Objectives: Granulomatous myositis (GM) is defined by focal collections of activated macrophages that fuse to form multinucleated cells that aggregate into granulomas within skeletal muscle. This study aimed to elucidate the pathophysiology of GM by defining its specific transcriptomic profile. Methods: Bulk RNA sequencing was performed on 722 muscle biopsies, including 38 from patients with GM, other myopathies, and healthy comparators. Spatial transcriptomics and immunohistochemistry assays were used to identify the location of specific transcripts and proteins within the tissue. Results: The transcriptomic signature of GM muscle biopsies was characterized by high levels of IFNγ, IFNγ-inducible genes, and proinflammatory cytokine genes including IL1B, TNF, and TGFB1. The expression levels of 1293 specifically upregulated and 256 specifically downregulated genes were highly correlated with transcriptomic markers of disease activity. Support vector machine models using this gene set identified GM with an AUC of 99.6% (99.0%-99.9%) and an accuracy of 98.6% (98.2%-98.9%). Expression of CHIT1, the most significantly upregulated gene, strongly correlated with disease severity and was detected at the RNA and protein level in granulomas and giant cells. Conclusions: GM is transcriptomically characterized by a strong IFNγ signature and overexpression of proinflammatory cytokines, including IL1B, TNF, and TGFB1. Additionally, it exhibits a unique transcriptomic profile, including CHIT1, which correlates with disease activity.
Unbiased antibody profiling can identify the targets of an immune reaction. A number of likely pathogenic autoreactive antibodies have been associated with life-threatening SARS-CoV-2 infection; yet, many additional autoantibodies likely remain unknown. Here we present Molecular Indexing of Proteins by Self Assembly (MIPSA), a technique that produces ORFeome-scale libraries of proteins covalently coupled to uniquely identifying DNA barcodes for analysis by sequencing. We used MIPSA to profile circulating autoantibodies from 55 patients with severe COVID-19 against 11,076 DNA-barcoded proteins of the human ORFeome library. MIPSA identified previously known autoreactivities, and also detected undescribed neutralizing interferon lambda 3 (IFN-λ3) autoantibodies. At-risk individuals with anti- IFN-λ3 antibodies may benefit from interferon supplementation therapies, such as those currently undergoing clinical evaluation.
Objective To describe the disease specificity, clinical phenotype, and risk of cancer in dermatomyositis (DM) patients with autoantibodies against cell division cycle and apoptosis regulator protein 1 (anti‐CCAR1). Methods The frequency of anti‐CCAR1 autoantibodies was measured by enzyme‐linked immunosorbent assay in the serum of DM patients from 2 independent cohorts (Johns Hopkins and Stanford), with patients with several other rheumatic diseases and healthy controls used as comparators. Clinical features and the risk of cancer incidence relative to that in the general population were determined in anti‐CCAR1–positive DM patients. Results Anti‐CCAR1 antibodies were significantly associated with anti–transcriptional intermediary factor 1γ (anti‐TIF1γ) antibodies present in the serum of patients with DM: 80 (32%) of 252 anti‐TIF1γ–positive DM patients versus 14 (8%) of 186 anti‐TIF1γ–negative DM patients were positive for anti‐CCAR1 antibodies ( P < 0.001). Anti‐CCAR1 antibodies were not detected in any of the 32 serum samples from healthy controls, and were present at very low frequencies in the sera of patients with other rheumatic diseases: 1 (2.3%) of 44 patients with anti–hydroxymethylglutaryl‐coenzyme A reductase–positive necrotizing myopathy, 1 (2.3%) of 44 patients with inclusion body myositis, and 3 (6.5%) of 46 patients with systemic lupus erythematosus were positive for anti‐CCAR1 antibodies. Upon examining data on occurrence of cancer from the onset of DM onward, the observed number of cancers diagnosed in anti–TIF‐1γ–positive DM patients was significantly greater than expected in both cohorts, with a standardized incidence ratio (SIR) of 3.49 (95% confidence interval [95% CI] 2.39–4.92) in the Johns Hopkins cohort and a SIR of 4.54 (95% CI 3.04–6.52) in the Stanford cohort (each P < 0.001). DM patients who were both anti‐TIF1γ positive and anti‐CCAR1 positive had lower SIRs for cancer, with a SIR of 1.78 (95% CI 0.77–3.51) ( P = 0.172) in the Johns Hopkins cohort and a SIR of 1.61 (95% CI 0.44–4.13) ( P = 0.48) in the Stanford cohort. Conclusion Anti‐CCAR1 autoantibodies are specific for anti‐TIF1γ–positive DM. Their presence in anti‐TIF1γ–positive patients attenuates the risk of cancer to a level comparable to that seen in the general population. image
Dermatomyositis (DM), antisynthetase syndrome (AS), immune-mediated necrotizing myopathy (IMNM), and inclusion body myositis (IBM) are four major types of idiopathic inflammatory myopathy (IIM). Muscle biopsies from each type of IIM have unique transcriptomic profiles. MicroRNAs (miRNAs) target messenger RNAs (mRNAs), thereby regulating their expression and modulating transcriptomic profiles. In this study, 18 DM, 12 IMNM, 6 AS, 6 IBM, and 6 histologically normal muscle biopsies underwent miRNA profiling using the NanoString nCounter system. Eleven miRNAs were exclusively differentially expressed in DM compared to controls, seven miRNAs were only differentially expressed in AS, and nine miRNAs were specifically upregulated in IBM. No differentially expressed miRNAs were identified in IMNM. We also analyzed miRNA-mRNA associations to identify putative targets of differentially expressed miRNAs. In DM and AS, these were predominantly related to inflammation and cell cycle progression. Moreover, our analysis showed an association between miR-30a-3p, miR-30e-3p, and miR-199b-5p downregulation in DM and the upregulation of target genes induced by type I interferon. In conclusion, we show that muscle biopsies from DM, AS, and IBM patients have unique miRNA signatures and that these miRNAs might play a role in regulating the expression of genes known to be involved in IIM pathogenesis.
To describe the experience managing treatment-refractory immune-mediated necrotizing myopathies (IMNM) with high-dose cyclophosphamide (HiCy) therapy.Five patients with severe refractory IMNM who were treated with HiCy without stem cell rescue were identified. Their medical records were reviewed to assess demographic, clinical, and histologic characteristics as well as response to therapy.Three patients with anti-signal recognition particle (SRP) and 2 patients with anti-HMG-CoA reductase autoantibodies were included. The mean follow-up time after HiCy therapy was 37 ± 28 months. Two patients demonstrated substantial response, evidenced by improved muscle strength and decreased muscle enzymes after HiCy therapy; both of these patients were anti-SRP positive. Four patients experienced febrile neutropenia after HiCy therapy, one of which required a prolonged intensive care unit stay for infectious complications, from which they eventually recovered.These data suggest that HiCy therapy without stem cell rescue may be considered as an alternative for the treatment of refractory IMNM.
Autoantibodies in the inflammatory myopathies identify unique subsets of patients with distinctive clinical features and provide useful diagnostic and prognostic information. However the association of individual autoantibodies with specific muscle biopsy features has rarely been studied.
Objectives
To characterize muscle biopsy features associated with different autoantibodies in a cohort of patients with inflammatory myopathies from a single-center.
Methods
Patients from the Johns Hopkins Myositis Center cohort diagnosed with probable or definite dermatomyositis (according to Bohan and Peter criteria) who had both a muscle biopsy read at Johns Hopkins and a known myositis autoantibody were included in the study. For each biopsy, the presence/absence of the following features were recorded: perifascicular atrophy, mitochondrial dysfunction (>5 COX negative fibers), perivascular inflammation, primary inflammation (invasion of non-necrotic fibers by mononuclear cells) and necrotizing myopathy (defined as biopsies with necrosis/degeneration without perifascicular atrophy or primary inflammation). As a complementary analysis, biopsy features of all anti-Jo1 positive patients with either dermatomyositis or polymyositis were compared through Fisher9s exact test.
Results
37 patients with dermatomyositis were included in the study (79.5% definite). These included 11 with anti-Jo-1, 7 with anti-Mi-2, 11 with anti-TIF1-γ, 8 with anti-NXP2, and 5 with anti-PM-Scl. Biopsies from anti-Mi-2 positive patients showed frequent perifascicular atrophy (71.4%), perivascular inflammation (85.7%) and primary inflammation (57.1%); biopsies from anti-TIF1-γ positive patients showed frequent perifascicular atrophy (63.3%), mitochondrial dysfunction (55.6%) and perivascular inflammation (72.7%); biopsies from anti-NXP2 positive patients showed frequent perifascicular atrophy (50%) and biopsies from anti-PM-Scl positive patients showed frequent perivascular inflammation (80%) and primary inflammation (60%). Overall, 16.2% of DM patients had a necrotizing myopathy without perifascicular atrophy or primary inflammation. In the complementary analysis 7 Jo-1 polymyositis patients were identified. Biopsy features of anti-Jo1 patients with dermatomyositis and polymyositis were not statistically different (all p>0.05).
Conclusions
The majority of anti-PM-Scl and anti-Mi-2 patients have primary inflammation, previously considered the hallmark of PM. Most biopsies from anti-TIF1-γ positive patients reveal mitochondrial dysfunction. 16.2% of DM biopsies have necrotizing myopathy. Biopsy features are similar between dermatomyositis and polymyositis in anti-Jo1 positive patients.
References
Dalakas MC. Muscle biopsy findings in inflammatory myopathies. Rheumatic diseases clinics of North America 2002;28:779-98, vi. Mozaffar T, Pestronk A. Myopathy with anti-Jo-1 antibodies: pathology in perimysium and neighbouring muscle fibres. Journal of neurology, neurosurgery, and psychiatry 2000;68:472-8. Pestronk A. Acquired immune and inflammatory myopathies: pathologic classification. Current opinion in rheumatology 2011;23:595-604.
Ionotropic glutamate receptors are known to cluster at high concentration on the postsynaptic membrane of excitatory synapses, but the mechanism by which this occurs is poorly understood. Studies on the neuromuscular junction and central inhibitory synapses suggest that clustering of neurotransmitter receptors requires its interaction with a cytoplasmic protein. Recently, in vitro studies have shown that members of the N-methyl—aspartate (NMDA) class of glutamate receptors interact with a synapse-associated protein, SAP90 (PSD-95). However, evidence for the in vivo interaction of NMDA receptors with SAPs is still lacking. In the present study, we demonstrate the specific interaction between SAP102, a novel synapse-associated protein, and the NMDA receptor complex from the rat cortical synaptic plasma membranes using co-immunoprecipitation techniques. No association was observed between SAP102 and GluR1, a member of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate class of glutamate receptors. To identify the domain on the NMDA receptor responsible for this interaction, we constructed hexahistidine fusion proteins from different regions of the NR1a and NR2 subunits of the NMDA receptor. Immunoblot overlay experiments showed that while the C-terminal domain of the NR2 subunit displayed strong binding, the NR1a intracellular C-terminal tail did not interact with SAP102. The site of interaction was more precisely located to the last 20 amino acids of the NR2 subunit as indicated by the interaction of the synthetic peptide with SAP102. In summary, we demonstrate here for the first time an in vivo interaction between the native NMDA receptor complex and a synapse-associated protein. These results suggest that SAP102 may play an important role in NMDA receptor clustering and immobilization at excitatory synapses. Ionotropic glutamate receptors are known to cluster at high concentration on the postsynaptic membrane of excitatory synapses, but the mechanism by which this occurs is poorly understood. Studies on the neuromuscular junction and central inhibitory synapses suggest that clustering of neurotransmitter receptors requires its interaction with a cytoplasmic protein. Recently, in vitro studies have shown that members of the N-methyl—aspartate (NMDA) class of glutamate receptors interact with a synapse-associated protein, SAP90 (PSD-95). However, evidence for the in vivo interaction of NMDA receptors with SAPs is still lacking. In the present study, we demonstrate the specific interaction between SAP102, a novel synapse-associated protein, and the NMDA receptor complex from the rat cortical synaptic plasma membranes using co-immunoprecipitation techniques. No association was observed between SAP102 and GluR1, a member of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate class of glutamate receptors. To identify the domain on the NMDA receptor responsible for this interaction, we constructed hexahistidine fusion proteins from different regions of the NR1a and NR2 subunits of the NMDA receptor. Immunoblot overlay experiments showed that while the C-terminal domain of the NR2 subunit displayed strong binding, the NR1a intracellular C-terminal tail did not interact with SAP102. The site of interaction was more precisely located to the last 20 amino acids of the NR2 subunit as indicated by the interaction of the synthetic peptide with SAP102. In summary, we demonstrate here for the first time an in vivo interaction between the native NMDA receptor complex and a synapse-associated protein. These results suggest that SAP102 may play an important role in NMDA receptor clustering and immobilization at excitatory synapses.
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