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 activemyofibremyophagocytosis 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

Multiple bilateral asymmetrical deficiency of trunk muscles

Trunk muscles are an important source for pedicled and free flaps in reconstructive surgery. Unilateral deficiencies of trunk muscles are well known, either isolated or as part of Poland's syndrome. Bilateral muscular deficiencies and a "bilateral Poland anomaly” have also been sporadically reported, but this is rare. We report on an 82-year-old male cadaver with clinically obscure, asymmetric bilateral deficiencies of the majority of trunk muscles. There was a history of acute poliomyelitis in childhood. Histological examination of representative muscle samples of the trunk showed extensive muscle atrophy with fat and connective tissue replacement. This was compatible with the prior diagnosis of poliomyelitis. However, representative sections of the spinal cord failed to reveal the antecedent poliomyelitis. The possibility of subclinical bilateral deficiencies of trunk muscles has to be taken into account in patients with a history of poliomyelitis when planning reconstructions in cases of regional pedicled muscle transfers or free microvascular tissue transfers in reconstructive surger

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

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, PDGFRβ+ 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

Successful plasmapheresis and immunoglobulin treatment for severe lipid storage myopathy: Doing the right thing for the wrong reason

Three consecutive skeletal muscle biopsies during a several months time-frame, showing different degrees of neutral lipid storage. This is highlighted by Oil-red-O stains (D, E, F) and electron microscopy (G, H, I). Note the impact on mitochondrial morphology with so called 'parking lots (K, L). Zooming 'in and out' into the ultrastructure, using the nanotomy platform provides interesting detailled information (http://nanotomy.org). ​

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

SIL1 deficiency causes degenerative changes of peripheral nerves and neuromuscular junctions in fish, mice and human.

BACKGROUND: Marinesco-Sjögren Syndrome (MSS) is a rare neuromuscular condition caused by recessive mutations in the SIL1 gene resulting in the absence of functional SIL1 protein, a co-chaperone for the major ER chaperone, BiP. As BiP is decisive for proper protein processing, loss of SIL1 results in the accumulation of misshaped proteins. This accumulation likely damages and destroys cells in vulnerable tissues, leading to congenital cataracts, cerebellar ataxia, vacuolar myopathy and other MSS phenotypes. Whether the peripheral nervous system (PNS) is affected in MSS has not been conclusively shown. METHODS: To study PNS vulnerability in MSS, intramuscular nerves fibres from MSS patients and from SIL1-deficient mice (woozy) as well as sciatic nerves and neuromuscular junctions (NMJ) from these mice have been investigated via transmission electron microscopic and immunofluorescence studies accompanied by transcript studies and unbiased proteomic profiling. In addition, PNS and NMJ integrity were analyzed via immunofluorescence studies in an MSS-zebrafish model which has been generated for that purpose. RESULTS: Electron microscopy revealed morphological changes indicative of impaired autophagy and mitochondrial maintenance in distal axons and in Schwann cells. Moreover, changes of the morphology of NMJs as well as of transcripts encoding proteins important for NMJ function were detected in woozy mice. These findings were in line with a grossly abnormal structure of NMJs in SIL1-deficient zebrafish embryos. Proteome profiling of sciatic nerve specimens from woozy mice revealed altered levels of proteins implicated in neuronal maintenance suggesting the activation of compensatory mechanisms. CONCLUSION: Taken together, our combined data expand the spectrum of tissues affected by SIL1-loss and suggest that impaired neuromuscular transmission might be part of MSS pathophysiology

Enlarging the Nosological Spectrum of Hereditary Diffuse Leukoencephalopathy with Axonal Spheroids (HDLS)

Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is an autosomal dominant disease clinically characterized by cognitive decline, personality changes, motor impairment, parkinsonism and seizures. Recently, mutations in the colony-stimulating factor-1 receptor (CSF1R) gene have been shown to be associated with HDLS. We report clinical, neuropathological and molecular genetic findings of patients from a new family with a mutation in the CSF1R gene. Disease onset was earlier and disease progression was more rapid compared with previously reported patients. Psychiatric symptoms including personality changes, alcohol abuse and severe depression were the first symptoms in male patients. In the index, female patient, the initial symptom was cognitive decline. Magnetic resonance imaging (MRI) showed bilateral, confluent white matter lesions in the cerebrum. Stereotactic biopsy revealed loss of myelin and microglial activation as well as macrophage infiltration of the parenchyma. Numerous axonal swellings and spheroids were present. Ultrastructural analysis revealed pigment-containing macrophages. Axonal swellings were detected by electron microscopy not only in the central nervous system (CNS) but also in skin nerves. We identified a heterozygous mutation (c.2330G>A, p.R777Q) in the CSF1R gene. Through this report, we aim to enlarge the nosological spectrum of HDLS, providing new clinical descriptions as well as novel neuropathological findings from the peripheral nervous system