B-cell immune dysregulation with low soluble CD22 levels in refractory seronegative myasthenia gravis.

Myasthenia gravis (MG), primarily caused by acetylcholine receptor (AChR) autoantibodies, is a chronic autoimmune disorder causing severe muscle weakness and fatigability. In particular, seronegative MG constitutes 10%-15% of MG cases and presents diagnostic challenges especially in early-onset female patients who often show severe disease and resistance to immunosuppressive therapy. Furthermore, the immunopathology of seronegative MG remains unclear. Thus, in this study, we aimed to elucidate the pathogenic mechanism of seronegative MG using scRNA-seq analysis and plasma proteome analysis; in particular, we investigated the relationship between immune dysregulation status and disease severity in refractory seronegative MG. Employing single-cell RNA-sequencing and plasma proteome analyses, we analyzed peripheral blood samples from 30 women divided into three groups: 10 healthy controls, 10 early-onset AChR-positive MG, and 10 refractory early-onset seronegative MG patients, both before and after intravenous immunoglobulin treatment. The disease severity was evaluated using the MG-Activities of Daily Living (ADL), MG composite (MGC), and revised 15-item MG-Quality of Life (QOL) scales. We observed numerical abnormalities in multiple immune cells, particularly B cells, in patients with refractory seronegative MG, correlating with disease activity. Notably, severe MG cases had fewer regulatory T cells without functional abnormalities. Memory B cells were found to be enriched in peripheral blood cells compared with naïve B cells. Moreover, plasma proteome analysis indicated significantly lower plasma protein levels of soluble CD22, expressed in the lineage of B-cell maturation (including mature B cells and memory B cells), in refractory seronegative MG patients than in healthy donors or patients with AChR-positive MG. Soluble CD22 levels were correlated with disease severity, B-cell frequency, and RNA expression levels of CD22. In summary, this study elucidates the immunopathology of refractory seronegative MG, highlighting immune disorders centered on B cells and diminished soluble CD22 levels. These insights pave the way for novel MG treatment strategies focused on B-cell biology.

A retrospective multicenter study on clinical and serological parameters in patients with MuSK myasthenia gravis with and without general immunosuppression.

Introduction: Muscle-specific kinase (MuSK)- myasthenia gravis (MG) is caused by pathogenic autoantibodies against MuSK that correlate with disease severity and are predominantly of the IgG4 subclass. The first-line treatment for MuSK-MG is general immunosuppression with corticosteroids, but the effect of treatment on IgG4 and MuSK IgG4 levels has not been studied. Methods: We analyzed the clinical data and sera from 52 MuSK-MG patients (45 female, 7 male, median age 49 (range 17-79) years) from Italy, the Netherlands, Greece and Belgium, and 43 AChR-MG patients (22 female, 21 male, median age 63 (range 2-82) years) from Italy, receiving different types of immunosuppression, and sera from 46 age- and sex-matched non-disease controls (with no diagnosed diseases, 38 female, 8 male, median age 51.5 (range 20-68) years) from the Netherlands. We analyzed the disease severity (assessed by MGFA or QMG score), and measured concentrations of MuSK IgG4, MuSK IgG, total IgG4 and total IgG in the sera by ELISA, RIA and nephelometry. Results: We observed that MuSK-MG patients showed a robust clinical improvement and reduction of MuSK IgG after therapy, and that MuSK IgG4 concentrations, but not total IgG4 concentrations, correlated with clinical severity. MuSK IgG and MuSK IgG4 concentrations were reduced after immunosuppression in 4/5 individuals with before-after data, but data from non-linked patient samples showed no difference. Total serum IgG4 levels were within the normal range, with IgG4 levels above threshold (1.35g/L) in 1/52 MuSK-MG, 2/43 AChR-MG patients and 1/45 non-disease controls. MuSK-MG patients improved within the first four years after disease onset, but no further clinical improvement or reduction of MuSK IgG4 were observed four years later, and only 14/52 (26.92%) patients in total, of which 13 (93.3%) received general immunosuppression, reached clinical remission. Discussion: We conclude that MuSK-MG patients improve clinically with general immunosuppression but may require further treatment to reach remission. Longitudinal testing of individual patients may be clinically more useful than single measurements of MuSK IgG4. No significant differences in the serum IgG4 concentrations and IgG4/IgG ratio between AChR- and MuSK-MG patients were found during follow-up. Further studies with larger patient and control cohorts are necessary to validate the findings.

Mendelian randomization and colocalization analysis reveal novel drug targets for myasthenia gravis.

OBJECTIVE: Myasthenia gravis (MG) is a complex autoimmune disease affecting the neuromuscular junction with limited drug options, but the field of MG treatment recently benefits from novel biological agents. We performed a drug-targeted Mendelian randomization (MR) study to identify novel therapeutic targets of MG. METHODS: Cis-expression quantitative loci (cis-eQTL), which proxy expression levels for 2176 druggable genes, were used for MR analysis. Causal relationships between genes and disease, identified by eQTL MR analysis, were verified by comprehensive sensitivity, colocalization, and protein quantitative loci (pQTL) MR analyses. The protein-protein interaction (PPI) analysis was also performed to extend targets, followed by enzyme-linked immunosorbent assay (ELISA) to explore the serum level of drug targets in MG patients. A phenome-wide MR analysis was then performed to assess side effects with a clinical trial review assessing druggability. RESULTS: The eQTL MR analysis has identified eight potential targets for MG, one for early-onset MG and seven for late-onset MG. Further colocalization analyses indicated that CD226, CDC42BPB, PRSS36, and TNFSF12 possess evidence for colocalization with MG or late-onset MG. pQTL MR analyses identified the causal relations of TNFSF12 and CD226 with MG and late-onset MG. Furthermore, PPI analysis has revealed the protein interaction between TNFSF12-TNFSF13(APRIL) and TNFSF12-TNFSF13B(BLyS). Elevated TNFSF13 serum level of MG patients was also identified by ELISA experiments. This study has ultimately proposed three promising therapeutic targets (TNFSF12, TNFSF13, TNFSF13B) of MG. CONCLUSIONS: Three drug targets associated with the BLyS/APRIL pathway have been identified. Multiple biological agents, including telitacicept and belimumab, are promising for MG therapy.

Serum irisin levels in patients with myasthenia gravis.

OBJECTIVE: Myasthenia gravis (MG) is an autoimmune disorder whose main symptoms are muscle weakness and fatigue. Irisin is a novel skeletal muscle-derived myokine participating in several physiological and pathological processes. The initial objective of the project was to explore serum levels of irisin in patients with MG, as well as its correlation with disease severity. METHODS: We retrospectively evaluated serum levels of irisin in 77 MG patients and 57 healthy controls (HCs) by enzyme-linked immunosorbent assay. Further, clinical parameters were measured properly. RESULTS: Serum irisin levels were significantly elevated in MG patients compared with HCs (p < 0.001). Furthermore, serum irisin levels were associated with the myasthenia gravis activities of daily living score in ocular myasthenia gravis (OMG) patients (r = 0.476, p = 0.004), but there was no relationship to be considered of any relevant value in generalized myasthenia gravis (GMG) patients. Acetylcholine receptor antibody-positive MG patients had higher serum irisin levels compared with HCs. Thymoma, endotracheal intubation, or intensive care unit treatments subsequently were not found to have effect on serum levels of irisin, but tendencies of increase were observed in negative ones. CONCLUSIONS: Serum irisin levels were elevated in patients with MG, suggesting its possible involvement in MG. And irisin is expected to be a signal to evaluate the activities of daily living of OMG patients, while its effect needs further study.

Effect of Buzhong Yiqi decoction on anti-acetylcholine receptor antibody and clinical status in juvenile ocular myasthenia gravis.

ABSTRACT: Ocular myasthenia gravis (MG) is the mainly widespread type of MG among juveniles. Buzhong Yiqi decoction (BZ) is a well-known traditional Chinese medicine prescription for treating MG. It has rarely been discussed whether the concentration of anti-acetylcholine receptor (AChR) antibodies is related to the clinical status of juvenile ocular myasthenia gravis (JOMG) treated with BZ.The patients with JOMG who had more than once AChR-antibody (ab) test and treated with BZ were retrospectively identified from June 2013 to January 2020 in the first hospital in Shijiazhuang. The presence or absence of ocular symptoms was used to grade the effectiveness of treatment. Generalized estimating equations logistic regression analysis was used to evaluate the effect of AChR ab concentration on the clinical status of MG.A total of 549 AChR-ab tests were performed in 135 patients, and the corresponding clinical status was recorded. One hundred two patients received treatment with BZ only and 33 combined with immunosuppressive drugs. In the group receiving only BZ treatment, the anti-acetylcholine receptor ab concentration was positively correlated with the clinical score after treatment.The results suggest that BZ could affect the AChR-ab. Repeated AChR-ab assays can provide information about the clinical status. For JOMG patients who only receive Buzhong Yiqi Decoction treatment, this can support treatment decisions.

Laboratory Investigation of Hybrid IgG4 k/λ in MuSK Positive Myasthenia Gravis.

Myasthenia gravis with antibodies (Abs) against the muscle-specific tyrosine kinase (MuSK) is a rare autoimmune disorder (AD) of the neuromuscular junction (NMJ) and represents a prototype of AD with proven IgG4-mediated pathogenicity. Thanks to the mechanism of Fab-arm exchange (FAE) occurring in vivo, resulting MuSK IgG4 k/λ Abs increase their interference on NMJ and pathogenicity. The characterization of hybrid MuSK IgG4 as a biomarker for MG management is poorly investigated. Here, we evaluated total IgG4, hybrid IgG4 k/λ, and the hybrid/total ratio in 14 MuSK-MG sera in comparison with 24 from MG with Abs against acetylcholine receptor (AChR) that represents the not IgG4-mediated MG form. In both subtypes of MG, we found that the hybrid/total ratio reflects distribution reported in normal individuals; instead, when we correlated the hybrid/total ratio with specific immune-reactivity we found a positive correlation only with anti-MuSK titer, with a progressive increase of hybrid/total mean values with increasing disease severity, indirectly confirming that most part of hybrid IgG4 molecules are engaged in the anti-MuSK pathogenetic immune-reactivity. Further analysis is necessary to strengthen the significance of this less unknown biomarker, but we retain it is full of a diagnostic-prognostic powerful potential for the management of MuSK-MG.

Intrathymic Plasmablasts Are Affected in Patients With Myasthenia Gravis With Active Disease.

BACKGROUND AND OBJECTIVES: To investigate intrathymic B lymphopoiesis in patients with myasthenia gravis (MG) and explore thymus pathology associated with clinical impact. METHODS: Thymic lymphocytes from 15 young patients without MG, 22 adult patients without MG, 14 patients with MG without thymoma, and 11 patients with MG with thymoma were subjected to flow cytometry analysis of T follicular helper (Tfh), naive B, memory B, plasmablasts, CD19+B220high thymic B cells, B-cell activating factor receptor, and C-X-C chemokine receptor 5 (CXCR5). Peripheral blood mononuclear cells of 16 healthy subjects and 21 untreated patients with MG were also analyzed. Immunologic values were compared, and correlations between relevant values and clinical parameters were evaluated. RESULTS: The frequencies of circulating and intrathymic plasmablasts were significantly higher in patients with MG than controls. On the other hand, the frequency of CD19+B220high thymic B cells was not increased in MG thymus. We observed a significant increase in CXCR5 expression on plasmablasts in MG thymus and an increased frequency of intrathymic plasmablasts that was correlated with preoperative disease activity. The frequency of intrathymic Tfh cells was significantly lower in patients who received immunosuppressive (IS) therapy than those without IS therapy. However, there was no significant difference in the frequency of intrathymic plasmablasts irrespective of IS therapy. DISCUSSION: Our findings confirmed a correlation between increased frequency of intrathymic plasmablasts and disease activity before thymectomy. We postulate that activated intrathymic plasmablasts endow pathogenic capacity in MG.

Differential Expression of miRNA in the Peripheral Blood Mononuclear Cells in Myasthenia Gravis with Muscle-Specific Receptor Tyrosine Kinase Antibodies.

To determine if differential profile of miRNAs in peripheral blood mononuclear cells (PBMCs) could be identified in muscle-specific receptor tyrosine kinase antibody positive myasthenia gravis (MuSK-MG) and linked to disease stage, a case-control method was used to compare the difference in miRNA expression profiles of PBMCs using next generation sequencing (NGS) in MuSK-MG patients and healthy controls (HCs). Six significant miRNAs from the discovery set were then validated using RT-qPCR in 11 MuSK-MG patients and 10 HCs. A unique miRNA prediction algorithm was used to predict the target genes of differentially expressed miRNAs and a network of miRNA gene pathways. Compared with HCs, 101 differentially expressed miRNAs were screened in MuSK-MG, of which 5 miRNAs were upregulated, and 96 miRNAs were downregulated. The top six differentially expressed molecules were selected for verification; four of them (miR-340-5p, miR-106b-5p, miR-27a-3p, and miR-15a-3p) were significantly different. The network analysis of miRNA gene pathways revealed that differentially expressed miRNAs were involved in a complex set of biological processes. Clinically, the four miRNAs that were validated are not correlated to MuSK antibody titers and quantitative myasthenia gravis score. Four miRNAs that were validated in this study have specificity to distinguish MuSK-MG from HCs.

Melanoma Cell Adhesion Molecule Expressing Helper T Cells in CNS Inflammatory Demyelinating Diseases.

BACKGROUND AND OBJECTIVE: To elucidate the relationship between melanoma cell adhesion molecule (MCAM)-expressing lymphocytes and pathogenesis of CNS inflammatory demyelinating diseases (IDDs). METHODS: Patients with multiple sclerosis (MS) (n = 72) and neuromyelitis optica spectrum disorder (NMOSD, n = 29) were included. We analyzed the frequency and absolute numbers of MCAM+ lymphocytes (memory helper T [mTh] cells, naive helper T cells, CD8+ T cells, and B cells) in the peripheral blood (PB) and the CSF of patients with MS and NMOSD, treated with/without disease-modifying drugs (DMDs) or steroids, using flow cytometry. RESULTS: The frequency of MCAM+ cells was higher in the mTh cell subset than that in other lymphocyte subsets. A significant increase in the frequency and the absolute number of MCAM+ mTh cells was observed in the PB of patients with NMOSD, whereas no increase was observed in the PB of patients with MS. The frequency of CSF MCAM+ mTh cells was higher in relapsing patients with MS and NMOSD than that in the control group. Although there was no difference in the frequencies of MCAM+ lymphocytes among the DMD-treated groups, fingolimod decreased the absolute number of MCAM+ lymphocytes. DISCUSSION: MCAM+ mTh cells were elevated in the CSF of relapsing patients with MS and in both the PB and CSF of patients with NMOSD. These results indicate that MCAM contributes to the pathogenesis of MS and NMOSD through different mechanisms. MCAM could be a therapeutic target of CNS IDDs, and further study is needed to elucidate the underlying mechanism of MCAM in CNS IDD pathogenesis.

Acetylcholine receptor antibodies in a patient with sensory neuropathy.

Antibodies to acetylcholine receptor (AChR) are detected in the vast majority of patients with generalized myasthenia gravis (MG) and are rarely detected in significant titer in other autoimmune diseases. We report a patient with an axonal predominately sensory neuropathy for over 12 years with persistent binding and modulating AChR antibodies as well as striational muscle antibodies with no evidence of MG or any neoplastic disease.

Myasthenia gravis associated with renal cell carcinoma: a paraneoplastic syndrome or just a coincidence.

BACKGROUND: Myasthenia gravis (MG) can occur as a paraneoplastic phenomenon associated with thymoma. The association of MG with renal cell carcinoma (RCC) is not clear. Herein, we describe six cases of MG associated with RCC. METHODS: There were 283 patients diagnosed with MG admitted to our hospital from 2014 to 2019. Among them, 6 patients also had RCC. None of them had immune checkpoint inhibitor therapies. We performed a retrospective clinical data collection and follow-up studies of these 6 patients. RESULTS: These 6 patients with an average MG onset age of 61.3 ± 13.3 years, were all positive for anti-acetylcholine receptor antibodies. MG symptoms appeared after RCC resection in 3 cases. RCC was discovered after the onset of MG in 2 cases, and synchronously with MG in 1 case. After nephrectomy, the MG symptoms showed a stable complete remission in 1 case. Among them, four patients met the diagnostic criteria of possible paraneoplastic neurological syndromes. CONCLUSIONS: Except for thymoma, patients with MG should pay attention to other tumors including RCC. MG may be a paraneoplastic syndrome of RCC, and further studies are needed to elucidate the relationship.

A Stable Cell Line Expressing Clustered AChR: A Novel Cell-Based Assay for Anti-AChR Antibody Detection in Myasthenia Gravis.

Cell-based assays (CBAs) and radioimmunoprecipitation assay (RIPA) are the most sensitive methods for identifying anti-acetylcholine receptor (AChR) antibody in myasthenia gravis (MG). But CBAs are limited in clinical practice by transient transfection. We established a stable cell line (KL525) expressing clustered AChR by infecting HEK 293T cells with dual lentiviral vectors expressing the genes encoding the human AChR α1, ß1, δ, ϵ and the clustering protein rapsyn. We verified the stable expression of human clustered AChR by immunofluorescence, immunoblotting, and real-time PCR. Fluorescence-activated cell sorting (FACS) was used to detect anti-AChR antibodies in 103 MG patients and 58 healthy individuals. The positive results of MG patients reported by the KL525 was 80.6% (83/103), 29.1% higher than the 51.4% (53/103) of RIPA. 58 healthy individuals tested by both the KL525 CBA and RIPA were all negative. In summary, the stable expression of clustered AChR in our cell line makes it highly sensitive and advantageous for broad clinical application in CBAs.

Sexual dysfunction in patients with myasthenia gravis.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune disease that causes fatigueable muscle weakness. Sexual dysfunction (SD) is a common condition, but the association between SD and MG remains poorly understood. METHODS: An observational study was conducted to explore SD incidence and risk factors in MG patients. The study enrolled 158 MG patients and 161 age- and sex-matched healthy individuals. SD was investigated using the Female Sexual Function Inventory (FSFI), the abridged International Index of Erectile Function-5 (IIEF-5), and the Chinese Index of Premature Ejaculation-5 (CIPE-5). The mental health was evaluated using Hamilton Depression Scale (HAMD) and Hamilton Anxiety Scale (HAMA). RESULTS: A total of 52 male patients and 106 female patients were finally included. The average age of these patients was 41.82 ± 10.44 years. The incidence of female SD was significantly higher in MG patients (48.11%) than in healthy people (22.64%) (P < 0.001). The incidence of SD in male MG patients was also higher. Age and depression were significantly correlated with decreased libido, wakefulness, lubrication, orgasm, and satisfaction scores, indicating that these are risk factors for SD. Age (OR:1.13, CI%:1.06-1.21, P < 0.001) and HAMD scores (OR:1.53, CI%:1.0-2.13, P = 0.011) are independent risk factors for SD of MG patients. CONCLUSION: SD is a common problem in MG, and its severity does not change with the severity of the disease. Age and depression are risk factors for sexual dysfunction.

Cholinergic hyperactivity in patients with myasthenia gravis with MuSK antibodies: A neurophysiological study.

OBJECTIVE: Patients with myasthenia gravis associated with muscle-specific tyrosine kinase antibodies (MuSK-MG) often manifest signs of cholinergic hyperactivity with standard doses of acetylcholinesterase inhibitors (AChE-Is). Aim of the study was to investigate whether repetitive compound muscle action potential (R-CMAP), the neurophysiological correlate of cholinergic hyperactivity, was present in MuSK-MG irrespective of AChE-I treatment. METHODS: Patients with confirmed diagnosis of MuSK-MG were consecutively enrolled during follow-up visits, from January 2019 to April 2020. All these subjects underwent the same neurophysiological protocol, including motor nerve conduction studies and repetitive nerve stimulation. In patients taking pyridostigmine, neurophysiological testing was performed at least 12 hours after the last dose. For comparison, the presence of R-CMAP was investigated in 20 consecutive acetylcholine receptor antibody positive myasthenia gravis (AChR-MG) patients. RESULTS: We enrolled 25 MuSK-MG patients (20 females), aged 16-79 years at the study time, with disease duration ranging 0.6-48.8 years (median: 17.7 years). R-CMAP was detected in 12/25 (48%) MuSK-MG cases and in none of the AChR-MG controls (p = 0.0003). In the MuSK-MG population, a history of muscle cramps and fasciculations, during low-dose pyridostigmine therapy, was significantly more frequent in R-CMAP positive than in R-CMAP negative patients (100% vs 31%, p = 0.001). At the time of the study, the proportion of patients still symptomatic for MG was higher among R-CMAP positive cases (92% vs 23%, p = 0.0005). CONCLUSIONS: Cholinergic hyperactivity is a relatively common finding in MuSK-MG patients, independent of AChE-I treatment, and may constitute an intrinsic feature of the disease. SIGNIFICANCE: R-CMAP detection can represent a useful diagnostic clue for MuSK-MG and predicts poor tolerance to AChE-Is.

Myasthenia gravis exacerbation in association with antibody overshoot following plasmapheresis.

INTRODUCTION/AIM: Antibody overshoot following therapeutic plasmapheresis (PLEX) is defined by subsequent increase in antibody to levels exceeding those prior to removal. It has been infrequently described in the past, and its influence on the clinical course of myasthenia gravis (MG) remains unclear. METHODS: This was a retrospective analysis of five patients with generalized MG treated with PLEX. RESULTS: All five patients possessed antibodies against acetylcholine receptor (AChR-Ab). After undergoing 3 to 12 PLEX treatment sessions, AChR-Ab titer increased to a median of 1292% of the baseline level. The median interval from the last PLEX session to peak AChR-Ab detection was 6 wk. In four patients, AChR-Ab overshoot was associated with a clinical deterioration. DISCUSSION: The AChR-Ab overshoot may occur following PLEX. In patients who deteriorate following PLEX treatment, the presence of antibody overshoot may serve as additional guidance for treatment adjustment.

High levels of serum interleukin-6 are associated with disease activity in myasthenia gravis.

Myasthenia gravis (MG), a neuromuscular junction disorder, is caused by pathogenic autoantibodies. Interleukin-6 (IL-6) plays important roles in T helper 17 (Th17), T follicular helper (Tfh), and B cell activations as well as in antibody production. This study aimed to evaluate the clinical significance of serum IL-6 level as a biomarker of disease activity in patients with anti-acetylcholine receptor (AChR) antibody-positive MG. In the present study, serum IL-6 levels were measured in 93 treatment-naïve patients with anti-AChR antibody-positive MG and compared with those in 101 controls. Moreover, correlations between serum IL-6 levels and clinical characteristics were analyzed. Serum IL-6 levels were significantly higher in patients with anti-AChR antibody-positive MG than in controls (median [interquartile range], 2.5 [1.5-8.3] pg/mL vs. 1.5 [1.5-3.2] pg/mL, P < 0.001). The serum levels were correlated with the MG Foundation of America clinical classification (Spearman's ρ = 0.27; P < 0.01) and decreased following immunosuppressive treatment in parallel with disease activity (P = 0.01). In conclusion, IL-6 is involved in the pathogenesis of anti-AChR antibody-positive MG and could be a therapeutic target in MG.

Natural killer cells promote the differentiation of follicular helper T cells instead of inducing apoptosis in myasthenia gravis.

Recent evidence has shown that natural killer (NK) cells have an immunoregulatory function in the pathogenesis of myasthenia gravis (MG). In this study, the phenotype and function of NK cell subsets in peripheral blood of new-onset MG (N-MG) and stable MG (S-MG) patients were explored. Circulating CD56dim and CD56bright NK cells were increased and decreased, respectively, in patients with N-MG and S-MG compared with healthy control (HC). Moreover, all circulating NK cell subsets from N-MG patients showed significantly lower expression of activating receptor NKG2D and production of Interferon (IFN) -γ than that from HC. The killing effects of NK cells on CD4+ T cells and Tfh cells were impaired in MG patients, whereas, they promoted the differentiation and activation of Tfh cells. These data indicated that the immune-regulation of NK cells on CD4+ T cells and Tfh cells in MG patients was abnormal, which may contribute to the immune-pathological mechanism of MG.