Franco Salerno

Complete stable remission and autoantibody specificity in myasthenia gravis

Objectives: Patients with myasthenia gravis (MG) are subgrouped as acetylcholine receptor (AChR)–positive, muscle-specific kinase (MuSK)–positive, and AChR/MuSK-negative MG (or double negative [DN]) on the basis of autoantibody assay. We investigated the relationships between autoantibody specificity, main clinical features, and outcome of the disease, in particular the occurrence of complete stable remission (CSR), by means of a retrospective study on a cohort of 677 Italian patients with MG. Methods: A total of 517 (76%) patients with AChR-positive MG, 55 (8%) patients with MuSK-positive MG, and 105 (16%) patients with DN MG were included in the study. Kaplan-Meier and Cox proportional hazard regression analyses were used to evaluate associations between baseline characteristics, antibody specificity, and CSR. Results: Clinical stage at onset and at maximal worsening was more severe for MuSK-positive patients: bulbar impairment at maximal worsening was found in 83.6% of MuSK-positive patients compared with 58.6% of AChR-positive patients and 43.8% of DN patients (p < 0.001). Baseline characteristics of AChR-positive and DN patients were similar. CSR was observed in 3.6% of MuSK-positive patients compared with 22.2% of AChR-positive and 21.9% of DN patients. In the whole MG cohort, onset before age 40 (hazard ratio [HR] = 1.96, 95% confidence interval [CI] 1.27–3.02, p = 0.002) and ocular and generalized clinical stages at maximal worsening were associated with CSR (ocular, HR = 8.05, 95% CI 1.88–34.53, p = 0.005; generalized, HR = 3.71, 95% CI 1.16–11.90, p = 0.023; bulbar, HR = 3.16, 95% CI 1.00–10.05, p = 0.051). Conclusions: MuSK antibodies identify a clinically distinguishable, more severe form of MG since the disease onset, with a lower occurrence of CSR. These features should be considered by the clinician in the management of this particular form of MG.

Sleep breathing disorders in 40 Italian patients with Myotonic dystrophy type 1

The aim of this study was to estimate the prevalence and nature of sleep breathing disorders in Myotonic dystrophy type 1 (DM1). We wanted to determine whether there is a relationship between sleep breathing disorders and clinical parameters such as pulmonary function, degree of neuromuscular impairment, daytime sleepiness, and fatigue. This will help assess the prevalence of DM1 patients requiring nocturnal ventilatory treatments. We studied a random sample of 40 unrelated patients and found that 22/40 patients had obstructive sleep apnoea. Of these 22 patients, five showed also periodic breathing and four showed sleep hypoventilation. Nine patients were put on nocturnal ventilation following clinical and instrumental evaluations. Our study reveals that obstructive sleep apnoea is very common in these patients, but cannot be predicted on the basis of clinical-neurological features and diurnal functional respiratory tests. Our data emphasize that a periodical evaluation by polysomnography should be mandatory to ascertain, and treat if necessary, the presence of obstructive sleep apnoea, periodic breathing or nocturnal hypoventilation.

Autophagy, inflammation and innate immunity in inflammatory myopathies.

Autophagy has a large range of physiological functions and its dysregulation contributes to several human disorders, including autoinflammatory/autoimmune diseases such as inflammatory myopathies (IIMs). In order to better understand the pathogenetic mechanisms of these muscular disorders, we sought to define the role of autophagic processes and their relation with the innate immune system in the three main subtypes of IIM, specifically sporadic inclusion body myositis (sIBM), polymyositis (PM), dermatomyositis (DM) and juvenile dermatomyositis (JDM). We found that although the mRNA transcript levels of the autophagy-related genes BECN1, ATG5 and FBXO32 were similar in IIM and controls, autophagy activation in all IIM subgroups was suggested by immunoblotting results and confirmed by immunofluorescence. TLR4 and TLR3, two potent inducers of autophagy, were highly increased in IIM, with TLR4 transcripts significantly more expressed in PM and DM than in JDM, sIBM and controls, and TLR3 transcripts highly up-regulated in all IIM subgroups compared to controls. Co-localization between autophagic marker, LC3, and TLR4 and TLR3 was observed not only in sIBM but also in PM, DM and JDM muscle tissues. Furthermore, a highly association with the autophagic processes was observed in all IIM subgroups also for some TLR4 ligands, endogenous and bacterial HSP60, other than the high-mobility group box 1 (HMGB1). These findings indicate that autophagic processes are active not only in sIBM but also in PM, DM and JDM, probably in response to an exogenous or endogenous ‘danger signal’. However, autophagic activation and regulation, and also interaction with the innate immune system, differ in each type of IIM. Better understanding of these differences may lead to new therapies for the different IIM types.

Adult-onset leukodystrophies from respiratory chain disorders: do they exist?

Respiratory chain disorders (RCDs) have been included in the differential diagnosis of adult-onset leukodystrophies. Here, we first report a 32-year-old female with an atypical, adult-onset, non-syndromic RCD due to a mitochondrial DNA deletion and manifesting as complicated ataxia. A ‘leukodystrophic’ pattern was found on brain MRI, but it was neither isolated nor predominant because of the presence of overt basal ganglia and infratentorial lesions, which led us to the proper diagnosis. Subsequently, we evaluated our series of patients with RCDs in order to verify whether a ‘leukodystrophic’ pattern with little or no involvement of deep grey structures and brainstem may be found in adult-onset RCDs, as reported in children. Among 52 patients with adult-onset RCDs, no case with a ‘leukodystrophic’ pattern was found, apart from three cases with a classical phenotype of mitochondrial neurogastrointestinal encephalopathy. In addition, no case of RCDs was found among six cases of adult-onset leukodystrophy of unknown origin and at least one feature suggestive of mitochondrial disease. The review of the literature was in agreement with these findings. Thus, we provide evidence that, unlike in children, RCDs should not be included in the differential diagnosis of adult-onset leukodystrophies, except when there are additional MRI findings or clinical features which unequivocally point towards a mitochondrial disorder.

Botulinum toxin type A affects the transcriptome of cell cultures derived from muscle biopsies of controls and spastic patients

Botulin toxin (BTX) is widely used for treating skeletal muscle spasticity. Experimental reports on BTX treatment were mainly focused on the neuromuscular junction, while relatively little is known about toxin effects on the muscle cell itself. We investigated possible impact of BTX type A on skeletal muscle cell transcriptome by microarray analysis in muscle-derived cell cultures (fibroblasts, myoblasts and myotubes) from controls and spastic patients, and results were then validated at transcript and protein level. BTX-A treatment of control cells induced major changes in the myogenic component of the transcriptome, whereas the same treatment had a negligible effect in the fibrogenic component. BTX-A treatment of cell cultures from spastic patients induced an increased number of genes differentially expressed both in the fibrogenic and myogenic components. Specifically, BTX-A had a major effect on cell cycle-related genes in myoblasts, on muscle contraction-related genes in myotubes, and on extracellular matrix-related genes in fibroblasts from spastic patients. Our findings show that in vitro BTX-A treatment differentially affects transcript expression in muscle cells from spastic patients compared to those from controls suggesting a direct effect of BTX-A on muscle-specific functional pathways.

Up-regulation of Toll-like receptors 7 and 9 and its potential implications in the pathogenic mechanisms of LMNA-related myopathies

ABSTRACT Laminopathies are a heterogeneous group of diseases, caused by mutations in lamin A/C proteins. The most common laminopathy (LMNA-related myopathies, LMNA-RM) affects skeletal and cardiac muscles; muscle histopathology is variable, ranging from mild unspecific changes to dystrophic features, sometimes with inflammatory evidence. Whether the genetic defect might activate innate immune components, leading to chronic inflammation, myofiber necrosis and fibrosis, is still unknown. By qPCR, a significant up-regulation of Toll-like receptor (TLR) 7 and 9 transcripts was found in LMNA-RM compared to other myopathic and non-myopathic muscles. A marked TLR7/9 staining was observed on LMNA-RM blood vessels and muscle fibers and, when present, on infiltrating cells, mainly macrophages, scattered in the tissue or localized close to degenerated muscle fibers and connective tissue. Our results recognize innate immunity as a player in LMNA-RM pathogenesis. Modulation of TLR7/9 signaling pathways and decrease of macrophage-mediated inflammation might be potential therapeutic strategies in LMNA-RM management. Abbreviations: DMD, Duchenne muscular dystrophy; EDMD2, Emery-Dreifuss muscular dystrophy type 2; FSHD, facio-scapulo-humeral muscular dystrophy; LGMD1B, limb-girdle muscular dystrophy type 1B; LMNA-CMD, LMNA-related congenital muscular dystrophy; LMNA-RM, LMNA-related myopathies; sIBM, sporadic inclusion body myositis; TLR, Toll-like receptor

P.21.1 Autophagy as a link between immunity and inflammation in idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of immune-mediated muscle disorders characterized by muscle inflammation and weakness. They comprise inclusion body myositis (IBM), polymyositis (PM), juvenile dermatomyositis (JDM) and adult dermatomyositis (DM). Autophagic pathway impairment has been well documented in sporadic IBM, where it has been suggested to be responsible for the accumulation of multiple-protein aggregates, typical of the myopathy. The main candidates responsible for this impairment are suggested to be TLRs. We evaluated the autophagic process also in PM and DM, in particular the interaction between autophagosome maturation and innate immune system. LC3 and other autophagic molecules, together with TLR3, TLR4, HSP60 and HMGB1/2 were analyzed in IIM and control muscles by qPCR, immunohistochemistry and immunoblot. Myoblasts and myotubes from PM, DM and control muscle biopsies were analyzed to evaluate changes in TLR and HMGB1/2 expression after induction or inhibition of autophagy. Gene expression analysis showed a dysregulation of autophagy in all IIM subgroups. A tight correlation between autophagy and innate immunity in myopathic muscles compared to controls was highlighted by confocal microscopy; activation of stress response and the presence of bacterial infection were observed connected with an abnormal accumulation of autophagosomes in IIM myofibers. These findings demonstrate the involvement of the autophagic process in the pathogenesis of all subtypes of IIMs; it is not clear if this mechanism represents a consequence or a cause of the induction of immune response and inflammation. Autophagic machinery components might represent a possible target for new therapeutic approaches for IIM.

Idiopathic Inflammatory Myopathies: A Review of Immunopathological Features and Current Models of Pathogenesis

The idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of chronic systemic disorders characterized by muscle inflammation and progressive muscle weakness. The major clinical variants are dermatomyositis (DM) including a distinct juvenile (JDM) subtype, polymyositis (PM), and inclusion body myositis (IBM) (Engel & Hohlfeld, 2004). IBM is divided into sporadic IBM (sIBM), the most common muscle disease starting after age 50 years, occurring mainly in men and leading to severe disability, and hereditary inclusion body myopathy, characterized by pathologic alterations resembling those of sIBM except for a lack of muscle inflammation (hence “myopathy” instead of “myositis”) (Askanas & Engel, 1998). DM may occur in children or adults and is considered a humorally-mediated microangiopathy, while PM occurs mainly after the second decade of life and is a T cell-mediated disease characterized by cytotoxic attack against non-necrotic muscle fibers (Dalakas, 2011c). For all IIM forms, both target antigens and triggering factors for autoimmune response remain unknown. A growing body of evidence suggests that genetically susceptible individuals probably develop an idiopathic inflammatory myopathy in response to particular environmental stimuli (Feldman et al., 2008; Needham & Mastaglia, 2007; O’Hanlon et al., 2006; O’Hanlon & Miller, 2009; Rider et al., 2010; Sarkar et al., 2005; Vegosen et al., 2007).