis of MG.

Introduction: We validated the scale for myasthenia gravis (MG) developed at the Neurological Institute Foundation of Milan (INCB-MG scale). Methods: A total of 174 patients were evaluated with the INCB-MG and compared with the MG Composite (MGC) as the gold standard. Dimensionality, reliability, and validity of the INCB-MG scale were studied by principal component factor analysis, Cronbach alpha, and Pearson correlation coefficients; interobserver reliability was calculated by the weighted Cohen K coefficient. Results: Generalized and bulbar INCB-MG subscales were unidimensional with excellent consistency; the INCB-MG and MGC scales were strongly correlated. Fatigability assessment was correlated with the INCB-MG generalized, bulbar, and respiratory subscales. Conclusions: The INCB-MG scale is a reliable tool to assess MG and is strongly correlated with the MGC. The INCB-MG scale is a valid tool for every day practice and should be further investigated for its application in clinical trials. Muscle Nerve 53: 32–37, 2016

The pool size ratio measured by quantitative magnetization transfer MRI is hypothesized to closely reflect myelin density, but their relationship has so far been confirmed mostly in ex vivo conditions. We investigate the correspondence between this parameter measured in vivo at 7.0 T, with Black Gold II staining for myelin fibres, and with myelin basic protein and beta-tubulin immunofluorescence in a hybrid longitudinal study of C57BL/6 and SJL/J mice treated with cuprizone, a neurotoxicant causing relatively selective myelin loss followed by spontaneous remyelination upon treatment suspension. Our results confirm that pool size ratio measurements correlate with myelin content, with the correlation coefficient depending on strain and staining method, and demonstrate the in vivo applicability of this MRI technique to experimental mouse models of multiple sclerosis.

Gut microorganisms (microbiota) live in symbiosis with the host and influence human nutrition, metabolism, physiology, and immune development and function. The microbiota prevents pathogen infection to the host, and in turn the host provides a niche for survival. The alteration of gut bacteria composition (dysbiosis) could contribute to the development of immune-mediated diseases by influencing the immune system activation and driving the pro- and anti-inflammatory responses in order to promote or counteract immune reactions. Probiotics are nonpathogenic microorganisms able to interact with the gut microbiota and provide health benefits; their use has recently been exploited to dampen immunological response in several experimental models of autoimmune diseases. Here, we focus on the relationships among commensal bacteria, probiotics, and the gut, describing the main interactions occurring with the immune system and recent data supporting the clinical efficacy of probiotic administration in rheumatoid arthritis, multiple sclerosis, and myasthenia gravis (MG) animal models. The encouraging results suggest that selected strains of probiotics should be evaluated in clinical trials as adjuvant therapy to restore the disrupted tolerance in myasthenia gravis.

Probiotics beneficial effects on the host are associated with regulation of the intestinal microbial homeostasis and with modulation of inflammatory immune responses in the gut and in periphery. In this study, we investigated the clinical efficacy of two lactobacillus and two bifidobacterium probiotic strains in experimental autoimmune myasthenia gravis (EAMG) and experimental autoimmune encephalomyelitis (EAE) models, induced in Lewis rats. Treatment with probiotics led to less severe disease manifestation in both models; ex vivo analyses showed preservation of neuromuscular junction in EAMG and myelin content in EAE spinal cord. Immunoregulatory transcripts were found differentially expressed in gut associated lymphoid tissue and in peripheral immunocompetent organs. Feeding EAMG animals with probiotics resulted in increased levels of Transforming Growth Factor-β (TGFβ) in serum, and increased percentages of regulatory T cells (Treg) in peripheral blood leukocyte. Exposure of immature dendritic cells to probiotics induced their maturation toward an immunomodulatory phenotype, and secretion of TGFβ. Our data showed that bifidobacteria and lactobacilli treatment effectively modulates disease symptoms in EAMG and EAE models, and support further investigations to evaluate their use in autoimmune diseases.

This paper presents the Italian guidelines for autoantibody testing in myasthenia gravis that have been developed following a consensus process built on questionnaire-based surveys, internet contacts and discussions during dedicated workshops of the sponsoring Italian Association of Neuroimmunology (AINI). Essential clinical information on myasthenic syndromes, indications and limits of antibody testing, instructions for result interpretation and an agreed laboratory protocol (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motoneuron loss in the CNS. In G93A-SOD1 mice, motoneuron degeneration is associated with proliferative restorative attempts of ependymal stem progenitor cells (epSPCs), usually quiescent in the spinal cord. The aims of the study were to demonstrate that epSPCs isolated from the spinal cord of G93A-SOD1 mice express neurogenic potential in vitro, and thus gain a better understanding of epSPC neural differentiation properties. For this purpose, we compared the ability of epSPCs from asymptomatic and symptomatic G93A-SOD1 and WT SOD1 transgenic mice to proliferate and differentiate into neural cells. Compared to control cells, G93A-SOD1 epSPCs differentiated more into neurons than into astrocytes, whereas oligodendrocyte proportions were similar in the two populations. G93A-SOD1 neurons were small and astrocytes had an activated phenotype. Evaluation of microRNAs, specific for neural cell fate and cell-cycle regulation, in G93A-SOD1 epSPCs showed that miR-9, miR-124a, miR-19a and miR-19b were differentially expressed. Expression analysis of the predicted miRNA targets allowed identification of a functional network in which Hes1, Pten, Socs1, and Stat3 genes were important for controlling epSPC fate. Our findings demonstrate that G93A-SOD1 epSPCs are a source of multipotent cells that have neurogenic potential in vitro, and might be a useful tool to investigate the mechanisms of neural differentiation in relation to miRNA expression whose modulation might constitute new targeted therapeutic approaches to ALS. © 2013 Elsevier Inc.

(19)F-MRI offers unique opportunities to image diseases and track cells and therapeutic agents in vivo. Herein we report a superfluorinated molecular probe, herein called PERFECTA, possessing excellent cellular compatibility, and whose spectral properties, relaxation times, and sensitivity are promising for in vivo (19)F-MRI applications. The molecule, which bears 36 equivalent (19)F atoms and shows a single intense resonance peak, is easily synthesized via a simple one-step reaction and is formulated in water with high stability using trivial reagents and methods.

The thymus is the main site of immune sensitization to AChR in myasthenia gravis (MG). In our previous studies we demonstrated that Toll-like receptor (TLR) 4 is over-expressed in MG thymuses, suggesting its involvement in altering the thymic microenvironment and favoring autosensitization and autoimmunity maintenance processes, via an effect on local chemokine/cytokine network. Here, we investigated whether TLR4 signaling may favor abnormal cell recruitment in MG thymus via CCL17 and CCL22, two chemokines known to dictate immune cell trafficking in inflamed organs by binding CCR4. We also investigated whether TLR4 activation may contribute to immunodysregulation, via the production of Th17-related cytokines, known to alter effector T cell (Teff)/regulatory T cell (Treg) balance. We found that CCL17, CCL22 and CCR4 were expressed at higher levels in MG compared to normal thymuses. The two chemokines were mainly detected around medullary Hassall's corpuscles (HCs), co-localizing with TLR4(+) thymic epithelial cells (TECs) and CCR4(+) dendritic cells (DCs), that were present in higher number in MG thymuses compared to controls. TLR4 stimulation in MG TECs increased CCL17 and CCL22 expression and induced the production of Th17-related cytokines. Then, to study the effect of TLR4-stimulated TECs on immune cell interactions and Teff activation, we generated an in-vitro imaging model by co-culturing CD4(+) Th1/Th17 AChR-specific T cells, naive CD4(+)CD25(+) Tregs, DCs and TECs from Lewis rats. We observed that TLR4 stimulation led to a more pronounced Teff activatory status, suggesting that TLR4 signaling in MG thymic milieu may affect cell-to-cell interactions, favoring autoreactive T-cell activation. Altogether our findings suggest a role for TLR4 signaling in driving DC recruitment in MG thymus via CCL17 and CCL22, and in generating an inflammatory response that might compromise Treg function, favoring autoreactive T-cell pathogenic responses.

Background Among the various stem cell populations used for cell therapy, adult mesenchymal stromal cells (MSCs) have emerged as a major new cell technology. These cells must be tracked after transplantation to monitor their migration within the body and quantify their accumulation at the target site. This study assessed whether rat bone marrow MSCs can be labelled with superparamagnetic iron oxide (SPIO) nanoparticles and perfluorocarbon (PFC) nanoemulsion formulations without altering cell viability and compared magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) results from iron-labelled and fluorine-labelled MSCs, respectively. Methods Of MSCs, 2 × 106 were labelled with Molday ION Rhodamine-B (MIRB) and 2 × 106 were labelled with Cell Sense. Cell viability was evaluated by trypan blue exclusion method. Labelled MSCs were divided into four samples containing increasing cell numbers (0.125 × 106, 0.25 × 106, 0.5 × 106, 1 × 106) and scanned on a 7T MRI: for MIRB-labelled cells, phantoms and cells negative control, T1, T2 and T2* maps were acquired; for Cell Sense labelled cells, phantoms and unlabelled cells, a 19F non-localised single-pulse MRS sequence was acquired. Results In total, 86.8% and 83.6% of MIRB-labelled cells and Cell Sense-labelled cells were viable, respectively. MIRB-labelled cells were visible in all samples with different cell numbers; pellets containing 0.5 × 106 and 1 × 106 of Cell Sense-labelled cells showed a detectable 19F signal. Conclusions Our data support the use of both types of contrast material (SPIO and PFC) for MSCs labelling, although further efforts should be dedicated to improve the efficiency of PFC labelling.

To develop a preliminary version of a disease-specific, patient-reported disability assessment instrument for myasthenia gravis (MG) based on the International Classification of Functioning, Disability and Health (ICF): the MG-DIS.Five consecutive steps were taken: literature review and selection of outcome measures; linking of measures' concepts to ICF categories and selection of those reported by 30% of the instruments; comparison of linking results with a previous selection of MG-relevant ICF categories; patient interview; development of questions based on retained ICF categories.Thirty-one papers containing 21 different outcome measures were found: 13 ICF categories were linked to them. Fifty-five items were retained after the comparison with the list of MG-specific categories, and were used for patient interview. Thirteen interviews were conducted before saturation of data was reached and the final list was composed of 42 categories: based upon them, 44 questions were developed.The preliminary version of the MG-DIS contains more information than each single MG-specific tool, in particular, for the component of environmental factors. Further research is needed to test its psychometric properties.It is important that patient-reported outcome is incorporated in MG patient's assessment. MG features can be evaluated with ICF-based methods. An MG-specific patient-reported disability assessment instrument can be used to monitor changes of functioning in patients on MG-specific treatments, and can be used in clinical trials as outcome measure.

Introduction: Acquired myasthenia gravis (MG) is a rare disease and according to the EU Register of designated Orphan Medicinal Products only two drugs are registered as orphan drugs for MG while no orphan drug for MG has been approved by the US Food and Drug Administration. The aim of this review is to discuss the role and mechanism of action of the recognized orphan drugs for MG and of other therapies potentially useful for MG treatment, which should be considered as orphan drugs. Areas covered: In the context of an updated overview of MG pathogenesis, the authors discuss current MG treatments available in clinical practice, orphan drugs for MG according to the EU Orphan Drugs Registry and emerging therapies, among which are therapies exclusively developed for MG. Expert opinion: New effective drugs for MG are needed to reduce side effects, mainly those related to steroids or to chronic immunosuppression, and to manage patients not responsive to common treatments. Hence, in recent years drugs potentiall...

The authors have analyzed single nucleotide polymorphisms in the thiopurine S-methyltransferase (TPMT) gene in the context of efficacy and toxicity of azathioprine (AZA) to determine possible genotype-phenotype correlations between TPMT allelic variants and response to AZA treatment in 76 Italian patients with myasthenia gravis. They confirm known intronic and exonic TPMT polymorphisms that do not correlate with AZA responses and demonstrate a novel intronic polymorphism in a patient intolerant to AZA. Most importantly, they show that of the 22 AZA-intolerant patients, all 5 who carried mutations of the intolerance-linked haplotype TPMT*3A also carried the intronic T140+114A (rs3931660), all 3 mutations being part of a new haplotype designated TMPT*3E. TPMT*3E was not observed in unresponsive or responsive patients. The association of TPMT*3E with AZA intolerance and its frequency must be ascertained in larger, ethnically different cohorts. Nevertheless, in view of the highly significant association (Psim = 0.0026) between TPMT*3E and AZA intolerance in the study, this new haplotype should be taken into consideration in pharmacogenetic profiling for AZA.

Clinical registries may facilitate research on myasthenia gravis (MG) in several ways: as a source of demographic, clinical, biological, and immunological data on large numbers of patients with this rare disease; as a source of referrals for clinical trials; and by allowing rapid identification of MG patients with specific features. Physician-derived registries have the added advantage of incorporating diagnostic and treatment data that may allow comparison of outcomes from different therapeutic approaches, which can be supplemented with patient self-reported data. We report the demographic analysis of MG patients in two large physician-derived registries, the Duke MG Patient Registry, at the Duke University Medical Center, and the INNCB MG Registry, at the Istituto Neurologico Carlo Besta, as a preliminary study to assess the consistency of the two data sets. These registries share a common structure, with an inner core of common data elements (CDE) that facilitate data analysis. The CDEs are concordant with the MG-specific CDEs developed under the National Institute of Neurological Disorders and Stroke Common Data Elements Project.

Multiple Sclerosis (MS) is an inflammatory disease with neurodegenerative alterations, ultimately progressing to neurological handicap. Therapies are effective in counteracting inflammation but not neurodegeneration. Biomarkers predicting disease course or treatment response are lacking. We investigated whether altered gene and protein expression profiles were detectable in the peripheral blood of 78 relapsing remitting MS (RR-MS) patients treated by disease-modifying therapies. A discovery/validation study on RR-MS responsive to glatiramer acetate identified 8 differentially expressed genes: ITGA2B, ITGB3, CD177, IGJ, IL5RA, MMP8, P2RY12, and S100β. A longitudinal study on glatiramer acetate, Interferon-β, or Fingolimod treated RR-MS patients confirmed that 7 out of 8 genes were downregulated with reference to the different therapies, whereas S100β was always upregulated. Thus, we identified a peripheral gene signature associated with positive response in RR-MS which may also explain drug immunomodulatory effects. The usefulness of this signature as a biomarker needs confirmation on larger series of patients.

Myasthenia gravis (MG) is a chronic autoimmune disease caused by the immune attack of the neuromuscular junction. Antibodies directed against the acetylcholine receptor (AChR) induce receptor degradation, complement cascade activation, and postsynaptic membrane destruction, resulting in functional reduction in AChR availability. Besides anti-AChR antibodies, other autoantibodies are known to play pathogenic roles in MG. The experimental autoimmune MG (EAMG) models have been of great help over the years in understanding the pathophysiological role of specific autoantibodies and T helper lymphocytes and in suggesting new therapies for prevention and modulation of the ongoing disease. EAMG can be induced in mice and rats of susceptible strains that show clinical symptoms mimicking the human disease. EAMG models are helpful for studying both the muscle and the immune compartments to evaluate new treatment perspectives. In this review, we concentrate on recent findings on EAMG models, focusing on their utility and limitations.

Myasthenia gravis (MG) is a T-cell dependent autoimmune disorder of the neuromuscular junction, characterised by muscle weakness and fatigability. Autoimmunity is thought to initiate in the thymus of acetylcholine receptor (AChR)-positive MG patients; however, the molecular mechanisms linking intra-thymic MG pathogenesis with autoreactivity via the circulation to the muscle target organ are poorly understood. Using whole-transcriptome sequencing, we compared the transcriptional profile of peripheral blood mononuclear cells from AChR-early onset MG (AChR-EOMG) patients with healthy controls: 178 coding transcripts and 229 long non-coding RNAs, including 11 pre-miRNAs, were differentially expressed. Among the 178 coding transcripts, 128 were annotated of which 17% were associated with the ‘infectious disease’ functional category and 46% with ‘inflammatory disease’ and ‘inflammatory response-associated’ categories. Validation of selected transcripts by qPCR indicated that of the infectious disease-related transcripts, ETF1, NFKB2, PLK3, and PPP1R15A were upregulated, whereas CLC and IL4 were downregulated in AChR-EOMG patients; in the ‘inflammatory’ categories, ABCA1, FUS, and RELB were upregulated, suggesting a contribution of these molecules to immunological dysfunctions in MG. Data selection and validation were also based on predicted microRNA-mRNA interactions. We found that miR-612, miR-3654, and miR-3651 were increased, whereas miR-612-putative AKAp12 and HRH4 targets and the miR-3651-putative CRISP3 target were downregulated in AChR-EOMG, also suggesting altered immunoregulation. Our findings reveal a novel peripheral molecular signature in AChR-EOMG, reflecting a critical involvement of inflammatory- and infectious disease-related immune responses in disease pathogenesis.

The recommendations for clinical research standards published in 2000 by a task force of the Medical Scientific Advisory Board (MSAB) of the Myasthenia Gravis Foundation of America (MGFA) were largely successful in introducing greater uniformity in the recording and reporting of MG clinical trials. Recognizing that changes in clinical trial design and implementation may increase the likelihood that new therapies are developed for MG, the MGFA MSAB Task Force here presents updated recommendations for the design and implementation of clinical trials in MG, including (a) the use of a quantitative measure, such as the MG-Composite, that is weighted for clinical significance and incorporates patient reported outcomes; (b) consideration of nontrial strategies; and (c) development of biomarkers that support mechanistic studies of pharmacotherapies. The hope is that these updated task force recommendations will expedite the development and acceptance of more effective and less noxious therapies for MG.

In the current study, we investigated whether naturally occurring CD4+CD25+ T cells, separated by immunomagnetic anti-CD4 and anti-CD25 Abs from naive animals, are able to protect from experimental autoimmune myasthenia gravis (EAMG) and modify the progression of ongoing disease when administered to Torpedo californica acetylcholine receptor (AChR)-immunized Lewis rats. Even though CD4+CD25+ and CD4+CD25high T cell frequencies were similar in the spleens and lymph nodes of EAMG and healthy rats, we observed that CD4+CD25+ T cells isolated from the spleens of naive animals inhibited in vitro the Ag-induced proliferation of T cell lines specific to the self-peptide 97–116 of the anti-AChR subunit (R97-116), an immunodominant and myasthenogenic T cell epitope, whereas CD4+CD25+ T cells purified from the spleens of EAMG rats were less effective. CD4+CD25+ T cells from EAMG rats expressed less forkhead box transcription factor P3 but more CTLA-4 mRNA than healthy rats. Naive CD4+CD25+ T cells, obtained from naive rats and administered to T. californica AChR-immunized animals according to a preventive schedule of treatment, reduced the severity of EAMG, whereas their administration 4 wk postinduction of the disease, corresponding to the onset of clinical symptoms (therapeutic treatment), was not effective. We think that the exogenous administration of CD4+CD25+ naive T cells prevents the early events underlying the induction of EAMG, events linked to the T cell compartment (Ag recognition, epitope spreading, and T cell expansion), but fails to ameliorate ongoing EAMG, when the IgG-mediated complement attack to the AChR at the neuromuscular junction has already taken place.

Background: Genetic and environmental factors are thought to contribute to the etiology of the autoimmune disease myasthenia gravis (MG). Viral involvement has long been suspected, but direct evidence of involvement has not been found. We recently reported that Toll-like receptor 4 (TLR4)—a key activator of innate immunity—was overexpressed in the thymus of some patients with MG, suggesting that thymic infection by pathogens might be involved in MG pathogenesis. We searched for evidence of intrathymic infection in patients with MG. Methods: Twenty-seven MG thymuses (6 involuted, 7 hyperplastic, 5 thymitis, and 9 thymoma) previously tested for TLR4 expression, 18 nonpathologic control thymuses, and 10 pathologic control thymuses from patients without MG (8 thymoma and 2 hyperplastic) were analyzed for cytomegalovirus, varicella-zoster virus, herpes simplex virus types 1 and 2, eubacteria, respiratory syncytial virus, and enteroviruses using PCR techniques. Immunohistochemistry and double immunofluorescence were used to detect enterovirus capsid protein VP1 in thymic specimens and analyze TLR4 expression in VP1-positive cells. Results: Poliovirus was detected in 4 MG thymuses (14.8%; 2 thymitis and 2 thymoma). No virus was detected in any control thymus. A linear correlation between plus and minus strand poliovirus RNA levels was observed in all 4 thymuses, suggesting persistent thymic infection. VP1 protein was detected in the cytoplasm of CD68-positive macrophages scattered through thymic medulla in all PV-positive thymuses. VP1 and TLR4 colocalized in infected cells. Conclusions: Poliovirus-infected macrophages are present in thymus of some patients with myasthenia gravis, suggesting a viral contribution to the intrathymic alterations leading to the disease.

We investigated the effect of IgG immunoadsorption (IA) on cytokine network in patients with treatment-resistant Myasthenia Gravis (MG) and Lambert-Eaton Syndrome (LEMS). We observed upregulation of interleukin (IL)-10, an anti-inflammatory and B cells growth factor, and reduction of pro-inflammatory factors such as IL-18 and IL-17, in both MG and LEMS after IA. Our observation suggests that the massive removal of antibodies might induce modifications of the cytokine balance linked to T and B cells mediated autoimmunity.

The idiopathic inflammatory myopathies (IIMs) dermatomyositis, polymyositis, and inclusion body myositis are characterized by myofiber degeneration and inflammation. The triggering factors of muscle autoaggression in these disorders are unknown, but infiltrating T cells may be activated locally and proliferate in situ. T-cell polarization involving reorientation of cytoskeleton and microtubule-organizing centers mediated by motor proteins may occur within inflammatory cells in the muscle. We therefore analyzed ubiquitous and neuronal kinesin superfamily (KIF) members KIF-5, dynein, and KIF4 in IIM muscle biopsies and in activated peripheral blood lymphocytes from healthy donors. Only KIF-4 was altered. Transcript levels were significantly higher in IIM muscle than in controls, and KIF4 inflammatory cells were found in IIM muscles. In polymyositis and inclusion body myositis, KIF4 cells were mainly located around individual muscle fibers, whereas in dermatomyositis, they were also near blood vessels. KIF4 cells were not specific to any immune lineage, and some were Ki67. In peripheral blood lymphocytes stimulated with mitogens, interleukin 2 or anti-CD3/CD28 antibodies, KIF4 expression was upregulated, and the protein was localized in the cytoplasm in association with lysosome-associated membrane protein 1 and perforin lysosomal vesicles. These results imply that KIF4 is associated with activated T cells, irrespective of their functional phenotype, and that it is likely involved in cytoskeletal modifications associated with in situ T-cell activation in IIM.

Neural stem cells (NSCs) transplantation has been proposed as a means of restoring damaged brain tissue, a possibility rendered more likely by reports of low NSCs immunogenicity in various experimental models because of low expression of MHC class I and II as well as co-stimulatory molecules. We investigated the immunogenicity of a human NSC line grown in normal culture conditions and in the presence of pro-inflammatory cytokines IFN-gamma and tumor necrosis factor alpha by one-way mixed lymphocyte reaction (MLR) experiments with peripheral blood lymphocytes from eight HLA-incompatible donors. NSCs stimulated lymphocyte proliferation in almost all donors tested, with stimulation indices in the range of the low-end distribution curve of MLR between donors. The healthy subject that gave negative MLR results was the best compatible donor with respect to NSC haplotype. Since we observed low MLR responses overall, we studied if NSCs might exert any immunomodulatory activity. We detected transcription and release of the immunomodulatory molecule transforming growth factor beta (TGF-beta)-1; moreover, the addition of TGF-beta1 in MLR experiments down-regulated proliferative responses. To further confirm the immunological potential of human NSCs, we studied xenogeneic recognition of NSCs by immunocompetent cells derived from C57BL/6 mice, showing that NSCs can elicit an allo(xeno) response ex vivo. Our data indicate that NSCs have low but not negligible immunogenic potential that is sufficient to activate peripheral lymphocytes. Secretion of TGF-beta1 might balance the immunogenicity of NSCs. Nevertheless, the possibility that allo-NSCs grafting might induce in the long term an immune activation, thus vanishing their therapeutical effect, should not be overlooked and deserves further investigation.