Flavia Scuderi

MHC class I, MHC class II and intercellular adhesion molecule-1 (ICAM-1) expression in inflammatory myopathies

We investigated the relationship between the MHC‐I, MHC‐II and intercellular adhesion molecule‐1 (ICAM‐1) expression on myofibres and the presence of inflammatory cells in muscle specimens of 18 patients with inflammatory myopathies (nine polymyositis, seven dermatomyositis, two inclusion body myositis). We observed MHC‐I expression in muscle fibres, infiltrating mononuclear cells and endothelial cells in every specimen. In seven patients, some muscle fibres were MHC‐II‐positive for the DR antigen, while the DP and DQ antigens were absent. ICAM‐1 expression, detected in seven patients, was found in clusters of myofibres, associated with a marked MHC‐1 positivity and a widespread mononuclear infiltration. Most of the ICAM‐1‐positive fibres were regenerating fibres. Furthermore, some fibres expressed both ICAM‐I and DR antigens near infiltrating cells. This finding could support the hypothesis that myofibres may themselves be the site of autosensitization.

Anti-P110 Autoantibodies Identify a Subtype of “Seronegative” Myasthenia Gravis with Prominent Oculobulbar Involvement

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness and pathogenetic autoantibodies directed against the nicotinic acetylcholine receptor (seropositive myasthenia gravis; SPMG). Nearly 15% to 20% of MG patients do not have these antibodies (seronegative myasthenia gravis; SNMG), but several evidence indicate that these patients have circulating pathogenic autoantibodies directed against other muscle antigens. Using the TE671 rhabdomyosarcoma cell line as an antigen source, we analyzed sera from 63 SNMG and 26 SPMG patients and 26 healthy blood donors by FACS analysis. We found that 40 of 63 SNMG patients and only 1 of 26 SPMG patients had IgG binding to the TE671 cell line. None of the sera bound to the unrelated MRC5 cell line. To identify the antigen, we analyzed sera immunoreactivity in more detail by immunoprecipitation of biotinylated membrane proteins from TE671 cells. When the immunoprecipitated proteins were separated by SDS-PAGE electrophoresis and then transferred to nitrocellulose membranes, we found that SNMG IgG identify a band corresponding to a protein with a molecular weight of 110 kDa (P110), which is not recognized by seropositive MG sera. This anti-P110 immunoreactivity is significantly associated with a distinct clinical picture characterized by a prominent involvement of ocular and bulbar muscles, with frequent respiratory problems (p < 0.005), and is recognized by a specific antimuscle specific kinase (MuSK) antiserum. In a recent article, the presence of anti-MuSK antibodies was described in SNMG. Our results confirm the presence of these antibodies in SNMG and suggest that anti-P110/MuSK autoantibodies identify a subtype of SNMG in which the different pathogenesis induces the distinct clinical picture.

Anti-MuSK antibodies: Correlation with myasthenia gravis severity

The authors measured anti–muscle-specific tyrosine kinase (anti-MuSK) antibodies (Abs) in 83 serum samples from 40 patients and evaluated their correlation with myasthenia gravis severity and treatment response. Ab concentrations were often reduced by immunosuppression but not after thymectomy. Both in individual cases and in the whole population, a correlation between Ab levels and disease severity was found.

A genome-wide association study of myasthenia gravis

IMPORTANCE 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. OBJECTIVE To identify genetic variants that alter susceptibility to myasthenia gravis, we performed a genome-wide association study. DESIGN, SETTING, AND PARTICIPANTS DNA was obtained from 1032 white individuals from North America diagnosed as having acetylcholine receptor antibody-positive myasthenia gravis and 1998 race/ethnicity-matched control individuals from January 2010 to January 2011. These samples were genotyped on Illumina OmniExpress single-nucleotide polymorphism arrays. An independent cohort of 423 Italian cases and 467 Italian control individuals were used for replication. MAIN OUTCOMES AND MEASURES We calculated P values for association between 8,114,394 genotyped and imputed variants across the genome and risk for developing myasthenia gravis using logistic regression modeling. A threshold P value of 5.0×10(-8) was set for genome-wide significance after Bonferroni correction for multiple testing. RESULTS In the overall case-control cohort, we identified association signals at CTLA4 (rs231770; P=3.98×10(-8); odds ratio, 1.37; 95% CI, 1.25-1.49), HLA-DQA1 (rs9271871; P=1.08×10(-8); odds ratio, 2.31; 95% CI, 2.02-2.60), and TNFRSF11A (rs4263037; P=1.60×10(-9); odds ratio, 1.41; 95% CI, 1.29-1.53). These findings replicated for CTLA4 and HLA-DQA1 in an independent cohort of Italian cases and control individuals. Further analysis revealed distinct, but overlapping, disease-associated loci for early- and late-onset forms of myasthenia gravis. In the late-onset cases, we identified 2 association peaks: one was located in TNFRSF11A (rs4263037; P=1.32×10(-12); odds ratio, 1.56; 95% CI, 1.44-1.68) and the other was detected in the major histocompatibility complex on chromosome 6p21 (HLA-DQA1; rs9271871; P=7.02×10(-18); odds ratio, 4.27; 95% CI, 3.92-4.62). Association within the major histocompatibility complex region was also observed in early-onset cases (HLA-DQA1; rs601006; P=2.52×10(-11); odds ratio, 4.0; 95% CI, 3.57-4.43), although the set of single-nucleotide polymorphisms was different from that implicated among late-onset cases. CONCLUSIONS AND RELEVANCE Our genetic data provide insights into aberrant cellular mechanisms responsible for this prototypical autoimmune disorder. They also suggest that clinical trials of immunomodulatory drugs related to CTLA4 and that are already Food and Drug Administration approved as therapies for other autoimmune diseases could be considered for patients with refractory disease.

HLA class II allele analysis in MuSK-positive myasthenia gravis suggests a role for DQ5

Myasthenia gravis (MG) is caused by autoantibodies targeting, in most cases, the acetylcholine receptor (AChR-MG). Different disease subtypes are distinguished on the basis of clinical characteristics and thymus pathology. In 40% of patients with anti-AChR negative generalized MG, the disease appears to be mediated by antibodies against the muscle specific kinase (MuSK-MG).1 We evaluated HLA-DRB1*, DQA1*, and DQB1* allele profile in MuSK-MG in comparison with a control population and non-thymoma early onset AChR-MG (AChR-EOMG). We chose to compare these two clinical entities as they share a high prevalence in women and a proportion of MuSK-MG patients have early onset disease. ### Patients. Our study includes consecutive unrelated patients, all with generalized MG. Patients gave informed consent to inclusion in the study, which was approved by the local Ethics Committee. The MuSK-MG group included 37 patients (8 men/29 women, onset age: 6–62 years), the thymus was normal for age in 10 patients who underwent thymectomy, thyroid autoimmunity was associated in 4/37 cases (10.5%). The AChR-EOMG group comprised 28 patients (4 men/24 women, onset age: 9–39 years), thymic hyperplasia was found in 24/26 thymectomized cases, and different autoimmune disorders were associated in 8/28 (28.6%). All patients were from Central Italy, with Italian ancestors. For …

Skeletal muscle cells: from local inflammatory response to active immunity

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.

Constitutive and cytokine-induced expression of MHC and intercellular adhesion molecule-1 (ICAM-1) on human myoblasts

We studied the expression of MHC-I and MHC-II molecules and ICAM-1 in cultured human myoblasts in response to IL-1beta, IL-4, IL-6, IFN-gamma and LPS. IFN-gamma, LPS and IL-4 greatly increase MHC-I molecule expression. MHC-II molecule expression is induced only by IFN-gamma. Membrane ICAM-1 and mRNA expression are absent under basal conditions, but can be induced by IFN-gamma, IL-1beta, IL-4, LPS and IL-6 with different efficiencies and time-courses. Soluble ICAM-1 secretion can be induced to a different extent by all cytokines. Our study shows that the expression of adhesion-related molecules in muscle is finely regulated by these cytokines.

Myasthenia gravis, thymectomy, and antiacetylcholine receptor antibody

SummaryThe antiacetylcholine receptor antibody was titered in the serum of 63 patients with myasthenia gravis (MG) and 20 control healthy subjects. The titer was significantly high in 92% of MG patients in contrast with none of the controls and no correlation was found with the thymus pathology and the severity of the disease. The titer decreased after thymectomy almost steadily with the improvement of the myasthenic signs. The role of the antibody in the pathogenesis of the disease is discussed.ZusammenfassungDie Antikörper gegen Acetylcholin-Rezeptoren wurden quantitativ im Serum von 63 Patienten mit Myasthenia Gravis und von 20 gesunden Kontrollpersonen bestimmt. Der Titer war bei 92% der Myasthenie Gravis-Patienten signifikant erhöht, jedoch bei keinem der Kontrollfälle. Es wurde keine Korrelation zwischen dem Titer einerseits und den pathologischanatomischen Besonderheiten beziehungsweise dem klinischen Schweregrad der Erkrankung andererseits festgestellt. Der Titer nahm nach Thymektomie ziemlich geradlinig ab, parallel zur klinischen Besserung der Myasthenie. Es wird die Rolle der Antikörper bei der Pathogenese der Erkrankung diskutiert.

Characterization of B cells in muscle-specific kinase antibody myasthenia gravis.

Objective: To characterize B-cell subsets in patients with muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). Methods: In accordance with Human Immunology Project Consortium guidelines, we performed polychromatic flow cytometry and ELISA assays in peripheral blood samples from 18 patients with MuSK MG and 9 healthy controls. To complement a B-cell phenotype assay that evaluated maturational subsets, we measured B10 cell percentages, plasma B cell–activating factor (BAFF) levels, and MuSK antibody titers. Immunologic variables were compared with healthy controls and clinical outcome measures. Results: As expected, patients treated with rituximab had high percentages of transitional B cells and plasmablasts and thus were excluded from subsequent analysis. The remaining patients with MuSK MG and controls had similar percentages of total B cells and naïve, memory, isotype-switched, plasmablast, and transitional B-cell subsets. However, patients with MuSK MG had higher BAFF levels and lower percentages of B10 cells. In addition, we observed an increase in MuSK antibody levels with more severe disease. Conclusions: We found prominent B-cell pathology in the distinct form of MG with MuSK autoantibodies. Increased BAFF levels have been described in other autoimmune diseases, including acetylcholine receptor antibody–positive MG. This finding suggests a role for BAFF in the survival of B cells in MuSK MG, which has important therapeutic implications. B10 cells, a recently described rare regulatory B-cell subset that potently blocks Th1 and Th17 responses, were reduced, which suggests a potential mechanism for the breakdown in immune tolerance in patients with MuSK MG.

Effect of pro-inflammatory/anti-inflammatory agents on cytokine secretion by peripheral blood mononuclear cells in rheumatoid arthritis and systemic lupus erythematosus.

We studied a well-selected population of patients with active rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) without immunosuppressive therapy. Control and patient peripheral blood mononuclear cells (PBMC) were incubated with IL-1 g, IL-10, TGF- g or LPS for 20 h and the in vitro basal and stimulated secretions of IL-6, TNF- f, IL-1 g and IL-1ra were measured by ELISA. We found that in the SLE patients the basal secretion of IL-6 was significantly lower and that of IL-1ra significantly higher than in control subjects, while in the RA group the basal IL-1ra secretion was higher than in healthy subjects. SLE and RA PBMC responded to LPS and IL-1 g reaching higher cytokine secretion values than controls. The in vitro response of SLE and RA PBMC to TGF g was normal, while that to IL-10 was defective: IL-10 was able to stimulate the production of IL-6 and IL-1ra in PBMC from normal subjects, but it was unable to enhance IL-6 secretion in RA cells and it was also completely ineffective in inducing IL-1ra secretion in both SLE and RA PBMC. Our work add new data useful for the evaluation of IL-10 and IL-1ra as therapeutic agents in rheumatic diseases.