The pathogenesis of dermatomyositis associated to MDA5 autoantibodies: An in vitro and in vivo study

Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212 1

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J. Aouizerate , M. De Antonio , B. Bader-Meunier , C. Bodemer , C. Barnerias 4, I. Desguerre 4, R. Gherardi 1, J. Charuel 5, F. Authier 6, C. Gitaux 4 1 INSERM U955-Team 10, Creteil, France; 2 Garches-Necker-Mondor-Hendaye Reference Center for Neuromuscular 3 Necker Hospital, Paris, France; Diseases, Creteil, France; 4 Garches-Necker-Mondor-Hendaye Reference Center for Neuromuscular Diseases, Paris, France; 5 CHU Pitié-Salpêtrière, Paris, France; 6 Paris Est-Creteil University, Paris, France Myositis-associated autoantibodies are useful biomarkers to classify Juvenile Dermatomysitis (JDM). Anti-NXP2 antibodies are present in 20% of JDM (Tansley, 2014) and may be associated with risk of calcinosis and less inflammation on the histological study in small series (Pinal-Fernandez, 2015). Clinical outcome and histological features of NXP2 patients remain unknown. We retrospectively assessed clinical, biological and histological findings from 25 JDM patients diagnosed from 2013 to 2015 in both Pediatric rheumatology and dermatology centers. Systematic auto-antibodies screening (Mi2, MDA5, TIF1-gamma, NXP2, SAE, Ro52, Jo1, PL7, PL12, EJ, SRP, Ku, PM-Scl, Scl70,) and myopathological study of deltoid muscle biopsy (including immunohistochemistry for endothelial cells (CD31/PECAM), regenerating myofibers (CD56/NCAM), inflammatory cells, anti-human major histocompatibility complex (MHC) class I (HLA-ABC), class II (HLA-DR), and C5b-9 were performed. All biopsies were reviewed using the score tool in JDM (Varsani, 2015). Autoantibodies were detected in 16/25 JDM patients, including anti-NXP2 in 10/25, anti-MDA5 in 2/25 and anti-Tif1gamma in 4/25. Patients with NXP2 auto-antibodies (NXP2+ group) exhibited more severe clinical presentation: MMT, CMAS scores, CPK, vasculopathy-related features (gastrointestinal involvement, p = 0,007) leading to more aggressive treatment (plasmapheresis and immunoadsorption) and increased number of lines of treatment. Regarding the histological features, we observed typical lesions of dermatomyositis in all cases: inflammatory and perimysial myopathy with ischemic lesions, perifascicular atrophy and necrosis, expression of HLA ABC class I, C5b9 capillary and sarcolemmal deposits. NXP2+ group displayed more frequent ischaemic punch-out vacuoles, microinfarcts (p = 0,022) and capillary dropout (p = 0,01). The vascular domain is more severe in NXP2DM+ (p = 0,001) (Varsani score). http://dx.doi.org/10.1016/j.nmd.2016.06.214

P.195 Diagnostic utility of MxA expression for dermatomyositis A. Uruha 1, A. Nishikawa 2, R. Tsuburaya 2, S. Suzuki 3, I. Nishino 2 1 Medical Genome Center, NCNP, Tokyo, Japan; 2 National Institute of Neuroscience, NCNP, Tokyo, Japan; 3 Keio University School of Medicine, Tokyo, Japan Myxovirus resistance A (MxA), which can reflect production of type 1 interferon, is considered to play an important role in the pathogenesis of dermatomyositis (DM). However, its diagnostic utility is not sufficiently validated. To show the sensitivity and specificity of MxA expression on muscle tissues, we performed immunohistochemical analysis of MxA in muscle samples of a range of idiopathic inflammatory myopathies (IIM) and other neuromuscular disorders. We enrolled 154 consecutive patients with IIM, including 34 DM (according to the ENMC criteria, 12 definite, 18 probable, and 4 possible DM), 8 polymyositis, 16 anti-ARS antibody-associated myositis, 46 immune-mediated necrotizing myopathy (24 with anti-SRP antibodies, 6 with anti-HMGCR antibodies, and 16 without anti-SRP, anti-HMGCR, or anti-ARS antibodies), and 50 inclusion body myositis patients. As controls, we also studied 51 patients with muscular dystrophies and 26 patients with neuropathies. The sensitivity and specificity of cytoplasmic MxA expression were 71% (24/34) and 98% (118/120 with IIM). While the high specificity was almost comparable to those of perifascicular atrophy (PFA) and membrane attack complex (MAC) deposition on endomysial capillaries, the sensitivity was significantly higher than those of them (P = 0.01): PFA showed 47% sensitivity

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(16/34) and 98% specificity (118/120) and capillary MAC deposition showed 35% sensitivity (12/34) and 93% specificity (112/120). In probable DM patients (typical skin rash but lack of PFA), 44% (8/18) showed cytoplasmic MxA expression, which tended to be higher than capillary MAC deposition showing 17% (3/18, P = 0.07). In the patients with muscular dystrophy or neuropathy, no muscle samples showed positive findings of MxA, PFA, and MAC except for only one neuropathy patient’s sample having capillary MAC deposition. This study has confirmed that cytoplasmic MxA expression is a more sensitive marker for a pathologic diagnosis of DM. http://dx.doi.org/10.1016/j.nmd.2016.06.215

P.196 In vitro activation of type I interferon pathway reproduces the characteristics damages observed in dermatomyositis patients L. Ladislau 1, X. Suárez-Calvet 2, S. Toquet 3, C. Benjamim 4, V. Mouly 3, G. Boutler-Browne 3, O. Benveniste 3, Y. Allenbach 3 1 Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 2 Universitat Autònoma de Barcelona, Barcelona, Spain; 3 UPMC, Paris, France; 4 UFRJ, Rio de Janeiro, Brazil The type I interferons (IFN-I) that includes IFN-α and IFN-β are key cytokines involved in innate immune response to viral infection. Almost all cells can produce IFN-I, express IFN-I receptor (IFNAR) and induce the transcription of IFN stimulated genes (ISG), which have anti-proliferative and immunomodulatory activities. Idiopathic inflammatory myopathies (IIM) are acquired auto-immune diseases. Among the IIM, Dermatomyositis (DM) is characterized by skin lesions, muscle specific pathologic features combining inflammatory infiltration with HLA-ABC over-expression and a vasculopathy. It is known that DM patients express up-regulated ISG in muscle fibers, endothelial cells (EC), skin tissues and peripheral blood. However, the effect of the IFN-I on myoblasts (MB), myotubes (MT) and EC has not been well determined. Therefore, the aim of this study is determine if the MB, MT and EC present functional changes when exposed to IFN-I. We analyzed in vitro the effect of IFN-I on MB and EC differentiation and on MT. Recombinant IFN-I, IFN-α and IFN-β were used. In addition, Poly (I:C) (PIC), an agonist of TLR3 receptor was used. During MB differentiation we observed that, PIC, IFN-α and IFN-β abolished myotube formation and decreased myogenin (MyoG) expression. In differentiated MT all stimuli induced ISG up-regulation. Moreover, IFN-a, IFN-β and PIC dramatically reduced myotube surface area and up-regulated atrophic genes. All associated with a decreased MyoG expression. All stimuli increased expression of HLA-ABC in MT. In vitro EC exposure to IFN-I induced a decrease in cell proliferation and also ISG up-regulation. In addition, IFN-I disrupted the vascular network organization, suggesting a defect in angiogenesis. In conclusion, in vitro stimulation of the IFN pathway reproduces both muscular and vascular damages observed in vivo in DM patients. This emphasizes the key role of the IFN-I pathway in the pathophysiology of DM, leading to new avenues for therapeutic approaches. http://dx.doi.org/10.1016/j.nmd.2016.06.216

P.197 The pathogenesis of dermatomyositis associated to MDA5 autoantibodies: An in vitro and in vivo study X. Suárez-Calvet 1, S. Toquet 1, C. Danel 2,3, A. Couvelard 2,3, P. Roland 4, Y. Uzunhan 5, E. Balada 6, A. Selva-O’Callaghan 6, Y. Allenbach 1, O. Benveniste 1 1 Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS 974, CNRS FRE 3617, Center of Research in Myology, Paris, France; 2 INSERM U1149, DHU Unity, Paris Diderot University, Paris, France; 3 Department of Pathology, Bichat Hospital, Paris, France; 4 UF Immunologie Autoimmunité et Hypersensibilités, APHP Hopital Bichat-Claude Bernard, Paris, France; 5 Avicennes University Hospital, Bobigny, France; 6 Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona, Barcelona, Spain

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Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212

Dermatomyositis (DM) is a heterogeneous disease in which its pathogenesis is not fully understood but it has been considered that vascular injury and type I interferons (IFN-I) play an important role in the muscle pathology. DM patients with anti-MDA5 autoantibodies have poor prognosis that is associated to an aggressive interstitial lung disease and a vasculopathy with severe skin ulcers. Although clinically they have absent or mild muscle involvement, we recently reported that the muscle of these patients also display IFN-I signature but in a less degree compared with classical DM patients. MDA5 autoantibodies are useful as a biomarker but the role of these autoantibodies and the pathogenesis of the disease is completely unknown. The aim of this study is to elucidate the possible mechanisms involved in the pathogenesis of DM with anti-MDA5 antibodies. To achieve this, we analyzed the histopathology of the lungs of these patients, the effect of anti-MDA5 in vitro on possible target cells (muscle, endothelial and pulmonary cells) and the effect of the autoantibodies in vivo through passive transfer experiments in mice. Histopathological analysis of the lungs of MDA5 patients showed a significant presence of inflammatory infiltrates (T, B cells and macrophages) that were more abundant in more severely affected areas. In vitro experiments showed that MDA5+ plasma reduces the fusion index and the surface area of human myotubes. Tube formation assays on endothelial cells showed a disruption of the vascular network organization, suggesting a possible negative effect on angiogenesis. In addition, we observed a reduction in endothelial cells proliferation. Ongoing experiments will help to elucidate the role of anti-MDA5 abs and could provide the rationale for the future use of new treatments for these patients. http://dx.doi.org/10.1016/j.nmd.2016.06.217

P.198 Distribution and severity of weakness in patients with polymyositis and dermatomyositis: Different pathophysiology, different affected muscle groups H. Durmus, F. Deymeer, Y. Parman, P. Oflazer-Serdarog˘lu Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey

A. Ishiyama 1, I. Shibuya 1, Y. Motohashi 1, E. Takeshita 1, H. Komaki 1, K. Sugai 1, M. Sasaki 1, S. Mitsuhashi 2, S. Noguchi 2, I. Nonaka 2, I. Nishino 2 1 National Center Hospital, National Center of Neurology and Psychiatry, Tokyo, Japan; 2 National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan The purpose of our study is to determine whether signal abnormalities of muscle and fascia in lower limbs are correlated to clinical course in children with juvenile dermatomyositis (JDM). Nine children (5 boys and 4 girls, range 2–11 years) with clinically diagnosed muscle biopsy-proven JDM were included. Pretreatment MRI examinations in lower limbs were evaluated. Muscle location was divided into three groups in thigh: anterior compartment, posterior compartment and adductors, and into two groups in calf: anterior and posterior compartment. The grading of signal abnormalities was divided into five groups based on short tI inversion recovery (STIR): grade 0, no muscle signal abnormality; grade 1, 1–25% muscle involvement; grade 2, 26–50%; grade 3, 51–75%; and grade 4, 76–100%. Assessment of perimuscular fascial signal intensity abnormality was subjectively divided into absent or present. Signal abnormalities of muscle compartments were floridly increased in all children. All children were affected at the anterior compartment and adductors of the thigh showing grade 3 to 4 signal intensity changes and the posterior compartment showing grade 1 to 2. The anterior compartment of calf was affected in 4 patients, showing grade 3 to 4. Fascial signal changes were also seen in 4 children who diagnosed earlier than 6 months after the appearance of muscle symptoms while no children showed the fascial signal changes later than 6 months. The signal abnormalities of anterior muscle of thigh were significantly higher than the posterior thigh. However, the signal abnormalities were not associated with the time between symptom onset and patient presentation. Fascial signal changes indicate that fasciitis is a common lesion of JDM and suggest that the fascial involvement is the primary site of inflammatory cell infiltration. The findings of fasciitis on STIR may contribute to presume the clinical course, especially earlier than 6 months after onset, in patients with JDM. http://dx.doi.org/10.1016/j.nmd.2016.06.219

In this study, we aimed to compare the distribution and severity of muscle weakness in patients with polymyositis (PM) and dermatomyositis (DM) using manual muscle testing, which may help to distinguish the two conditions, particularly in cases with dermatomyositis sine dermatitis, and also may help planning physiotherapy. Polymyositis and dermatomyositis are both rare acquired inflammatory myopathies, presenting with similar features, such as subacute onset, proximal, symmetric muscle weakness and elevated serum CK levels. Despite these similarities, the two diseases have different immunopathological mechanisms. Clinical and laboratory findings of 58 patients diagnosed with dermatomyositis or polymyositis according to the Peter and Bohan criteria at the Department of Neurology, Istanbul Faculty of Medicine between 1992 and 2015 were retrospectively evaluated. SPSS version 13 was used for statistical analysis. Thirty-one patients were diagnosed with PM and 27 with DM. Thirty patient were female. The mean age of onset was 41.8 ± 15.4 years (ranging between 17 and 74 years). All of our patients had symmetrical proximal dominant weakness. Swallowing difficulties were more frequent in PM as compared to DM (p = 0.03). PM patients had more profound weakness at the point of the maximum disease severity (p < 0.001). Neck flexors, deltoid, pectoralis major, triceps, iliopsoas muscles, hip adductors and flexors were more severely affected in PM (p = 0.001, p = 0.03, p = 0.02, p = 0.008, p = 0.002, p = 0.002, p = 0.002, respectively). The only two muscles which were more markedly affected in DM than PM were wrist extensors and hamstrings (p = 0.002 and p = 0.03). Our results suggest that detailed manual muscle testing may help in the differential diagnosis of PM and DM. http://dx.doi.org/10.1016/j.nmd.2016.06.218

P.199 Signal abnormalities of muscle and fascia in muscular MRI imaging at pretreatment stage in children with juvenile dermatomyositis

NEUROMUSCULAR DISORDERS – GENERAL P.200 Assessment of intra-oral structure and swallowing function in pediatric neuromuscular disorders H. Kılınç, N. Bulut, I. Alemdarog˘lu, O. Yılmaz, H. Topalog˘lu, A. Karaduman Hacettepe University, Ankara, Turkey Swallowing disorders which may be a problem for pediatric patients with neuromuscular disorders (NMDs) in different functional stages may lead to life-threatening problems such as aspiration pneumonia, airway obstruction and malnutrition. The aim of our study was to investigate problems related to swallowing disorders and reveal structural impairments of intra-oral region in pediatric patients with NMDs. Forty-five patients with NMD who had no complaints (Functional Oral Intake Scale Score = 7) related to swallowing problems were included in the study. Intra oral examination including structural evaluation of tongue, lips, jaw, soft and hard palate was performed. Swallowing problems were examined with a questionnaire by asking patients and their caregivers. Three Ounce Water Swallow test was also applied to all patients. The mean age of patients was 10.22 ± 3.32 years. Sustained wheezing (n = 15, 33%), recurrent pulmonary infection (n = 5, 11%), coughing during feeding (n = 9, 20%), feeling of food stuck in the throat during feeding (n = 8, 18%), voice impairment after feeding (n = 5, 11%), fatigue during mastication (n = 12, 27%), macroglossia (n = 6, 13%), fasciculation of tongue (n = 4, 9%) and excessive high palate (n = 6, 13%) were determined. Besides, 5 patients (11%) have failed in Three Ounce Water Swallow Test. We tried to uncover the frequency and types of intra-oral structural impairments and problems related to swallowing in pediatric patients with NMDs with this study. Early diagnosis