P.14.6 Evidence for a direct role of anti-signal recognition particle antibodies in the pathogenesis of necrotizing myopathies C. Bloch Queyrat 1 , L. Drouot 2 , L. Arouche 3 , G. Butler Browne 3 , O. Boyer 1 , O. Benveniste 1 1 Pitie ´-Salpe ˆtrie `re Hospital, Internal Medicine 1, Paris, France; 2 Inserm U905, Rouen, France; 3 Inserm U974, Paris, France Anti-SRP auto-antibodies are associated with necrotizing myopathy (SRP-NM). We have previously shown (Arthritis Rheum. 2011 Mar 11. doi: 10.1002/art.30344) that serum titers of anti-SRP Abs closely correlate with CK level and muscle weakness in patients. The aim of this study is to investigate a possible direct role of anti-SRP Abs in the development of this form of myopathy. Muscle deposits of anti-SRP Abs were assessed by immunohistochem- istery, by both light- and electron-microscopy, using different sources of anti-SRP Abs. Cytopathic effect of anti-SRP Abs was assessed on cultured primary human muscle cells (myotubes). Finally, Anti-SRP + serum (400 lL) or affinity chromatography-purified IgGs from an anti- SRP + patient (2 mg) were injected intraperitonealy daily for 14 days to C57BL/6 mice. Controls were serum or purified IgGs from healthy donor. Similar histological aspects were found by light microscopy after incu- bation with the different sources of anti-SRP Abs. Immuno-stainings appeared as a punctuated intracellular signal within the endomysium with reinforcement at the membranes of myocytes. Preincubation of anti-SRP with excess recombinant SRP54 led to signal disappearance. Immunogold staining followed by electron-microscopy revealed immunoreactivity located in the endoplasmic reticulum. When cultured with purified anti- SRP Abs, myotubes showed a dramatic decrease of their diameter as com- pared to those cultured with control polyclonal IgG or no Abs, leading to a 60% decrease in the surface they covered (p = 0.003, Kruskal–Wallis test with Monte–Carlo resampling). In vivo injection of anti-SRP Abs resulted significant decrease in muscle strength as attested by rotarod results (25 s. vs. 130 s (p < 0.01)). These results provide the first experimental evidence that anti-SRP Abs may play a direct role in the pathogenesis of necrotizing myopathies. http://dx.doi:10.1016/j.nmd.2013.06.614 P.14.7 Anti- SRP antibody induces muscle cell lysis through complement mediated pathway A. Rojana-udomsart 1 , C. Mitrpant 2 , C. Bundell 3 , P. Hollingsworth 3 , F.L. Mastaglia 1 1 University of Western Australia, Centre for Neuromuscular and Neuro- logical Disorders, Perth, Australia; 2 Faculty of Medicine, Siriraj Hospital, Department of Biochemistry, Bangkok, Thailand; 3 Pathwest, Queen Eliz- abeth II Medical Centre, Department of Clinical Immunology, Perth, Australia The signal recognition particle (SRP), an intracytoplasmic complex of ribonucleoprotein, binds to the endoplasmic reticulum (ER) localising sig- nal sequences of elongating polypeptide chains during their synthesis and translocates nascent peptides to the ER membrane. Anti-SRP antibodies to one or more of the components have been associated with a necrotising autoimmune myopathy (NAM) characterised by severe subacute progres- sive proximal muscle weakness with very high serum CK levels. The mus- cle biopsies from such patients show abundant necrotic and regenerating fibres but only a sparse inflammatory infiltrate. These findings suggest that cytotoxic T cells and cell-mediated muscle fibre destruction are not involved. The possibility of a humoral mechanism has been proposed but this has not been investigated. In the present study, we utilised an in vitro human myoblast culture system to investigate a possible anti- body-mediated cytotoxic mechanism of cell death. Anti-SRP positive sera from NAM patients or anti-SRP negative sera from healthy controls were incubated with myoblasts in the presence or absence of fresh complement. In the absence of complement, there was no difference in cell viability between two groups. In contrast, when complement was added, the sur- vival of myoblasts incubated with anti-SRP positive serum was signifi- cantly reduced compared to cultures incubated with anti-SRP negative sera. In anti-SRP positive sera, we also observed complement deposition onto the membrane of normal human myoblast. Complement deposition was also observed on biopsies from three NAM patients who muscle sec- tions were available. We conclude that one of the pathological pathways involved in muscle cell death in anti-SRP positive NAM is antibody- dependent complement mediated cell lysis. However, the possibility that more than one mechanism might be involved cannot be excluded and fur- ther studies would be helpful in understanding the pathogenesis of anti- SRP associated NAM. http://dx.doi:10.1016/j.nmd.2013.06.615 P.14.8 Ethnic origins influence the natural history modelling of necrotizing myop- athy with anti-SRP antibodies C. Bloch Queyrat 1 , A. Rigolet 2 , Y. Allenbach 2 , L. Musset 3 , J.M. Treluyer 1 , S. Urien 1 , O. Benveniste 2 1 Cochin Hospital, Unite ´ de Recherche Clinique Paris-Decartes, Paris, France; 2 Pitie ´-Salpe ˆtrie `re Hospital, Internal Medicine, Paris, France; 3 Pitie ´-Salpe ˆtrie `re Hospital, Immunology, Paris, France Anti-SRP auto-antibodies are associated with necrotizing myopathy (SRP-NM). Our purpose is to describe the natural history of the disease in addition to its progression under treatment. We conducted a retrospective study on 32 patients. At each visit, the strength of the patients was assessed in 8 muscle groups by the MRC scale in 5 points. The evolution of strength is described by a model which sup- poses that the score is an equilibrium between a disease generation process (zero-order process) and a first-order elimination of this process (“turn- over model”). The random effects are simultaneously estimated (inter-sub- jects and residual variabilities). Thirty-two patients were included (median age: 40), 17 were of Cauca- sian and 15 of African origins. The median duration of follow-up was 74 months (34 months without treatment and 38 under treatment). The median speed of progression of the disease before treatment was À0.77 points of MRC per year. This speed was 10 times superior in non-Cauca- sian patients (p < 0.001). Age at diagnosis, sex, CK levels, score of necrosis in muscle biopsy, presence of an interstitial lung disease or a heart disorder were not factors of influence. All treated patients (n = 27) received predni- sone (1 mg/kg/day), combined to methotrexate (MTX) or azathioprine (Azat, n = 6), intravenous immunoglobulins (IVIg, n = 15), or rituximab (RTX, n = 6). The speed of recovery of the muscular strength was +0.7 points MRC per year. The MTX or Azat were the most effective, with no benefit in adding IVIg. The effect of corticoids was unquantifiable because it was always combined with one of the previous drugs. This study describes, for the first time, the natural history of SRP-NM and shows that slowly progressive forms (sometimes taken for LGMD) are more frequently observed in Caucasian patients. The rate of muscular recovery seems to be better when patients receive MTX or Azat in addi- tion to corticosteroids. http://dx.doi:10.1016/j.nmd.2013.06.616 P.14.9 Functional redundancy of MyD88-dependent signaling pathways in a murine model of histidyl-tRNA synthetase-induced myositis Abstracts / Neuromuscular Disorders 23 (2013) 738–852 815