Emanuela Bartoccioni

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.

Constitutive and cytokine-induced production of interleukin-6 by human myoblasts.

Several recent studies have shown that some inflammatory myopathies are autoimmune diseases. It is possible that certain alterations in the muscle-immune cell microenvironment and in the local production of cytokines could take part in the pathogenesis of inflammatory myopathies. In the present study we investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the secretion of interleukin-6 (IL-6) by myoblasts. Purified human myoblasts from normal subjects and from patients with polymositis were cultured in the presence of TNF-alpha and IFN-gamma at two concentrations (100 and 200 U/ml), alone or in combination, for 12, 24 and 48 h. The supernatants were collected and the IL-6 concentrations tested by ELISA (Genzyme). We found that myoblasts secrete IL-6 constitutively. The secretion of IL-6 was greatly increased by TNF-alpha; the increase was both time- and dose-dependent. IFN-gamma caused a moderate increase in IL-6 secretion, but this effect was not significant, despite a slight positive trend over time. There was no synergism in the effect of IFN-gamma and TNF-alpha. It is known that inflammatory myopathies are characterized by mononuclear cell infiltration and muscle regeneration: myoblasts are present in infiltrated tissues. Thus, the local production of cytokines that characterizes the inflammatory reaction, could stimulate myoblasts to secrete IL-6, which might add to the pro-inflammatory effects of IL-6 produced by activated macrophages and T cells.

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.

Ocular myasthenia: diagnostic and therapeutic problems

ABSTRACT— Forty‐eight patients with purely ocular myasthenia were studied. Tensilon test was positive in 46 patients (95%); decremental response from limb muscles was present in 24 patients (50%); anti‐AChR antibodies were detected in 20 patients of 44 (45.5%). Tweny‐two patients underwent thymectomy, 18 were given corticosteroids, 42 received AChE drugs. At the end of the observation period, 8% of the patients were in remission, 67% were improved, 25% were unchanged. In our experience, the diagnosis of ocular myasthenia relies mainly on clinical data; AChE drugs are not very effective in extrinsic ocular muscles; indications for thymectomy should be restricted to thymoma cases and, perhaps, to patients in the early stages of the disease, within the first year of onset; corticosteroids are effective in most cases, but relapses after withdrawal are not uncommon.

Juvenile myasthenia gravis with prepubertal onset

Juvenile myasthenia gravis (JMG) with prepubertal onset is an uncommon disease. We studied 19 patients with age at onset ranging from 1.5 to 9.2 years and compared their clinical characteristics and response to therapy with 114 cases with MG onset after the prepubertal age, up to 20 years. Neither sex prevalence nor autoimmune diseases other than MG were found in younger patients. Although ocular myasthenia was more frequent than in later-onset JMG, children with generalized symptoms were often severely affected and respiratory involvement was present in 8/19 patients. Anti-acetylcholine receptor antibodies were detected at a lower rate and, in contrast with results in older patients, seronegativity was more frequent among children with generalized disease. Three out of six patients with onset before the age of five showed spontaneous remission. Nine prepubertal patients underwent thymectomy and, as most of them also received immunosuppressive therapy, the influence of surgery on disease outcome remains unclear; in no case was thymoma found. This is in contrast to the good results after thymectomy and the presence of thymoma in the later-onset group. Eleven patients in the prepubertal series were treated with immunosuppressive therapy. At the end of follow-up, most patients were in good condition. The frequency of immunosuppressive therapy and the rate of good therapeutic results did not differ from those observed in older patients.

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 …

Seronegative myasthenia gravis: comparison of neurophysiological picture in MuSK+ and MuSK− patients

The aim of this study was to compare the neurophysiological and clinical pictures of a large sample of seronegative myasthenia gravis (SNMG) patients with and without anti‐MuSK antibodies. Fifty‐two consecutive SNMG patients were retrospectively evaluated. They had undergone an extended neurophysiological evaluation: repetitive nerve stimulation (RNS), single fiber EMG (SFEMG), and electromyography (EMG) with nerve conduction study. A muscle biopsy was performed in 11 of 52 patients, the edrophonium test in 44 of 52 patients and anti‐AChR antibodies and anti‐MuSK antibodies were tested in all patients. Anti‐MuSK antibodies were detected in 25 SNMG patients (48.1%). The number of women in the MuSK+ group was significantly higher (P = 0.01) than in the MuSK− group. Seronegative MuSK+ patients are more severely affected and the deficit often involves the bulbar and the respiratory muscles. No statistically significant differences were observed in the edrophonium test between MuSK+ and MuSK− groups. The RNS test was abnormal in a significantly higher number of MUSK− patients than MUSK+ patients (P < 0.00001). With regard to SFEMG data, MuSK− patients were characterized to have more severe neurophysiological pattern. Our observations showed several differences between the clinical and neurophysiological pictures of MUSK+ and MUSK− patients.

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.