Anti-Ku + myositis: an acquired inflammatory protein-aggregate myopathy.

Myositis with anti-Ku-autoantibodies is a rare inflammatory myopathy associated with various connective tissue diseases. Histopathological studies have identified inflammatory and necrotizing aspects, but a precise morphological analysis and pathomechanistic disease model are lacking. We therefore aimed to carry out an in-depth morpho-molecular analysis to uncover possible pathomechanisms. Muscle biopsy specimens from 26 patients with anti-Ku-antibodies and unequivocal myositis were analyzed by immunohistochemistry, immunofluorescence, transcriptomics, and proteomics and compared to biopsy specimens of non-disease controls, immune-mediated necrotizing myopathy (IMNM), and inclusion body myositis (IBM). Clinical findings and laboratory parameters were evaluated retrospectively and correlated with morphological and molecular features. Patients were mainly female (92%) with a median age of 56.5 years. Isolated myositis and overlap with systemic sclerosis were reported in 31%, respectively. Isolated myositis presented with higher creatine kinase levels and cardiac involvement (83%), whereas systemic sclerosis-overlap patients often had interstitial lung disease (57%). Histopathology showed a wide spectrum from mild to pronounced myositis with diffuse sarcolemmal MHC-class I (100%) and -II (69%) immunoreactivity, myofiber necrosis (88%), endomysial inflammation (85%), thickened capillaries (84%), and vacuoles (60%). Conspicuous sarcoplasmic protein aggregates were p62, BAG3, myotilin, or immunoproteasomal beta5i-positive. Proteomic and transcriptomic analysis identified prominent up-regulation of autophagy, proteasome, and hnRNP-related cell stress. To conclude, Ku + myositis is morphologically characterized by myofiber necrosis, MHC-class I and II positivity, variable endomysial inflammation, and distinct protein aggregation varying from IBM and IMNM, and it can be placed in the spectrum of scleromyositis and overlap myositis. It features characteristic sarcoplasmic protein aggregation on an acquired basis being functionally associated with altered chaperone, proteasome, and autophagy function indicating that Ku + myositis exhibit aspects of an acquired inflammatory protein-aggregate myopathy.

"Amyopathic" MDA5-positive dermatomyositis with severe lung involvement presenting with net myositic morphological features - insights from an autopsy study.

Anti-MDA5-positive dermatomyositis (MDA5-DM) often presents with extramuscular, especially pulmonary and skin manifestations, and apparent clinical signs of frank myositis can be missing (so called amyopathic DM). We hereby present two male patients who died from respiratory failure during the course of MDA5-DM. While overt signs of myositis or any skin involvement were absent at admission to hospital we noticed conspicuous inflammatory alterations in various skeletal muscles morphologically, showing different degrees of affection. Furthermore, pathological changes of the lungs compatible with rapid progressive interstitial lung disease and characteristic cutaneous vasculoocclusive features were identified at autopsy. This observation shows that muscles and skin are subclinically affected in a widespread fashion, hence subtle signs of muscle involvement should be sought after in anti-MDA5-positive patients with predominant lung affection to ensure adequate treatment.

Morphological and molecular comparison of HIV-associated and sporadic inclusion body myositis.

OBJECTIVE: The molecular characteristics of sporadic inclusion body myositis (sIBM) have been intensively studied, and specific patterns on the cellular, protein and RNA level have emerged. However, these characteristics have not been studied in the context of HIV-associated IBM (HIV-IBM). In this study, we compared clinical, histopathological, and transcriptomic patterns of sIBM and HIV-IBM. METHODS: In this cross-sectional study, we compared patients with HIV-IBM and sIBM based on clinical and morphological features as well as gene expression levels of specific T-cell markers in skeletal muscle biopsy samples. Non-disease individuals served as controls (NDC). Cell counts for immunohistochemistry and gene expression profiles for quantitative PCR were used as primary outcomes. RESULTS: 14 muscle biopsy samples (7 HIV-IBM, 7 sIBM) of patients and 6 biopsy samples from NDC were included. Clinically, HIV-IBM patients showed a significantly lower age of onset and a shorter period between symptom onset and muscle biopsy. Histomorphologically, HIV-IBM patients showed no KLRG1+ or CD57+ cells, while the number of PD1+ cells did not differ significantly between the two groups. All markers were shown to be significantly upregulated at gene expression level with no significant difference between the IBM subgroups. CONCLUSION: Despite HIV-IBM and sIBM sharing important clinical, histopathological, and transcriptomic signatures, the presence of KLRG1+ cells discriminated sIBM from HIV-IBM. This may be explained by longer disease duration and subsequent T-cell stimulation in sIBM. Thus, the presence of TEMRA cells is characteristic for sIBM, but not a prerequisite for the development of IBM in HIV+ patients.

Eosinophilic fasciitis (Shulman syndrome)-recognition of the histological spectrum allows for new insights into possible pathomechanisms.

OBJECTIVES: EF is a rare disease characterized by fibrosis and inflammation of the fascia, scleroderma-like skin indurations and optional blood eosinophilia. We aimed to expand the knowledge about its aetiology and pathogenesis. METHODS: Biopsy specimens from 16 EF patients were assessed by histology, immunohistochemistry and quantitative reverse transcription PCR in comparison with anti-Mi-2+ DM patients and non-disease controls. RESULTS: Histologically, EF shows mild to severe inflammation at the muscle-fascia interface, with frequent involvement of the underlying muscle tissue, though varying in degree. CD206+ macrophages predominate and eosinophils are detected within the fascia in the majority of cases, however in quite small numbers, and seen infrequently within the muscle. Activators of the so-called Th2-M2 pathway like STAT6 and IL-4 are upregulated leading to high expression levels of CD206. Activators of the so-called Th1-M1 pathway like STAT1 and IFN-γ (IFNG) are also upregulated, though not translating into a significant upregulation of the effector molecule COX2. Interestingly, activators or chemoattractants of eosinophils show no significant upregulation in EF compared with DM. EF shows features of perifascicular pathology comparable to DM, with upregulation of MHC class I and II; however, this is not accompanied by perifascicular atrophy or any signs of a type I IFN response or hypoxia-mediated processes. CONCLUSIONS: Our findings highlight a specific immune phenotype of leucocyte infiltrates in EF along features of perifascicular pathology similar to DM, while there is no evidence of hypoxia-mediated or type I IFN-associated processes with perifascicular fibre atrophy, indicating different pathomechanisms of muscle involvement.

Endoplasmic reticulum-stress and unfolded protein response-activation in immune-mediated necrotizing myopathy.

Patients suffering from immune-mediated necrotizing myopathies (IMNM) harbor, the pathognomonic myositis-specific auto-antibodies anti-SRP54 or -HMGCR, while about one third of them do not. Activation of chaperone-assisted autophagy was described as being part of the molecular etiology of IMNM. Endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR)-stress accompanied by activation of the unfolded protein response (UPR) often precedes activation of the protein clearance machinery and represents a cellular defense mechanism toward restoration of proteostasis. Here, we show that ER/SR-stress may be part of the molecular etiology of IMNM. To address this assumption, ER/SR-stress related key players covering the three known branches (PERK-mediated, IRE1-mediated, and ATF6-mediated) were investigated on both, the transcript and the protein levels utilizing 39 muscle biopsy specimens derived from IMNM-patients. Our results demonstrate an activation of all three UPR-branches in IMNM, which most likely precedes the activation of the protein clearance machinery. In detail, we identified increased phosphorylation of PERK and eIF2a along with increased expression and protein abundance of ATF4, all well-documented characteristics for the activation of the UPR. Further, we identified increased general XBP1-level, and elevated XBP1 protein levels. Additionally, our transcript studies revealed an increased ATF6-expression, which was confirmed by immunostaining studies indicating a myonuclear translocation of the cleaved ATF6-form toward the forced transcription of UPR-related chaperones. In accordance with that, our data demonstrate an increase of downstream factors including ER/SR co-chaperones and chaperones (e.g., SIL1) indicating an UPR-activation on a broader level with no significant differences between seropositive and seronegative patients. Taken together, one might assume that UPR-activation within muscle fibers might not only serve to restore protein homeostasis, but also enhance sarcolemmal presentation of proteins crucial for attracting immune cells. Since modulation of ER-stress and UPR via application of chemical chaperones became a promising therapeutic treatment approach, our findings might represent the starting point for new interventional concepts.

Association Between SARS-CoV-2 Infection and Immune-Mediated Myopathy in Patients Who Have Died.

Importance: Myalgia, increased levels of creatine kinase, and persistent muscle weakness have been reported in patients with COVID-19. Objective: To study skeletal muscle and myocardial inflammation in patients with COVID-19 who had died. Design, Setting, and Participants: This case-control autopsy series was conducted in a university hospital as a multidisciplinary postmortem investigation. Patients with COVID-19 or other critical illnesses who had died between March 2020 and February 2021 and on whom an autopsy was performed were included. Individuals for whom informed consent to autopsy was available and the postmortem interval was less than 6 days were randomly selected. Individuals who were infected with SARS-CoV-2 per polymerase chain reaction test results and had clinical features suggestive of COVID-19 were compared with individuals with negative SARS-CoV-2 polymerase chain reaction test results and an absence of clinical features suggestive of COVID-19. Main Outcomes and Measures: Inflammation of skeletal muscle tissue was assessed by quantification of immune cell infiltrates, expression of major histocompatibility complex (MHC) class I and class II antigens on the sarcolemma, and a blinded evaluation on a visual analog scale ranging from absence of pathology to the most pronounced pathology. Inflammation of cardiac muscles was assessed by quantification of immune cell infiltrates. Results: Forty-three patients with COVID-19 (median [interquartile range] age, 72 [16] years; 31 men [72%]) and 11 patients with diseases other than COVID-19 (median [interquartile range] age, 71 [5] years; 7 men [64%]) were included. Skeletal muscle samples from the patients who died with COVID-19 showed a higher overall pathology score (mean [SD], 3.4 [1.8] vs 1.5 [1.0]; 95% CI, 0-3; P < .001) and a higher inflammation score (mean [SD], 3.5 [2.1] vs 1.0 [0.6]; 95% CI, 0-4; P < .001). Relevant expression of MHC class I antigens on the sarcolemma was present in 23 of 42 specimens from patients with COVID-19 (55%) and upregulation of MHC class II antigens in 7 of 42 specimens from patients with COVID-19 (17%), but neither were found in any of the controls. Increased numbers of natural killer cells (median [interquartile range], 8 [8] vs 3 [4] cells per 10 high-power fields; 95% CI, 1-10 cells per 10 high-power fields; P < .001) were found. Skeletal muscles showed more inflammatory features than cardiac muscles, and inflammation was most pronounced in patients with COVID-19 with chronic courses. In some muscle specimens, SARS-CoV-2 RNA was detected by reverse transcription-polymerase chain reaction, but no evidence for a direct viral infection of myofibers was found by immunohistochemistry and electron microscopy. Conclusions and Relevance: In this case-control study of patients who had died with and without COVID-19, most individuals with severe COVID-19 showed signs of myositis ranging from mild to severe. Inflammation of skeletal muscles was associated with the duration of illness and was more pronounced than cardiac inflammation. Detection of viral load was low or negative in most skeletal and cardiac muscles and probably attributable to circulating viral RNA rather than genuine infection of myocytes. This suggests that SARS-CoV-2 may be associated with a postinfectious, immune-mediated myopathy.

Inflammatory features in sporadic late-onset nemaline myopathy are independent from monoclonal gammopathy.

Sporadic late-onset nemaline myopathy (SLONM) is a rare adult-onset non-hereditary disease with subacute proximal muscle and often axial muscle weakness, characterized by the presence of nemaline bodies in skeletal muscle biopsies. Considering its association with concurrent monoclonal gammopathy of undetermined significance (MGUS), the disease is classified into two major subtypes (1) SLONM without MGUS (SLONM-noMGUS) and (2) with MGUS (SLONM-MGUS) association. SLONM associated with HIV infection (SLONM-HIV) is also reported. SLONM-MGUS has been shown to be associated with poorer prognosis and required aggressive treatment including high-dose melphalan and autologous stem cell transplantation. The approach is currently debatable as recent reports suggested effectiveness of intravenous immunoglobulin as initial treatment with indifference of overall survival despite the presence of MGUS. Our study aimed to find an underlying basis by review of pathological features in 49 muscle biopsy proven-SLONM from two large tertiary centers in Japan and Germany (n = 49: SLONM-noMGUS = 34, SLONM-MGUS = 13, SLONM-HIV = 2). We compared pathological findings in SLONM-noMGUS and SLONM-MGUS and focused on the presence of any detectable inflammatory features by immunohistochemistry. The clinical and histological features in SLONM-noMGUS and SLONM-MGUS were not distinctively different except for more common regenerating fibers (>5% of myofibers) present in SLONM-MGUS (p < 0.01). HLA-ABC expression and fine granular p62 were observed in 66.7% and 78.3% of SLONM, respectively. The predominant inflammatory cells were CD68+ cells. The inflammatory cells showed positive correlations with the percentage of nemaline-containing fibers (p < 0.001). In conclusion, inflammatory features are present although rather mild in SLONM. This finding contributes to the hypothesis of an acquired inflammatory disease pathogenesis and opens the possibility to offer immunotherapy in SLONM with inflammatory features regardless of the monoclonal gammopathy status.

NanoString technology distinguishes anti-TIF-1γ+ from anti-Mi-2+ dermatomyositis patients.

Dermatomyositis (DM) is a systemic idiopathic inflammatory disease affecting skeletal muscle and skin, clinically characterized by symmetrical proximal muscle weakness and typical skin lesions. Recently, myositis-specific autoantibodies (MSA) became of utmost importance because they strongly correlate with distinct clinical manifestations and prognosis. Antibodies against transcription intermediary factor 1γ (TIF-1γ) are frequently associated with increased risk of malignancy, a specific cutaneous phenotype and limited response to therapy in adult DM patients. Anti-Mi-2 autoantibodies, in contrast, are typically associated with classic DM rashes, prominent skeletal muscle weakness, better therapeutic response and prognosis, and less frequently with cancer. Nevertheless, the sensitivity of autoantibody testing is only moderate, and alternative reliable methods for DM patient stratification and prediction of cancer risk are needed. To further investigate these clinically distinct DM subgroups, we herein analyzed 30 DM patients (n = 15 Mi-2+ and n = 15 TIF-1 γ+ ) and n = 8 non-disease controls (NDC). We demonstrate that the NanoString technology can be used as a very sensitive method to clearly differentiate these two clinically distinct DM subgroups. Using the nCounter PanCancer Immune Profiling Panel™, we identified a set of significantly dysregulated genes in anti-TIF-1γ+ patient muscle biopsies including VEGFA, DDX58, IFNB1, CCL5, IL12RB2, and CD84. Investigation of type I IFN-regulated transcripts revealed a striking type I interferon signature in anti-Mi-2+ patient biopsies. Our results help to stratify both subgroups and predict, which DM patients require an intensified diagnostic procedure and might have a poorer outcome. Potentially, this could also have implications for the therapeutic approach.

Inflammation, fibrosis and skeletal muscle regeneration in LGMDR9 are orchestrated by macrophages.

AIMS: Variable degrees of inflammation, necrosis, regeneration and fibrofatty replacement are part of the pathological spectrum of the dystrophic process in alpha dystroglycanopathy LGMDR9 (FKRP-related, OMIM #607155), one of the most prevailing types of LGMDs worldwide. Inflammatory processes and their complex interplay with vascular, myogenic and mesenchymal cells may have a major impact on disease development. The purpose of our study is to describe the specific immune morphological features in muscle tissue of patients with LGMDR9 to enable a better understanding of the phenotype of muscle damage leading to disease progression. METHODS: We have analysed skeletal muscle biopsies of 17 patients genetically confirmed as having LGMDR9 by histopathological and molecular techniques. RESULTS: We identified CD206+ MHC class II+ and STAT6+ immune-repressed macrophages dominating the endomysial infiltrate in areas of myofibre regeneration and fibrosis. Additionally, PDGFRß+ pericytes were located around MHC class II+ activated capillaries residing in close proximity to areas of fibrosis and regenerating fibres. Expression of VEGF was found on many regenerating neonatal myosin+ fibres, myofibres and CD206+ macrophages also co-expressed VEGF. CONCLUSION: Our results show characteristic immune inflammatory features in LGMDR9 and more specifically shed light on the predominant role of macrophages and their function in vascular organisation, fibrosis and myogenesis. Understanding disease-specific immune phenomena potentially inform about possibilities for anti-fibrotic and anti-inflammatory therapeutic strategies, which may complement Ribitol replacement and gene therapies for LGMDR9 that may be available in the future.

Systemic sclerosis-associated myositis features minimal inflammation and characteristic capillary pathology.

Systemic sclerosis represents a chronic connective tissue disease featuring fibrosis, vasculopathy and autoimmunity, affecting skin, multiple internal organs, and skeletal muscles. The vasculopathy is considered obliterative, but its pathogenesis is still poorly understood. This may partially be due to limitations of conventional transmission electron microscopy previously being conducted only in single patients. The aim of our study was therefore to precisely characterize immune inflammatory features and capillary morphology of systemic sclerosis patients suffering from muscle weakness. In this study, we identified 18 individuals who underwent muscle biopsy because of muscle weakness and myalgia in a cohort of 367 systemic sclerosis patients. We performed detailed conventional and immunohistochemical analysis and large-scale electron microscopy by digitizing entire sections for in-depth ultrastructural analysis. Muscle biopsies of 12 of these 18 patients (67%) presented minimal features of myositis but clear capillary alteration, which we termed minimal myositis with capillary pathology (MMCP). Our study provides novel findings in systemic sclerosis-associated myositis. First, we identified a characteristic and specific morphological pattern termed MMCP in 67% of the cases, while the other 33% feature alterations characteristic of other overlap syndromes. This is also reflected by a relatively homogeneous clinical picture among MMCP patients. They have milder disease with little muscle weakness and a low prevalence of interstitial lung disease (20%) and diffuse skin involvement (10%) and no cases of either pulmonary arterial hypertension or renal crisis. Second, large-scale electron microscopy, introducing a new level of precision in ultrastructural analysis, revealed a characteristic capillary morphology with basement membrane thickening and reduplications, endothelial activation and pericyte proliferation. We provide open-access pan-and-zoom analysis to our datasets, enabling critical discussion and data mining. We clearly highlight characteristic capillary pathology in skeletal muscles of systemic sclerosis patients.

Sequestosome-1 (p62) expression reveals chaperone-assisted selective autophagy in immune-mediated necrotizing myopathies.

Diffuse myofiber necrosis in the context of inflammatory myopathy is the hallmark of immune-mediated necrotizing myopathy (IMNM). We have previously shown that skeletal muscle fibers of IMNM patients may display nonrimmed vacuoles and sarcoplasmic irregularities. The dysfunctional chaperone activity has been linked to the defective assembly of skeletal muscle proteins and their degradation via lysosomes, autophagy and the proteasomal machinery. This study was undertaken to highlight a chaperone-assisted selective autophagy (CASA) pathway, functionally involved in protein homeostasis, cell stress and the immune response in skeletal muscle of IMNM patients. Skeletal muscle biopsies from 54 IMNM patients were analyzed by immunostaining, as well as by qPCR. Eight biopsies of sIBM patients served as pathological controls, and eight biopsies of nondisease control subjects were included. Alteration of autophagy was detectable in all IMNM biopsy samples highlighted via a diffuse sarcoplasmic staining pattern by p62 and LC3 independent of vacuoles. This pattern was at variance with the coarse focal staining pattern mostly confined to rimmed vacuoles in sIBM. Colocalization of p62 with the chaperone proteins HSP70 and αB-crystalline points to the specific targeting of misfolded proteins to the CASA machinery. Bcl2-associated athanogene 3 (BAG3) positivity of these fibers emphasizes the selectivity of autophagy processes and these fibers also express MHC class I sarcolemma. Expression of genes involved in autophagy and endoplasmic reticulum (ER) stress pathways studied here is significantly upregulated in IMNM. We highlight that vacuoles without sarcolemmal features may arise in IMNM muscle biopsies, and they must not be confounded with sIBM-specific vacuoles. Further, we show the activation of selective autophagy and emphasize the role of chaperones in this context. CASA occurs in IMNM muscle, and specific molecular pathways of autophagy differ from the ones in sIBM, with p62 as a unique identifier of this process.

Inflammation-induced fibrosis in skeletal muscle of female carriers of Duchenne muscular dystrophy.

Female carriers of DMD gene mutations may be symptomatic and show variable skeletal as well as cardiac muscle symptoms. Skeletal muscle can exhibit morphological alterations. However, inflammatory, degenerative and fibrotic changes as seen in Duchenne boys have not been specifically analysed yet, so we addressed the question whether skeletal muscle of female carriers show such alterations. Thirteen carriers with an age range of 3 to 50 years were studied retrospectively. Five out of 13 women had clinically affected relatives. Clinically, most women showed mild muscle weakness, while the CK levels were increased in nine of them. Histomorphological analyses highlighted the typical mosaic pattern of dystrophin-positive and -negative fibres. Regenerating fibres were diffusely scattered and focally pronounced, while endo- and perimysial fibrosis was a variable but constant feature. Infiltration of CD206+TGFß+ macrophages and scattered T cells was noted in the endomysium. TGFb and CCL18, were significantly increased. However, gene expression of markers involved in Th1/Th2 immunity did not reach statistical significance compared to non-diseased controls. In summary, skeletal muscle of clinically manifest female DMD gene mutation carriers shows mild fibrosis and increased regeneration associated with endomysial CD206+TGFß+ and STAT6+ macrophages, which are most likely involved in fibrotic remodelling.

Idiopathic inflammatory myopathy: Interrater variability in muscle biopsy reading.

OBJECTIVE: To determine interrater variability in diagnosing individual muscle biopsy abnormalities and diagnosis. METHODS: We developed a scoring tool to analyze consensus in muscle biopsy reading of an ad hoc workgroup of international experts. Twenty-four samples from patients with suspected idiopathic inflammatory myopathy (IIM) were randomly selected, providing sections that were stained with standard histologic and immunohistochemical methods. Sections were made available on an online platform, and experts were queried about myopathologic features within 4 pathologic domains: muscle fibers, inflammation, connective tissue, and vasculature. A short clinical presentation of cases was included, and experts were asked to give a tentative diagnosis of polymyositis, dermatomyositis, inclusion-body myositis, antisynthetase syndrome-related myositis, immune-mediated necrotizing myopathy, nonspecific myositis, or other disease. Fleiss κ values, scoring interrater variability, showed the highest agreement within the muscle fiber and connective tissue domains. RESULTS: Despite overall low κ values, moderate agreement was achieved for tentative diagnosis, supporting the idea of using holistic muscle biopsy interpretation rather than adding up individual features. CONCLUSION: The assessment of individual pathologic features needs to be standardized and harmonized and should be measured for sensitivity and specificity for subgroup classification. Standardizing the process of diagnostic muscle biopsy reading would allow identification of more homogeneous patient cohorts for upcoming treatment trials.

Biomarkers in Inflammatory Myopathies-An Expanded Definition.

Biomarkers as parameters of pathophysiological conditions can be of outmost relevance for inflammatory myopathies. They are particularly warranted to inform about diagnostic, prognostic, and therapeutic questions. As biomarkers become more and more relevant in daily routine, this review focusses on relevant aspects particularly addressing myopathological features. However, the level of evidence to use them in daily routine at presence is low, still since none of them has been validated in large cohorts of patients and rarely in independent biopsy series. Hence, they should be read as mere expert opinions. The evaluation of biomarkers as well as key biological parameters is an ongoing process, and we start learning about relevance of them, as we must recognize that pathophysiology of myositis is biologically incompletely understood. As such this approach should be considered an essay toward expansion of the definition "biomarker" to myositis, an emerging field of interest in biomedical research.

Proteomic Profiling Unravels a Key Role of Specific Macrophage Subtypes in Sporadic Inclusion Body Myositis.

Unbiased proteomic profiling was performed toward the identification of biological parameters relevant in sIBM, thus giving hints about the pathophysiological processes and the existence of new reliable markers. For that purpose, skeletal muscle biopsies from 13 sIBM and 7 non-diseased control patients were analyzed with various methods, including liquid chromatography coupled to tandem mass spectrometry (four patients). Subsequent data analysis identified key molecules further studied in a larger cohort by qPCR, immunostaining, and immunofluorescence in situ. Proteomic signature of muscle biopsies derived from sIBM patients revealed the chaperone and cell surface marker CD74, the macrophage scavenger molecule CD163 and the transcription activator STAT1 to be among the highly and relevantly expressed proteins suggesting a significant contribution of immune cells among the myofibers expressing these markers. Moreover, in silico studies showed that 39% of upregulated proteins were involved in type I or mixed type I and type II interferon immunity. Indeed, further studies via immunohistochemistry clearly confirmed the prominent involvement of the key type I interferon signature-related molecules, ISG15 as well as IRF8 with MHC class II+ myofibers. Siglec1 colocalized with CD163+ macrophages and MHC class II molecules also co-localized with CD74 on macrophages. STAT1 co-localized with Siglec1+ macrophages in active myofibre myophagocytosis while STAT6 colocalized with endomysial macrophages. These combined results show involvement of CD74, CD163, and STAT1 as key molecules of macrophage activation being crucially involved in mixed and specific type I interferon, and interferon gamma associated-pathways in sIBM. On a more general note, these results also highlight the type of immune-interaction between macrophages and myofibers in the etiopathology of sIBM.

A new case expanding the mutation and phenotype spectrum of TMEM5-related alpha-dystroglycanopathy.

Dystroglycanopathies are a diverse group of neuromuscular disorders caused by aberrant glycosylation of alpha-dystroglycan. TMEM5 is one of many glycosyltransferases recently described to be associated with alpha-dystroglycanopathies. We report the case of a 15-year-old boy suffering from a congenital muscular dystrophy with elevated serum creatine kinase levels and an almost complete absence of alpha-dystroglycan in muscle biopsy. The clinical course was milder than any previously reported case and did not include brain or eye defects. Standard next-generation sequencing analysis revealed a homozygous mutation in the donor splice site region of exon 5 in TMEM5 (c.914+6 T>G). Available in-silico prediction tools anticipated a reduced efficiency of the splice site. Subsequent cDNA sequencing confirmed the expression of a truncated transcript of TMEM5 lacking exon 5, hence leading to an in-frame deletion in the exostosin domain of the protein. This report expands the clinical and mutation spectrum of alpha-dystroglycanopathies.

Necrosis in anti-SRP+ and anti-HMGCR+myopathies: Role of autoantibodies and complement.

OBJECTIVE: To characterize muscle fiber necrosis in immune-mediated necrotizing myopathies (IMNM) with anti-signal recognition particle (SRP) or anti-3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR) antibodies and to explore its underlying molecular immune mechanisms. METHODS: Muscle biopsies from patients with IMNM were analyzed and compared to biopsies from control patients with myositis. In addition to immunostaining and reverse transcription PCR on muscle samples, in vitro immunostaining on primary muscle cells was performed. RESULTS: Creatine kinase levels and muscle regeneration correlated with the proportion of necrotic fibers (r = 0.6, p < 0.001). CD68+iNOS+ macrophages and a Th-1 immune environment were chiefly involved in ongoing myophagocytosis of necrotic fibers. T-cell densities correlated with necrosis but no signs of cytotoxicity were detected. Activation of the classical pathway of the complement cascade, accompanied by deposition of sarcolemmal immunoglobulins, featured involvement of humoral immunity. Presence of SRP and HMGCR proteins on altered myofibers was reproduced on myotubes exposed to purified patient-derived autoantibodies. Finally, a correlation between sarcolemmal complement deposits and fiber necrosis was observed (r = 0.4 and p = 0.004). Based on these observations, we propose to update the pathologic criteria of IMNM. CONCLUSION: These data further corroborate the pathogenic role of anti-SRP and anti-HMGCR autoantibodies in IMNM, highlighting humoral mechanisms as key players in immunity and myofiber necrosis.

Mutations in Subunits of the Activating Signal Cointegrator 1 Complex Are Associated with Prenatal Spinal Muscular Atrophy and Congenital Bone Fractures.

Transcriptional signal cointegrators associate with transcription factors or nuclear receptors and coregulate tissue-specific gene transcription. We report on recessive loss-of-function mutations in two genes (TRIP4 and ASCC1) that encode subunits of the nuclear activating signal cointegrator 1 (ASC-1) complex. We used autozygosity mapping and whole-exome sequencing to search for pathogenic mutations in four families. Affected individuals presented with prenatal-onset spinal muscular atrophy (SMA), multiple congenital contractures (arthrogryposis multiplex congenita), respiratory distress, and congenital bone fractures. We identified homozygous and compound-heterozygous nonsense and frameshift TRIP4 and ASCC1 mutations that led to a truncation or the entire absence of the respective proteins and cosegregated with the disease phenotype. Trip4 and Ascc1 have identical expression patterns in 17.5-day-old mouse embryos with high expression levels in the spinal cord, brain, paraspinal ganglia, thyroid, and submandibular glands. Antisense morpholino-mediated knockdown of either trip4 or ascc1 in zebrafish disrupted the highly patterned and coordinated process of α-motoneuron outgrowth and formation of myotomes and neuromuscular junctions and led to a swimming defect in the larvae. Immunoprecipitation of the ASC-1 complex consistently copurified cysteine and glycine rich protein 1 (CSRP1), a transcriptional cofactor, which is known to be involved in spinal cord regeneration upon injury in adult zebrafish. ASCC1 mutant fibroblasts downregulated genes associated with neurogenesis, neuronal migration, and pathfinding (SERPINF1, DAB1, SEMA3D, SEMA3A), as well as with bone development (TNFRSF11B, RASSF2, STC1). Our findings indicate that the dysfunction of a transcriptional coactivator complex can result in a clinical syndrome affecting the neuromuscular system.