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The use of rituximab (RTX), an anti-CD20 monoclonal antibody (Ab), in refractory myasthenia gravis (MG) is associated with a better response in patients with Abs to the muscle-specific tyrosine kinase (MuSK) than in other MG subgroups. Anti-MuSK Abs are mostly IgG4 with proven pathogenicity and positive correlation with clinical severity. The rapid and sustained response to RTX may be related to MuSK Ab production by short-lived Ab-secreting cells derived from specific CD20+ B cells. Here, we investigated the long-term effects of RTX in nine refractory MuSK-MG patients with a follow-up ranging from 17 months to 13 years. In patients’ sera, we titrated MuSK-specific IgG (MuSK-IgG) and MuSK-IgG4, along with total IgG and IgG4 levels. Optimal response to RTX was defined as the achievement and maintenance of the status of minimal manifestations (MM)-or-better together with a ≥ 50% steroid reduction, withdrawal of immunosuppressants, and no need for plasma-exchange or intravenous immunoglobulin. After a course of RTX, eight patients improved, with optimal response in six, while only one patient did not respond. At baseline, MuSK-IgG and MuSK-IgG4 serum titers were positive in all patients, ranging from 2.15 to 49.5 nmol/L and from 0.33 to 46.2 nmol/L, respectively. MuSK Abs mostly consisted of IgG4 (range 63.80–98.86%). RTX administration was followed by a marked reduction of MuSK Abs at 2–7 months and at 12–30 months (p < 0.02 for MuSK-IgG and p < 0.01 for MuSK-IgG4). In patients with a longer follow-up, MuSK Ab titers remained suppressed, paralleling clinical response. In the patient who achieved long-term complete remission, MuSK-IgG4 was no longer detectable within 2 years, while MuSK-IgG remained positive at very low titers up to 10 years after RTX. In the patient who did not respond, MuSK-IgG and MuSK-IgG4 remained unchanged. In this patient series, total IgG and IgG4 transiently decreased (p < 0.05) at 2–7 months after RTX. The different trends of reducti

Myasthenia gravis is a chronic autoimmune disease characterized by autoantibody-mediated interference of signal transmission across the neuromuscular junction. We performed a genomewide association study (GWAS) involving 1,873 patients diagnosed with acetylcholine receptor antibody-positive myasthenia gravis and 36,370 healthy individuals to identify disease-associated genetic risk loci. Replication of the discovered loci was attempted in an independent cohort from the UK Biobank. We also performed a transcriptome-wide association study (TWAS) using expression data from skeletal muscle, whole blood, and tibial nerve to test the effects of disease-associated polymorphisms on gene expression. We discovered two signals in the genes encoding acetylcholine receptor subunits that are the most common antigenic target of the autoantibodies: A GWAS signal within the cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) gene and a TWAS association with the cholinergic receptor nicotinic beta 1 subunit (CHRNB1) gene in normal skeletal muscle. Two other loci were discovered on 10p14 and 11q21, and the previous association signals at PTPN22, HLA-DQA1/HLA-B, and TNFRSF11A were confirmed. Subgroup analyses demonstrate that early- and late-onset cases have different genetic risk factors. Genetic correlation analysis confirmed a genetic link between myasthenia gravis and other autoimmune diseases, such as hypothyroidism, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes. Finally, we applied Priority Index analysis to identify potentially druggable genes/proteins and pathways. This study provides insight into the genetic architecture underlying myasthenia gravis and demonstrates that genetic factors within the loci encoding acetylcholine receptor subunits contribute to its pathogenesis.

Objective: Genetic factors predisposing to late-onset myasthenia gravis (LOMG) have not been clearly defined yet. However, genome-wide association studies identified Human Leukocyte Antigen (HLA) Class II alleles as a hotspot in this disease subtype. The aim of this study was to analyze the correlations of HLA Class II alleles with clinical data and titin antibodies in this patient subgroup. Methods: This study consecutively enrolled anti-acetylcholine receptor antibody-positive, non-thymoma patients with generalized LOMG. All patients were of Italian ancestry. HLA-DRB1 and -DQB1 genotyping and serum titin antibody testing were performed in this population. Results: A total of 107 patients (females: 28/107, 26.2%; median age of onset: 68 years, range: 50-92) were included. We found a positive association with HLA-DRB1*07 (P = 1.1 × 10-5), HLA-DRB1*14 (P = 0.0251) and HLA-DQB1*02 (P = 0.0095). HLA-DRB1*03, HLA-DRB1*11, and HLA-DQB1*03 were protective alleles (P = 7.9 × 10-5, P = 0.0104, and P = 0.0067, respectively). By conditional haplotype analysis, HLA-DRB1*07-DQB1*02 was found to be the major risk haplotype (OR = 4.10; 95% C.I.: 2.80-5.99; P = 6.01 × 10-11). The mean age at onset was 73.4 years in DRB1*07 homozygotes, 69.7 years in heterozygotes, and 66.6 in non-carriers (P = 0.0488). DRB1*07 carriers and non-carriers did not differ in disease severity and response to therapy. Titin antibodies were detected in 61.4% of the cases, having no association with HLA alleles or specific clinical characteristics. Interpretation: In our study, we identified the HLA DRB1*07-DQB1*02 haplotype as a predisposing factor for the development of generalized LOMG in the Italian population.

Background: Multiple sclerosis (MS) is an autoimmune disease for which auto-antibodies fully validated as diagnostic and prognostic biomarkers are widely desired. Recently, an immunoreactivity against the inward rectifying potassium channel 4.1 (KIR4.1) has been reported in a large proportion of a group of MS patients, with amino acids 83–120 being the major epitope. Moreover, a strong correlation between anti-KIR4.183–120and anti-full-length-protein auto-antibodies titer was reported. However, this finding received limited confirmation. Objective: Validation of the diagnostic potential of anti-KIR4.183–120antibodies in 78 MS patients, 64 healthy blood donors, and 42 individuals with other neurological diseases. Methods: Analysis of anti-KIR4.183–120antibodies by enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum we produced as a new ELISA reliability control. Additionally, evaluation of reactivity against 293-T cells transiently transfected with full-length KIR4.1 by flow cytometry. Results: We found antibodies to KIR4.183–120only in 13 out of 78 (16.6%) MS patients; among these, only 2 were positive for anti-full-length KIR4.1 antibodies. Conclusion: Employing a new reliability control and a new cytofluorometric assay, we cannot support anti-KIR4.183–120auto-antibodies as a reliable biomarker in MS.

Background. Serological levels of free immunoglobulin light chains (FLCs), produced in excess of heavy chains during synthesis of immunoglobulins by plasma cells, can be considered a direct marker of B cell activity in different systemic inflammatory-autoimmune conditions and may represent a useful predictor of rituximab (RTX) therapeutic efficacy, as reported for rheumatoid arthritis and systemic lupus erythematosus. Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction with antibodies (abs) targeting the acetylcholine receptor (AChR) or the muscle-specific tyrosine kinase (MuSK), inducing muscle weakness and excessive fatigability. As MG course may be remarkably variable, we evaluated the possible use of FLCs as biomarkers of disease activity. Subjects and Methods. We assessed FLC levels in 34 sera from 17 AChR-MG and from 13 MuSK-MG patients, in comparison with 20 sera from patients with systemic autoimmune rheumatic diseases and 18 from healthy blood donors, along with titers of specific auto-abs and IgG subclass distribution. Results. We found a statistically significant increase in free. chains in both AChR-and MuSK-MG patients, while free lambda chain levels were increased only in AChR-MG. We also observed a significant reduction of both free kappa and lambda chains in 1/4 MuSK-MG patients along with specific abs titer, two months after RTX treatment. Conclusions. From our data, FLCs appear to be a sensitive marker of B cell activation in MG. Further investigations are necessary to exploit their potential as reliable biomarkers of disease activity.

Myasthenia graviswith antibodies to themuscle-specific tyrosine kinase(MuSK+ MG)is a rare diseasewith distinctive pathogenic mechanisms and clinical features. An acute onset and predominant bulbar muscle weakness are very commonand highly suggestive of the disease.On the other hand, amore indolent course, atypical ocular presentation, and signs of cholinergic hyperactivity may complicate the diagnosis. ThoughMuSK+ MGis still a severe disease, over the years we have observed a steady reduction in the rate of respiratory crisis and a significant improvement in the clinical outcome, both likely related to earlier diagnosis and timely treatment. Despite the improved management, MuSK+ MG patients tend to remain dependent on long-term immunosuppressive treatment and may develop permanent disabling weakness. In uncontrolled studies, B cell depletion with rituximab proved effective in most patientswith refractory disease, inducing prolonged clinical responses associatedwith a sustained reduction of serum antibody levels. Promising results fromexperimental studies and case reports suggest that both 3,4-diaminopyridine and albuterol may be effective as symptomatic agents.

Objectives To approximate the rate of familial myasthenia gravis and the coexistence of other autoimmune disorders in the patients and their families. Design Retrospective cohort study. Setting Clinics across North America. Participants The study included 1032 patients diagnosed with acetylcholine receptor antibody (AChR)-positive myasthenia gravis. Methods Phenotype information of 1032 patients diagnosed with AChR-positive myasthenia gravis was obtained from clinics at 14 centres across North America between January 2010 and January 2011. A critical review of the epidemiological literature on the familial rate of myasthenia gravis was also performed. Results Among 1032 patients, 58 (5.6%) reported a family history of myasthenia gravis. A history of autoimmune diseases was present in 26.6% of patients and in 28.4% of their family members. Discussion The familial rate of myasthenia gravis was higher than would be expected for a sporadic disease. Furthermore, a high proportion of patients had a personal or family history of autoimmune disease. Taken together, these findings suggest a genetic contribution to the pathogenesis of myasthenia gravis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease for which auto-antibodies fully validated as diagnostic and prognostic biomarkers are widely desired. Recently, an immunoreactivity against the inward rectifying potassium channel 4.1 (KIR4.1) has been reported in a large proportion of a group of MS patients, with amino acids 83-120 being the major epitope. Moreover, a strong correlation between anti-KIR4.183-120 and anti-full-length-protein auto-antibodies titer was reported. However, this finding received limited confirmation. OBJECTIVE: Validation of the diagnostic potential of anti-KIR4.183-120 antibodies in 78 MS patients, 64 healthy blood donors, and 42 individuals with other neurological diseases. METHODS: Analysis of anti-KIR4.183-120 antibodies by enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum we produced as a new ELISA reliability control. Additionally, evaluation of reactivity against 293-T cells transiently transfected with full-length KIR4.1 by flow cytometry. RESULTS: We found antibodies to KIR4.183-120 only in 13 out of 78 (16.6%) MS patients; among these, only 2 were positive for anti-full-length KIR4.1 antibodies. CONCLUSION: Employing a new reliability control and a new cytofluorometric assay, we cannot support anti-KIR4.183-120 auto-antibodies as a reliable biomarker in MS.

The co-occurrence of autoimmune diseases (ADs) in a large population of myasthenia gravis (MG) patients from a single center was evaluated. Our survey included 984 patients, 904 with anti-acetylcholine receptor antibodies and 80 with anti-muscle specific kinase antibodies. The anti-acetylcholine receptor positive population included patients with early-onset (age at onset ≤50 years), late-onset and thymoma-associated disease. Follow-up ranged 2-40 years. Two-hundred and fourteen ADs were diagnosed in 185 patients; 26 of them had two or more ADs in association with MG. Thyroid disorders were the most common and, together with vitiligo and thrombocytopenia, occurred in all disease subsets. Otherwise, there was a broad variability with partial overlap among patient groups. The highest rate of ADs was observed in early-onset patients, while clusters, i.e. 2 or more ADs other than MG in the same individual, were more common among thymoma cases. Thirty-four diseases were diagnosed at the same time, 88 occurred before and 92 after the onset of MG. On multivariate analysis, immunosuppressive treatment was the only independent variable which negatively influenced the risk of developing other ADs in our cohort.

Myasthenia gravis is a chronic, autoimmune, neuromuscular disease characterized by fluctuating weakness of voluntary muscle groups. Although genetic factors are known to play a role in this neuroimmunological condition, the genetic etiology underlying myasthenia gravis is not well understood.

Myasthenia gravis (MG) is a prototypical antibody-mediated disease characterized by muscle weakness and fatigability. Serum antibodies to the acetylcholine receptor and muscle-specific tyrosine kinase receptor (MuSK) are found in about 85% and 8% of patients respectively. We have previously shown that more than 70% of MG patients with MuSK antibodies share the HLA DQ5 allele. The aim of the present study was to analyze the T cell receptor (TCR) repertoire specific for recombinant human MuSK protein. We used the CDR3 TRBV-TRBJ spectratyping (immunoscope) to analyze the T cell response to MuSK from 13 DQ5+ MuSK-MG patients and from 7 controls (six DQ5+ MuSK negative subjects and one DQ5- DQ3+ MuSK positive patient). DQ5+ MuSK-MG patients but not controls used a restricted set of TCR VJ rearrangements in response to MuSK stimulation. One semiprivate (TRBV29-TRBJ2.5) rearrangement was found in 5/13 patients, while 4 other semiprivate (one in TRBV28-TRBJ2.1 and in TRBV3-TRBJ1.2, and two in TRBV28-TRBJ1.2) rearrangements were differently shared by 4/13 patients each and were absent in controls. When we sequenced the TRBV29-TRBJ2.5 rearrangement, we obtained 26 different sequences of the expected 130 bp length from 117 samples of the 5 positive patients: two common motifs GXGQET/TEHQET were shared in 4 patients as semiprivate motifs. Thus, the MuSK-specific T-cell response appears to be restricted in DQ5+ MuSK-MG patients, with a semiprivate repertoire including a common motif of TRBV29. This oligoclonal restriction of T cells will allow the identification of immunodominant epitopes in the antigen, providing therefore new tools for diagnosis and targeted therapy.

Increasing evidence suggests a contribution of epigenetic processes in promoting cancer and autoimmunity. Myasthenia gravis (MG) is an autoimmune disease mediated, in approximately 80% of the patients, by antibodies against the nicotinic acetylcholine receptor (AChR+). Moreover, epithelial tumours (thymomas) are present in about 10-20% of the patients, and there is indication that changes in DNA methylation might contribute to the risk and progression of thymomas. However, the role of epigenetics in MG is still not completely clarified. In the present study we investigated if a common polymorphism (-579G>T: rs1569686) in the promoter of the DNMT3B gene coding for the DNA methyltransferase 3B, an enzyme that mediates DNA methylation, increases the risk to develop MG or MG-associated thymomas. The study polymorphism was selected based on recent reports and a literature meta-analysis suggesting association with increased risk of various types of cancer. We screened 324 AChR+ MG patients (140 males and 184 females, mean age 56.0 ± 16.5 years) and 735 healthy matched controls (294 males and 441 females, mean age 57.3 ± 15.6 years). 94 of the total MG patients had a thymoma. While there was no association with the whole cohort of MG patients, we found a statistically significant association of the DNMT3B-579T allele (OR = 1.51; 95% CI=1.1-2.1, P = 0.01) and the TT homozygous genotype (OR = 2.59; 95% CI=1.4-4.9, P = 0.006) with the risk of thymoma. No association was observed in MG patients without thymoma, even after stratification into clinical subtypes. Present results suggest that the DNMT3B-579T allele might contribute to the risk of developing thymoma in MG patients, particularly in homozygous TT subjects.

Interleukin (IL)-6 is a multifunctional cytokine with a critical role in inflammatory, immunoregulatory and haemopoietic responses. Its receptor consists of an ubiquitously expressed membrane transducing element (gp130) and of the specific element IL-6R-alpha (gp80), present only on hepatocytes and some leukocyte subsets. IL-6R-alpha also exists as soluble protein (sIL-6R) that, in the presence of IL-6, forms a complex able to bind gp130 and, thanks to the mechanism called trans-signaling, transduces IL-6 effect through tyrosine phosphorylation and activation of the signal transducer and transcription activator (STAT)-3. The aim of this study was to analyze the bidirectional relationships between platelet aggregation and IL-6-dependent effects. While platelets do not produce IL-6, we found that resting platelets express gp130, but not gp80, on their membranes. Upon activation by thrombin or calcium ionophore A23187, but not by ADP, the IL-6R-alpha is released in soluble form, while cangrelor, the specific inhibitor of P2Y12 receptor, can partially inhibit sIL-6R release. This sIL-6R is biologically active and, in the presence of IL-6, can trigger IL-6 trans-signaling, inducing an autocrine activation loop (as measured by an increase in gp80 and gp130 content) and STAT3 phosphorylation. On the other hand, IL-6 trans-signaling has no effect on platelet degranulation or aggregation by itself, nor on thrombin-induced platelet aggregation. Our data add an important piece to the puzzle of thrombosis and inflammation: in the presence of IL-6, which can be produced by stressed endothelial cells, the platelet-derived IL-6 trans-signaling could be crucial for the evolution of inflammation within a damaged vessel.

The skeletal muscles are the major living component of the human body. They are constituted by stable cells, the myofibres, and by adult multipotent stem cells, the satellite cells, which can multiply to regenerate and repair the damaged tissues. Injections of DNA in muscle cells have been used to produce recombinant proteins with opposite goals: somatic reparation of genetic defects, which needs to elicit no inflammatory or immune response, and DNA vaccination, which needs a robust immune response. Because of possible therapeutical interventions, a growing body of information is being produced dealing with every aspect of the myofibres during inflammatory and autoimmune responses: skeletal muscle-antigen presenting cell (APC) interaction and intrinsic APC capabilities of myoblasts and myocytes, the response to released cytokines and their endogenous production, the regulation of Toll-like receptors and major histocompatibility complex expression. According to these data, the muscle tissue is now emerging no longer as a passive bystander, but more as an active player that, when correctly manipulated, can drive tolerance or immunization to these de novo produced proteins. In the present review, we summarize the recent developments on the control of muscle immune function.

We studied 111 myasthenic patients with thymoma followed over the last 20 years. Most patients were affected with severe myasthenia gravis (MG) and had been treated with immunosuppressive drugs. At the end of the follow-up, we observed good therapeutic results in 74% of patients, but a complete remission in only 10% of cases. The survival rate in thymoma patients was significantly lower than in nonthymoma cases. Clinical parameters, MG response to treatment and survival rate did not differ in patients with invasive and noninvasive thymic neoplasms. Patients with invasive thymoma showed slightly higher surgical mortality and a 10% incidence of tumor relapse.

Two cases of familial myasthenia gravis are reported. One patient is a typical case of autoimmune myasthenia with positive anti acetylcholine receptor antibodies, while in the second patient the impairment of neuromuscular transmission is likely to be due to antibodies directed against determinants other than the acetylcholine receptors.