Antisynthetase syndrome (ASSD) is a rare clinical condition that is characterized by the occurrence of a classic clinical triad, encompassing myositis, arthritis, and interstitial lung disease (ILD), along with specific autoantibodies that are addressed to different aminoacyl tRNA synthetases (ARS). Until now, it has been unknown whether the presence of a different ARS might affect the clinical presentation, evolution, and outcome of ASSD. In this study, we retrospectively recorded the time of onset, characteristics, clustering of triad findings, and survival of 828 ASSD patients (593 anti-Jo1, 95 anti-PL7, 84 anti-PL12, 38 anti-EJ, and 18 anti-OJ), referring to AENEAS (American and European NEtwork of Antisynthetase Syndrome) collaborative group’s cohort. Comparisons were performed first between all ARS cases and then, in the case of significance, while using anti-Jo1 positive patients as the reference group. The characteristics of triad findings were similar and the onset mainly began with a single triad finding in all groups despite some differences in overall prevalence. The “ex-novo” occurrence of triad findings was only reduced in the anti-PL12-positive cohort, however, it occurred in a clinically relevant percentage of patients (30%). Moreover, survival was not influenced by the underlying anti-aminoacyl tRNA synthetase antibodies’ positivity, which confirmed that antisynthetase syndrome is a heterogeneous condition and that antibody specificity only partially influences the clinical presentation and evolution of this condition.

Introduction: The endoplasmic reticulum (ER) stress-response, evoked in mice by the overexpression of class I major histocompatibility complex antigen (MHC-I), was proposed as a major mechanism responsible for skeletal muscle damage and dysfunction in autoimmune myositis. The present study was undertaken to characterize in more detail the ER stress-response occurring in myofibers of patients with inflammatory myopathies, focusing on the expression and distribution of Grp94, calreticulin and Grp75, three ER chaperones involved in immunomodulation. Methods: Muscle biopsies were obtained from seven healthy subjects and 29 myositis patients, who were subdivided into groups based on the morphological evidence of inflammation and/or sarcolemmal immunoreactivity for MHC-I. Biopsies were analyzed by means of immunohistochemistry and western blot using anti-Grp94, anti-calreticulin and anti-Grp75 specific antibodies. Parallel analyses on these ER chaperones were conducted in rabbit and/or murine skeletal muscle after experimental induction of regeneration or systemic inflammation. Results: Upregulation of Grp94 characterized regenerating myofibers of myositis patients (P = 0.03, compared with values detected in biopsies without signs of muscle regeneration) and developing and regenerating myofibers of mouse muscles. Conversely, levels of calreticulin and Grp75 increased about fourfold and twofold, respectively, in patient biopsies positive for sarcolemmal MHC-I immunoreactivity, compared with healthy subjects and patients negative for both inflammation and MHC-I labeling (P < 0.005). Differently from calreticulin, the Grp75 level increased significantly also in patient biopsies that displayed occasional sarcolemmal MHC-I immunoreactivity (P = 0.002), suggesting the interference of other mechanisms. Experimental systemic inflammation achieved in mice and rabbits by a single injection of bacterial lipopolysaccharide significantly increased Grp75 and calreticulin but not MHC-I expression in muscles. Conclusions: These results indicate that, in myositis patients, muscle regeneration and inflammation, in addition to MHC-I upregulation, do evoke an ER stress-response characterized by the increased expression of Grp94 and Grp75, respectively. The increase in the muscle Grp75 level in patients showing occasional immunoreactivity for sarcolemmal MHC-I might be considered further as a broader indicator of idiopathic inflammatory myopathy.

Idiopathic inflammatory myopathies (IIM), even though sharing common clinical manifestations, are characterized by diversified molecular pathogenetic mechanisms which may account for the partial inefficacy of currently used immunomodulatory drugs. In the last decades, the role of interferon (IFN) in IIM has been extensively elucidated thanks to genomic and proteomic studies which have assessed the molecular signature at the level of affected tissues or in peripheral blood across distinct IIM subtypes. A predominant type I IFN response has been shown in dermatomyositis (DM), being especially enhanced in anti-melanoma differentiation-associated gene 5 (MDA5)+ DM, while a type 2 IFN profile characterizes anti-synthetase syndrome (ASyS) and inclusion body myositis (IBM); conversely, a less robust IFN footprint has been defined for immune-mediated necrotizing myopathy (IMNM). Intracellular IFN signaling is mediated by the janus kinase/signal transducer and activator of transcription (JAK/STAT) through dedicated transmembrane receptors and specific cytoplasmic molecular combinations. These results may have therapeutic implications and led to evaluating the efficacy of new targeted drugs such as the recently introduced janus kinase inhibitors (JAKi), currently approved for the treatment of rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. In this review we aim to summarize the most significant evidence of IFN role in IIM pathogenesis and to describe the current state of the art about the ongoing clinical trials on IFN-targeting drugs, with particular focus on JAKi.

There is still a need for an efficient method for the isolation of extracellular vesicles (EVs) from human blood that provides a reliable yield with acceptable purity. Blood is a source of circulating EVs, but soluble proteins and lipoproteins hamper their concentration, isolation, and detection. This study aims to investigate the efficiency of EV isolation and characterization methods not defined as “gold standard”. EVs were isolated from human platelet-free plasma (PFP) of patients and healthy donors through size-exclusion chromatography (SEC) combined with ultrafiltration (UF). Then, EVs were characterized using transmission electron microscopy (TEM), imaging flow cytometry (IFC), and nanoparticle tracking analysis (NTA). TEM images showed intact and roundish nanoparticles in pure samples. IFC analysis detected a prevalence of CD63+ EVs compared to CD9+, CD81+, and CD11c+ EVs. NTA confirmed the presence of small EVs with a concentration of ~10¹⁰ EVs/mL that were comparable when stratifying the subjects by baseline demographics; conversely, concentration differed according to the health status across healthy donors and patients affected with autoimmune diseases (130 subjects in total, with 65 healthy donors and 65 idiopathic inflammatory myopathy (IIM) patients). Altogether, our data show that a combined EV isolation method, i.e., SEC followed by UF, is a reliable approach to isolate intact EVs with a significant yield from complex fluids, which might characterize disease conditions early.

Introduction: The aim of this study was to evaluate an extensive panel of cytokines involved in immune regulation during pregnancy in patients with systemic lupus erythematosus (SLE) and in healthy women. Methods: A total of 47 consecutive successful pregnancies in 46 SLE patients and 56 pregnancies in 56 matched healthy subjects, as controls, were prospectively studied. Serum interleukin (IL)-1-α, IL-1-β, IL-2, IL-6, IL-8, IL-10, IL-12p70, interferon (INF)-γ and tumor necrosis factor (TNF)-α were detected in sera obtained at the first and third trimester of pregnancy by a highly sensitive, multiplexed sandwich ELISA. Results: Medians (pg/ml) of serum levels of most helper T (Th)1-type cytokines were significantly lower in the third trimester compared with those observed in the first trimester of pregnancy in healthy women: INF-γ 2.0 vs 3.4, TNF-α 10.2 vs 11.5, IL-1-α 0.9 vs 1.1, IL-1-β 0.6 vs 1.0, IL-2 3.0 vs 3.5, and IL-12p70 4.9 vs 5.6 (P-values < 0.02 for all). By contrast, only the IL-1-α serum levels were lower in the third trimester compared with the first trimester in SLE patients (P = 0.006). IFN-γ/IL-6 and IFN-γ/IL-10 ratios were higher in controls than in SLE (P = 0.002, and P = 0.001, respectively); moreover, they were significantly reduced in the third compared to the first trimester of pregnancy in healthy women, but not in SLE. Conclusions: In SLE patients, Th1/Th2 cytokine serum level ratio does not decrease during pregnancy progression as much as in healthy pregnant women. This could account for the observation of a low frequency of disease flares in the third trimester of gestation.