This is a single-center, retrospective, observational cohort study including 48 patients who were diagnosis as ANM-SRP by clinical, serological, and pathological criterion. Demographic data, clinical features, and initial drug treatment information were collected (Supplementary materials). The procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation and approved by the Institutional Review Board. Informed consent for all examinations was obtained from the patients or their guardians.
Serum myositis antibody test
An immunoblot kit was used to determine the serum antibodies to SRP by detecting the 54kD subunit. The membrane strips were pretreated, incubated with patient serum, followed by enzyme binding. The results were scanned using EUROlineScan software, and were recorded as negative, weak positive (+), positive (++) and strong positive (+++). All the patients recruited were strong positive (+++).
Thirty-six patients underwent bilateral thigh MRI (tMRI) (3.0 T，GE 1.5 Sigma Twin Speed; GE Healthcare, Waukesha, WI, USA). Axial T1-weighted MRI was performed to evaluate the degree of fatty infiltration according to the modified Mercuri scale (0–5 scale). Axial Short T1 inversion recovery sequences were used to assess the degree of edema (0–5 scale) . Edema and fatty infiltration scores were calculated in the gluteus maximus at the pelvic level and thigh muscles (vastus intermedius, vastus medialis, vastus lateralis, rectus femoris, biceps femoris, semitendinosus, semimembranosus, adductor magnus, sartorius, long adductor, and gracilis) at the mid-thighs. We calculated and summed the total fatty infiltration and edema scores of the gluteus maximus and thigh muscles between adolescent patients and adult patients. Additionally, we compared the thigh MRI of 36 patients with 36 controls (autoimmune necrotizing myopathy patients with negative anti-SRP antibody) to observe the damage in the tMRI.
Muscle biopsies were taken from the biceps brachii or quadriceps femoris of all the patients. Serial frozen sections were stained with hematoxylin and eosin (HE), modified Gomori trichrome, periodic acid-Schiff, Oil Red O, adenosinetriphosphate (ATP) enzyme (pH 4.5 and 10.8), NADH-tetrazolium reductase, Succinate dehydrogenase (SDH), and cytochrome C oxidase (COX) stains. The sections were immunohistochemically stained with primary antibodies against human CD3, CD4, CD8, CD19, CD20, CD68, B cell activating factor (BAFF) and its receptor, major histocompatibility complex class -I (MHC-I), membrane attack complex (MAC), dystrophin, sarcoglycans, and dysferlin. The positive cellular expression of BAFF, BAFF-R and CD19 positive cells (numbers of BAFF, BAFF-R, CD19 positive cells / numbers of muscle fibers) were calculated per high-power microscopic field. Correlation of the positive cellular expression of BAFF, BAFF-R, CD19 positive cells was analyzed.
BAFF and BAFF-R measurement
Muscle tissue of 14 patients with ANM-SRP and four healthy control were sampled. RIPA lysate (including protease inhibitor and phosphatase inhibitor) was added to the tissue samples for homogenization, and the protein was extracted after lysis on ice for 30min. An equal amount of protein (80 μg) was suspended in loading buffer, denatured at 100°C for 5 minutes, and loaded on an SDS-PAGE gel. After being electrophoresed in 10% polyacrylamide gel, transferred electrophoretically to nitrocellulose membrane, the membrane was blocked with nonfat milk buffer for 1 hour and then incubated with the primary antibodies stay overnight at 4°C. The primary antibodies were IgG specific for BAFF (Abcam, ab16081, 1:500), BAFF-R (Abcam, ab5965, 1:500), Actin (Abcam, ab8226, 1:3000). After three 5-minute washes (20-mmol/L Tris, pH 7.6, 8g sodium chloride, 0.05% Tween-20), blots were incubated for 50 minutes with horseradish peroxidase–conjugated goat anti–rabbit IgG or rabbit anti-rat IgG(1:5000). After washing, bound IgG was detected autoradio-graphically by enhanced chemiluminescence (Quantity One v.4.6.2).
The primary outcome of this study was the achievement of complete or partial remission, the patients were divided into non-refractory and refractory groups according to the clinical follow-up indicators at 3, 6, and 12 months after treatment.
Patients who still had obvious myasthenia and high levels of serum CK on the basis of glucocorticoid therapy combined with other immunosuppressive agents or intravenous immunoglobulins (IVIg) in the study period were considered refractory. The therapeutic effect evaluation method adopted in this study is the modified Rankin Scale score (mRS). If patients’ muscle strength returned to normal or was close to normal, the mRS were 0-2 scores, they were placed in the non-refractory group. If there was still obvious limb weakness after 12 months of treatment, the mRS were 3-5 points, or recurrence, the patients were placed into the refractory group [8, 13]. We monitored the clinical symptoms, the muscle fatty infiltration and edema changes by tMRI for 3, 6, 12, 18 and 24 months after treatment. Clinical follow-up indicators were mRS , imaging indicators were the average change rates of thigh muscle fatty infiltration and edema (the scale gap of tMRI / interval time between before and after treatment) for 3, 6, 12, 18 and 24 months after treatment.
All analyses were performed using SPSS 17.0 software. Single factor and binary logistic regression analysis were used to compare the clinical, tMRI changes and pathological features between the two groups. The Mann-Whitney U or Kruskal-Wallis test was used for the continuous variables and Chi-square test for categorical variables. Multivariate logistic regression analysis was performed on related predictors for refractory disease. Explanatory variables were selected using a liberal criteria (p<0.10) for inclusion in the multivariate regression model. For all statistical analyses, significance was accepted as p<0.05.