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ObjectiveTo 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.MethodsMuscle 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.ResultsCreatine 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.ConclusionThese 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.

RESUME L’analyse anatomopathologique des myopathies inflammatoires peut offrir une multitude d’informations sur le diagnostic, mais aussi la pathogenie des differentes entites. Les methodes d’histologie modernes sont parfaitement complementaires des approches moleculaires d’analyse dites ‘large scale’ ou ‘omics’ qui peuvent aider a detecter des voies de pathogenie. Ces differentes voies peuvent indiquer le regroupement ou alors la distinction de certains sous-types de myosites. Il est aujourd’hui possible de definir des items qui caracterisent des sous-groupes de myosites selon certaines caracteristiques physiopathologiques. Cette approche permet de distinguer plus precisement les myosites sur le plan pathologique. Cela permet aussi de relier l’anatomopathologie aux auto-anticorps specifiques des myosites et d’etudier leurs roles dans le tissu musculaire. Ainsi, on peut distinguer cinq sous-groupes : les dermatomyosites, les myosites associees aux syndromes des anti-synthetases, plusieurs formes de myopathies necrosantes autoimmunes, la myosite a inclusions et les myosites non-specifiques.

Myalgia, increased levels of creatine kinase, and persistent muscle weakness have been reported in patients with COVID-19.To study skeletal muscle and myocardial inflammation in patients with COVID-19 who had died.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.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.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.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.

Background:Idiopathic inflammatory myopathies (IIM) are autoimmune diseases that mainly affect skeletal muscle, lung, skin and joints. IIM can be separated into dermatomyositis (DM), inclusion body myositis (IBM), antisynthetase syndrome (AS) and immune-mediated necrotizing myopathy (IMNM). Type I interferons (IFN) are known to play a crucial role in the etiopathogenesis of some of these entities such as DM.[1] Sialic acid binding Ig-like lectin 1 (SIGLEC1, CD169) is part of the type I IFN signature found in SLE and DM and is expressed on the cell surface of monocytes. Thus, analysis of SIGLEC1 expression by flow cytometry enables a straightforward assessment of the type I IFN signature. Its utility has been shown for juvenile and adult SLE and other rheumatic diseases but not in IIM.[2,3] The assessment of the type I IFN system in clinical practice is an unmet need and, in this context, SIGLEC1 might be useful.Objectives:To assess SIGLEC1 expression on monocytes by flow cytometry as a type I IFN biomarker in IIMMethods:Pediatric and adult patients with a clinical diagnosis of DM, AS, IMNM and IBM and at least one measurement of SIGLEC1 who have been treated at the Department of Rheumatology, Charité - Universitätsmedizin Berlin between 2015 and 2020 were included in this retrospective study. Control groups of healthy individuals (n=19) and SLE patients (n=30) were included. Disease activity was assessed by Physician Global Assessment (PGA) and Childhood Myositis Assessment Scale (CMAS). SIGLEC1 expression on monocytes was analyzed by flow cytometry. Cross-sectional analyses (n=74) were performed using Mann Whitney-U test (MWU) and two-level mixed-effects linear regression model was used for longitudinal analyses (n=26, 110 visits). This study was approved by the local ethics committee of the Charité - Universitätsmedizin Berlin.Results:74 patients (adult/juvenile DM: n=21/n=17; AS: n=19; IMNM: n=8; IBM: n=9) were included. In cross-sectional analysis, SIGLEC1 expression was significantly upregulated in adult and juvenile DM patients with moderate to severe disease activity (PGA≥5) compared with adult/juvenile DM patients with no to moderate disease activity (PGAConclusion:SIGLEC1 is a useful biomarker to identify an activated type I IFN system in IIM. Flow cytometry is used widely in laboratory medicine, which could facilitate the implementation of SIGLEC1 into clinical routine.References:[1]Gallay L, Mouchiroud G, Chazaud B. Interferon-signature in idiopathic inflammatory myopathies: Current Opinion in Rheumatology 2019;31:634–42. doi:10.1097/BOR.0000000000000653[2]Rose T, Grutzkau A, Hirseland H, et al. IFNalpha and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus. Ann Rheum Dis 2013;72:1639–45. doi:10.1136/annrheumdis-2012-201586[3]Stuckrad SL von, Klotsche J, Biesen R, et al. SIGLEC1 (CD169) is a sensitive biomarker for the deterioration of the clinical course in childhood systemic lupus erythematosus. Lupus 2020;:961203320965699. doi:10.1177/0961203320965699Figure 1.SIGLEC1 expression on monocytes in IIM subgroups and control groups; in IIM subgroups, patients with low disease activity (PGADisclosure of Interests:None declared

A 31-year-old female hairdresser whose parents were first degree cousins complained of episodic attacks of headache, vomiting, and dizziness for the past eight years after an uneventful childhood and adolescence. Four years ago, she developed progressive weakness, muscle pain and difficulties walking and lifting her arms that she could not work in her profession anymore. She lost hair, weight and became amenorrhoic. Finally, her muscle weakness required intensive care. Early on her CK was mildly elevated to 237 U/l (normal < 167), but later to 900 and 1800. By MRI, skeletal muscles showed minimal contrast enhancement.The clinically suspected diagnosis of myositis prompted repeated muscle biopsies, which disclosed non-specific myopathic changes, scattered necrotic muscle fibers without inflammation, protein aggregation, or vacuolation by light microscopy, but abnormally structured mitochondria with inclusions by electron microscopy, and treatment with steroids without any clinical improvement.A panel of 1131 mitochondrial genes revealed a homozygous mutation in the ETFDH gene.LEARNING OBJECTIVESThis presentation will enable the learner to: 1.Discuss MADD as a mitochondrial and lipid storage disease2.Recognize the myopathology of MADD

Inherited and acquired muscular weakness is caused by multiple conditions. While the inherited ones are mostly caused by mutations in genes coding for myopathic or neurogenic diseases, the acquired ones occur due to inflammatory, endocrine, or toxic etiologies. Precise diagnosis of a specific disease may be challenging and may require a multidisciplinary approach. What is the current place for a diagnostic biopsy of skeletal muscle? Diagnostic muscle biopsy lost in this context its first-tier place in the primary diagnostic workup for some diseases, but it is still mandatory for others. We here summarize conditions in which we believe a diagnostic sample is most relevant and mention those in which a biopsy may be secondary or can even be left out. We would like to stress that muscle biopsy nowadays has a new important place in description and definition of new diseases, for example, discovered by modern genetic approaches.

Objective Immune-mediated necrotizing myopathies (IMNM) may be associated with either anti–signal recognition protein (SRP) or anti–3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) antibodies (Abs), and the titer of these Abs is correlated with disease activity. We investigated whether anti-SRP and anti-HMGCR Abs could be involved in muscle damage. Methods Muscle biopsies of patients were analyzed for atrophy and regeneration by measuring fiber size and by performing immunostaining of neonatal myosin heavy chain. To further understand the role of the Abs in the pathology, we performed muscle cell coculture with the Abs. Atrophy and regeneration were evaluated based on the myotube surface area as well as gene and cytokine profiles. Results In muscle biopsies of patients with anti-SRP+ and anti-HMGCR+ Abs, a large number of small fibers corresponding to both atrophic and regenerating fibers were observed. In vitro, anti-SRP and anti-HMGCR Abs induced muscle fiber atrophy and increased the transcription of MAFbx and TRIM63. In addition, the muscle fiber atrophy was associated with high levels of inflammatory cytokines: tumor necrosis factor, interleukin (IL)-6, and reactive oxygen species. In the presence of anti-SRP or anti-HMGCR Abs, mechanisms involved in muscle regeneration were also impaired due to a defect of myoblast fusion. This defect was associated with a decreased production of IL-4 and IL-13. The addition of IL-4 and/or IL-13 totally rescued fusion capacity. Interpretation These data show that molecular mechanisms of atrophy and regeneration are affected and contribute to loss of muscle function occurring in IMNM. This emphasizes the potential interest of targeted therapies addressing these mechanisms. Ann Neurol 2017;81:538–548

ZusammenfassungEntzündliche Muskelerkrankungen stellen eine wichtige Gruppe der seltenen neuromuskulären Erkrankungen dar. Die Diagnose der Myositiden hat einen besonderen Stellenwert, da sie im Gegensatz zu vielen anderen hereditären neuromuskulären Erkrankungen kausal behandelt werden können. Daher ist eine rasche, genaue Diagnosestellung wichtig, um Patienten helfen zu können, bevor irreversible Defizite entstehen. Um Patienten mit der Diagnosestellung einer Myositis und deren Angehörigen bessere Informationen über die Erkrankung zu ermöglichen, hat sich vor einigen Jahren innerhalb der Selbsthilfe der Deutschen Gesellschaft für Muskelkranke e. V. die Diagnosegruppe Myositis gebildet (www.dgm.org). Zum interdisziplinären Austausch von Ärzten und Wissenschaftlern wurde 2015 das Myositis Netz gegründet, welches eine Plattform für wissenschaftliche Kooperationen bietet (www.myositis-netz.de). Hauptziele des Myositis Netz sind es, die Therapie bei Myositis zu optimieren und standardisieren und die Wissenschaft in diesem Bereich zu fördern. In dem vorliegenden Beitrag soll das Myositis Netz mit seinen Aufgaben, Zielen und Teilnehmern vorgestellt werden. Darüber hinaus werden die wesentlichen Fakten zur Diagnostik und Therapie der Myositis kompakt und übersichtlich dargestellt.

Microglia are resident immune cells in the central nervous system (CNS), which are essential for immune defence and critically contribute to neuronal functions during homeostasis. Until now, little is known about microglia biology in humans in part due to the lack of microglia-specific markers. We therefore investigated the expression of the purinergic receptor P2Y12 in human brain tissue. Compared to classical markers used to identify microglia such as Iba1, CD68 or MHCII, we found that P2Y12 is expressed on parenchymal microglia but is absent from perivascular or meningeal macrophages. We further demonstrate that P2Y12 expression is stable throughout human brain development, including fetal phases, and quantification of P2 Y12+ microglia revealed that the density of human microglia is constant throughout lifetime. In contrast, CD68 expression increases during aging in cerebellar but not in cortical microglia, indicating regional heterogeneity. CNS pathologies such as Alzheimer's disease or multiple sclerosis-but not schizophrenia-result in decreased P2Y12 immunoreactivity in plaque- or lesion-associated myeloid cells, whereas Iba1 expression remains detectable. Our results suggest that P2Y12 is a useful marker for the identification of human microglia throughout the lifespan. Moreover, P2Y12 expression might help to discriminate activated microglia and infiltrating myeloid cells from quiescent microglia in the human CNS. GLIA 2017;65:375-387.

A 6-year-old girl had reduced fetal movements, numerous apneic spells, muscle hypotonia, and developmental motor delay. Her muscle biopsy tissue showed variation in myofiber diameters, small minicores by electron microscopy, and near-uniformity of type I fibers. Although no mutations were detected in RYR1, SEPN1, and DMPK genes, the RAPSN gene revealed one known mutation, p.Asn88Lys, from the mother, and one novel mutation, p.Cys366Gly, from the father. Life-saving pyridostigmine treatment suppressed her apneic spells and improved her motor development.

ObjectiveTo determine interrater variability in diagnosing individual muscle biopsy abnormalities and diagnosis.MethodsWe 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.ResultsDespite 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.ConclusionThe 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.

International audience; Recent data suggest the implication of T, B and NK cells in the anti-synthetase syndrome (ASyS); nevertheless their role and activation states are poorly described. We performed deep immune-profiling using 37 markers on peripheral blood cells from 10 ASyS patients versus 17 healthy donors (HD) and 26 myositis control patients. We show decreased percentages of memory B cells in ASyS patients (mean ± SEM: ASyS = 13 ± 3%, HD = 37 ± 4% and 'myositis controls' = 32 ± 3), counterbalanced by increased percentages of naïve B cells. Interestingly, perifascicular infiltrations of memory B cells within muscle biopsies of ASyS patients suggest that they niche within the muscle.

Congenital myopathies (CM) are a large and heterogeneous group of disorders. Many new myopathies with congenital onset have recently been described phenotypically, and their molecular elucidation has rapidly ensued consecutively. CM reported between 2013 and 2017 and their corresponding gene defects have mostly been identified with modern molecular genetic techniques. Here, we report recently identified CM that have not been included in the 2017 gene table so far, of which some have been recognized with mutations in new genes and others have been recognized as variants of previously identified genes, associated with specific CM phenotypes.

Cerebral malaria is a life-threatening complication of malaria in humans, and the underlying pathogenic mechanisms are widely analyzed in a murine model of experimental cerebral malaria (ECM). Here, we show abrogation of ECM by hemocoel sporozoite-induced infection of a transgenic Plasmodium berghei line that overexpresses profilin, whereas these parasites remain fully virulent in transfusion-mediated blood infection. We, thus, demonstrate the importance of the clinically silent liver-stage infection for modulating the onset of ECM. Even though both parasites triggered comparable splenic immune cell expansion and accumulation of antigen-experienced CD8+ T cells in the brain, infection with transgenic sporozoites did not lead to cerebral vascular damages and suppressed the recruitment of overall lymphocyte populations. Strikingly, infection with the transgenic strain led to maintenance of CD115+Ly6C+ monocytes, which disappear in infected animals prone to ECM. An early induction of IL-10, IL-12p70, IL-6, and TNF at the time when parasites emerge from the liver might lead to a diminished induction of hepatic immunity. Collectively, our study reveals the essential role of early host interactions in the liver that may dampen the subsequent pro-inflammatory immune responses and influence the occurrence of ECM, highlighting a novel checkpoint in this fatal pathology.

Mutations in the mitochondrial complex IV assembly factor SURF1 represent a major cause of Leigh syndrome (LS), a rare fatal neurological disorder. SURF1-deficient animals have failed to recapitulate the neuronal pathology of human LS, hindering our understanding of the disease mechanisms. We generated induced pluripotent stem cells from LS patients carrying homozygous SURF1 mutations (SURF1 iPS) and performed biallelic correction via CRISPR/Cas9. In contrast to corrected cells, SURF1 iPS showed impaired neuronal differentiation. Aberrant bioenergetics in SURF1 iPS occurred already in neural progenitor cells (NPCs), disrupting their neurogenic potency. Cerebral organoids from SURF1 iPS were smaller and recapitulated the neurogenesis defects. Our data imply that SURF1 mutations cause a failure in the development of maturing neurons. Using NPC function as an interventional target, we identified SURF1 gene augmentation as a potential strategy for restoring neurogenesis in LS patients carrying SURF1 mutations.

International audience; 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.

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ObjectiveTo investigate the relevance of dysfunctional T cells in immune-mediated myopathies. We analyzed T-cell exhaustion and senescence, in the context of programmed cell death protein 1 (PD1)-related immunity in skeletal muscle biopsies from patients with immune-mediated necrotizing myopathy (IMNM), sporadic inclusion body myositis (sIBM), and myositis induced by immune checkpoint inhibitors (irMyositis).MethodsSkeletal muscle biopsies from 12 patients with IMNM, 7 patients with sIBM, and 8 patients with irMyositis were analyzed by immunostaining and immunofluorescence as well as by quantitative PCR. Eight biopsies from nondisease participants served as controls.ResultsCD3+CD8+ T cells in biopsies from IMNM, sIBM, and irMyositis were largely PD1-positive, while CD68+ macrophages were sparsely positive to the ligand of programmed cell death protein 1 (PD-L1). The sarcolemma of myofibers was PD-L2+ and was colocalized with major histocompatibility complex (MHC) class I. CD68+ macrophages were colocalized with PD-L2. Senescent T cells were strongly enriched in skeletal muscle of sIBM, revealing a distinct immunologic signature. Biopsies from patients with irMyositis showed mild signs of senescence and exhaustion.ConclusionPersistent exposure to antigens in IMNMs and sIBM may lead to T-cell exhaustion, a process controlled by the PD1 receptor and its cognate ligands PD-L1/PD-L2. To our knowledge, these data are the first evidence of presence of dysfunctional T cells and relevance of the PD1 pathway in IMNM, sIBM, and irMyositis. These findings may guide the way to a novel understanding of the immune pathogenesis of immune-mediated myopathies.

Background: Mutations in SLC25A4 (syn. ANT1, Adenine nucleotide translocase, type 1) are known to cause either autosomal dominant progressive external ophthalmoplegia (adPEO) or recessive mitochondrial myopathy, hypertrophic cardiomyopathy, and lactic acidosis. Methods and Results: Whole exome sequencing in a young man with myopathy, subsarcolemmal mitochondrial aggregations, cardiomyopathy, lactic acidosis, and L-2-hydroxyglutaric aciduria (L-2-HGA) revealed a new homozygous mutation in SLC25A4[c.653A>C, NM_001151], leading to the replacement of a highly conserved glutamine by proline [p.(Q218P); NP_001142] that most likely affects the folding of the ANT1 protein. No pathogenic mutation was found in L2HGDH, which is associated with “classic” L-2-HGA. Furthermore, L-2-HGDH enzymatic activity in the patient fibroblasts was normal. Long-range PCR and Southern blot confirmed absence of mtDNA-deletions in blood and muscle. Conclusion: The disturbed ADP/ATP transport across the inner mitochondrial membrane may lead to an accumulation of different TCA-cycle intermediates such as 2-ketoglutarate (2-KG) in our patient. As L-2-HG is generated from 2-KG we hypothesize that the L-2-HG increase is a secondary effect of 2-KG accumulation. Hence, our report expands the spectrum of laboratory findings in ANT1-related diseases and hints towards a connection with organic acidurias.

Neuropathology & applied neurobiology 46(4), 359-374 (2019). doi:10.1111/nan.12580, Published by Wiley-Blackwell, Oxford [u.a.]

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+TGFs+ 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+TGFβ+ and STAT6+ macrophages, which are most likely involved in fibrotic remodelling.

Antisynthetase syndrome (aSS) is characterized by the association of interstitial lung disease and myositis with anti–tRNA synthetase autoantibodies. Immune mechanisms leading to aSS could be initiated in the lungs, but the role of NK cells has not yet been studied. Both extensive NK cell phenotype and functions were compared between 33 patients and 26 controls. Direct and redirected polyfunctionality assays (degranulation and intracellular production of TNF-α and IFN-γ) were performed spontaneously or after IL-12 plus IL-18 stimulation in the presence of K562 or P815 target cells, respectively. NK cells from inactive patients showed normal phenotype, whereas active aSS revealed a differentiated NK cell profile, as indicated by increased CD57 and Ig-like transcript 2 and an inability to produce IFN-γ (p = 0.002) compared with controls. Importantly, active aSS was more specifically associated with a significant NKp30 decrease (p = 0.009), although levels of mRNA and intracellular protein were similar in aSS and healthy controls. This NKp30 decrease was strongly correlated with reduced NK cell polyfunctionality in both direct and redirected killing assays with anti-NKp30 Abs (p = 0.009 and p = 0.03, respectively), confirming its important impact in aSS. Histological studies revealed massive infiltrations of NK cells inside the lungs of aSS patients (148 versus 11/mm2). Taken together, these data suggest that NK cells and NKp30 could play a role in aSS pathogenesis.

We describe two siblings who were affected with early onset focal seizures, severe progressive postnatal microcephaly, muscular hypertonia, feeding problems and bouts of apnea, only minimal psychomotor development, as well as death in infancy and childhood. We identified compound heterozygous mutations in BRAT1 exons 5 (c.638_639insA) and 8 (c.1134+1G>A) in one affected child via next-generation sequencing of the disease-associated genome followed by phenotype-driven bioinformatic analysis. Sanger sequencing confirmed the presence of these mutations in both patients and a heterozygote status of the parents. Whereas the frameshift mutation (c.638_639insA) has been described in one family, the splice-site mutation (c.1134+1G>A) is novel. In contrast to all cases published so far, one of our patients showed a considerably milder clinical course with survival into childhood. Investigation of a skeletal muscle biopsy showed a severely reduced COX enzyme histochemical staining, indicating mitochondrial dysfunction. Our data expand the clinical and mutational spectrum of the BRAT1-associated phenotype. © 2016 Wiley Periodicals, Inc.

Deposits of the terminal-membrane-attack-complex (MAC) C5b-9 on perfascicular endomysial capillaries are generally regarded as diagnostic hallmark of dermatomyositis (DM). Although the pathophysiology is not clear, C5b-9 deposits on capillaries seem to be associated with microinfarctions and vascular damage. Here, we report on a series of 19 patients presenting with C5b-9 accumulation on endomysial capillaries in the absence of features for DM. To decipher differences in the capillary C5b-9 accumulation pattern between DM and non-DM cases, we assessed the extent of endomysial capillary C5b-9 deposits related to capillary density and extent of myofiber necrosis by immunohistochemistry in 12 DM and 8 control patients. We found similar numbers of C5b-9-positive myofibers in both DM and non-DM C5b-9(+) cases. The distribution pattern differed as DM cases showed significantly more perifascicular capillary C5b-9 deposits as compared to non-DM cases, which presented stronger endomysial capillary C5b-9 deposits in a diffuse pattern. While total capillary density was not differing, DM patients displayed significantly more C5b-9(+) necrotic fibers as compared to non-DM C5b-9(+). In summary, endomysial capillary C5b-9 deposits are present in a variety of non-DM cases, however with differing distribution pattern. In conclusion, capillary C5b-9(+) deposits should be assessed critically, taking into consideration the distribution pattern.

The anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody is specifically associated with dermatomyositis (DM). Nevertheless, anti–MDA5 + -patients experience characteristic symptoms distinct from classic DM, including severe signs of extramuscular involvement; however, the clinical signs of myopathy are mild or even absent. The morphological and immunological features are not yet described in adulthood. Data concerning the pathophysiology of anti-MDA5 DM are sparse; however, the importance of the interferon (IFN) type I pathway involved in DM has been shown. Our aim was to define morphological alterations of the skeletal muscle and the intrinsic immune response of anti–MDA5-positive DM patients. Immunohistological and RT-PCR analysis of muscle biopsy specimens from anti-MDA5 and classic DM were compared. Those with anti-MDA5 DM did not present the classic features of perifascicular fiber atrophy and major histocompatibility complex class I expression. They did not show significant signs of capillary loss; tubuloreticular formations were observed less frequently. Inflammation was focal, clustering around single vessels but significantly less intense. Expression of IFN-stimulated genes was up-regulated in anti-MDA5 DM; however, the IFN score was significantly lower. Characteristic features were observed in anti-MDA5 DM and not in classic DM patients. Only anti-MDA5 DM showed numerous nitric oxide synthase 2–positive muscle fibers with sarcoplasmic colocalization of markers of regeneration and cell stress. Anti–MDA5-positive patients demonstrate a morphological pattern distinct from classic DM.

Inflammatorische Myopathien sind zwar insgesamt relativ selten, stellen dabei jedoch aktuell die groste Gruppe potentiell behandelbarer Krankheiten der Muskulatur dar. Sie spielen im rheumatologischen Krankengut ebenso wie im neurologischen Krankengut sowohl im Kindes- wie auch im Erwachsenenalter eine signifikante Rolle, wobei das Management dieser Patienten haufig eine interdisziplinare Herangehensweise erfordert. Es handelt sich um eine ganz heterogene Gruppe von Erkrankungen, die traditionell in Dermatomyositis, Polymyositis, und spater, auch sporadische Einschluskorperchen Myositis gruppiert wurden. In den vergangenen Jahren sind nun eine Vielzahl von neuen Erkenntnissen zur Pathogenese, zu Autoantikorpern, zur Beteiligung verschiedener Organe usw. erhoben worden, so dass sich dieses simplifizierte Konzept nicht mehr aufrechterhalten lasst. Auf der Basis dieser aktuellen Erkenntnisse sind neue Klassifikationssysteme entwickelt und weiterentwickelt worden. Heute werden 6 Entitaten voneinander unterschieden: Dermatomyositis (DM), Polymyositis (PM), Autoimmun vermittelte Nekrotisierende Myositis (ANM), Anti-Synthetase-Syndrom-assoziierte Myositis (ASS-assoziierte Myositis) und unspezifische Myositis (NSM), sowie die sporadische Einschlusskorperchen Myositis (sIBM) ( Stenzel 2015 , Hoogendijk 2004 ). Diese Gruppen sind bis auf die sIBM auch im Kindesalter, dort jedoch mit einem anders gewichteten Verteilungsmuster der einzelnen Subentitaten als im Erwachsenenalter anzutreffen ( Rider 2013 ). Die Unterscheidung zwischen diesen Kategorien hat fundamentale Bedeutung fur die Prognose und das Therapieansprechen, so dass es auserst wichtig erscheint, sie korrekt zu identifizieren, zu benennen und von anderen Erkrankungen, die klinisch ahnlich erscheinen, abzugrenzen. In diesem Ubersichtsartikel mochten wir das aktuelle Wissen uber die erwahnten Entitaten unter klinisch-pathologischen Gesichtspunkten zusammenfassen und die Histopathologie dieser Erkrankungen illustrieren.

Typische Kennzeichen von metabolischen Myopathien sind belastungsinduzierte Muskelschmerzen, Muskelverkrampfungen, Belastungsintoleranz, Muskelschwache und Rhabdomyolyse bzw. Myoglobinurie. In einigen Fallen liegt auch eine proximal betonte Myopathie mit manifesten Paresen vor. Andere Organmanifestationen, wie Kardiomyopathie, Neuropathie oder ZNS-Beteiligung konnen vorhanden und differenzialdiagnostisch hilfreich sein. Die Erkrankungen konnen den Purin-, Glucose- und Glycogen- sowie den Lipidstoffwechsel und die mitochondriale Atmungskette betreffen. Ziel dieses Artikels ist es, die Abgrenzung der einzelnen metabolischen Myopathien untereinander und zu anderen hereditaren und den entzundlichen Muskelerkrankungen zu erleichtern.

// Tobias Ruck 1 , Stefan Bittner 2 , Ali Maisam Afzali 1 , Kerstin Gobel 1 , Sarah Glumm 1 , Peter Kraft 3 , Claudia Sommer 3 , Christoph Kleinschnitz 3 , Corinna Preuse 4 , Werner Stenzel 4 , Heinz Wiendl 1,* and Sven G. Meuth 1,* 1 Department of Neurology, University of Muenster, Muenster, Germany 2 Department of Neurology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany 3 Department of Neurology, University Hospital of Wuerzburg, Wuerzburg, Germany 4 Department of Neuropathology, Charite-Universitatsmedizin, Berlin, Germany * These authors have contributed equally to this work Correspondence to: Tobias Ruck, email: // Keywords : NKG2D, IL-15, polymyositis, idiopathic inflammatory myopathies, T cell activation, Pathology Section Received : June 08, 2015 Accepted : November 14, 2015 Published : December 04, 2015 Abstract NKG2D is an activating receptor on T cells, which has been implicated in the pathogenesis of autoimmune diseases. T cells are critically involved in idiopathic inflammatory myopathies (IIM) and have been proposed as specific therapeutic targets. However, the mechanisms underlying T cell-mediated progressive muscle destruction in IIM remain to be elucidated. We here determined the involvement of the NKG2D – IL-15 signaling pathway. Primary human myoblasts expressed NKG2D ligands, which were further upregulated upon inflammatory stimuli. In parallel, shedding of the soluble NKG2D ligand MICA (sMICA) decreased upon inflammation potentially diminishing inhibition of NKG2D signaling. Membrane-related expression of IL-15 by myoblasts induced differentiation of naive CD8 + T cells into highly activated, cytotoxic CD8 + NKG2D high T cells demonstrating NKG2D-dependent lysis of myoblasts in vitro . CD8 + NKG2D high T cell frequencies were increased in the peripheral blood of polymyositis (PM) patients and correlated with serum creatinine kinase concentrations, while serum sMICA levels were not significantly changed. In muscle biopsy specimens from PM patients expression of the NKG2D ligand MICA/B was upregulated, IL-15 was expressed by muscle cells, CD68 + macrophages as well as CD4 + T cells, and CD8 + NKG2D + cells were frequently detected within inflammatory infiltrates arguing for a local signaling circuit in the inflammatory muscle milieu. In conclusion, the NKG2D – IL-15 signaling pathway contributes to progressive muscle destruction in IIM potentially opening new therapeutic avenues.

ObjectiveTo report the clinicopathologic features and outcome of myositis in patients treated with immune checkpoint inhibitors (ICIs) (irMyositis).MethodsWe retrospectively analyzed patients diagnosed with irMyositis in tertiary centers in Paris, France, and Berlin, Germany, from January 2015 to July 2017. The main outcomes were clinical manifestations and muscle histology, which included major histocompatibility complex class I (MHC-I), C5b-9, CD3, CD4, CD8, CD20, CD68, programmed cell death protein 1 (PD-1), programmed cell death 1 ligand 1 (PD-L) 1, and programmed cell death 1 ligand 2 (PD-L2).ResultsTen patients with metastatic cancer were included; median age was 73 (range 56–87) years. Median follow-up duration was 48 (range 16–88) weeks. Six patients developed myositis during nivolumab therapy, 1 patient during pembrolizumab, 1 patient during durvalumab, and 2 patients during combined nivolumab and ipilimumab. Median delay between ICI initiation and myositis onset was 25 (range 5–87) days. Clinical manifestations were dominated by acute or subacute myalgia (8 patients) and limb-girdle (7), axial (7), and oculomotor (7) weakness. Four patients had evidence of myocarditis. In all patients, creatine kinase levels were elevated (median 2,668, range 1,059–16,620 U/L), while anti-acetylcholine receptor and myositis-associated antibodies were negative. Electrodiagnostic studies showed myopathic process without decrement in all patients. Muscle biopsy constantly showed multifocal necrotic myofibers, sarcolemmal MHC-I, and endomysial inflammation, consisting mainly of CD68+ cells expressing PD-L1 and CD8+ cells expressing PD-1. ICI treatment was withdrawn in all patients; 9 patients received immunosuppressive therapy, which consistently led to marked clinical improvement.ConclusionsirMyositis presents with remarkably homogeneous and unique clinicopathologic features, expanding the nosologic spectrum of inflammatory myopathies in patients with cancer. ICI withdrawal and treatment with corticosteroids improve outcome.

ObjectiveTo study the B-cell content, organization, and existence of distinct B-cell subpopulations in relation to the expression of type 1 interferon signature related genes in dermatomyositis (DM).MethodsEvaluation of skeletal muscle biopsies from patients with adult DM (aDM) and juvenile DM (jDM) by histology, immunohistochemistry, electron microscopy, and quantitative reverse-transcription PCR.ResultsWe defined 3 aDM subgroups—classic (containing occasional B cells without clusters), B-cell–rich, and follicle-like aDM—further elucidating IM B-lymphocyte maturation and immunity. The quantity of B cells and formation of ectopic lymphoid structures in a subset of patients with aDM were associated with a specific profile of cytokines and chemokines involved in lymphoid neogenesis. Levels of type 1 interferon signature related gene expression paralleled B-cell content and architectural organization and link B-cell immunity to the interferon type I signature.ConclusionThese data corroborate the important role of B cells in DM, highlighting the direct link between humoral mechanisms as key players in B-cell immunity and the role of type I interferon–related immunity.

A large number of viruses belonging to various families are able to cause central nervous system (CNS) infections and contribute significantly to burden of disease in humans globally. Most viral CNS infections are benign and self-limiting, and most remain undiagnosed. However, some viruses can cause severe inflammation, leading to morbidity and mortality, and result in severe long-term residual damage and neurologic dysfunction in patients. The potential of viruses to cause CNS inflammation greatly varies depending on host factors, such as age, sex, and genetic background, as well as viral factors. Despite the need for protection against viral invasion and replication, the extent of the immune response in the CNS is carefully regulated to prevent excessive inflammation and tissue destruction leading to irretrievable loss of neurons. Direct cytopathology is for many virus infections a major cause of neurologic symptoms; however, the antiviral immune response can in some instances contribute substantially to pathology. This chapter highlights a selection of clinically important neurotropic viruses that infect the CNS and cause neurologic diseases such as meningitis, encephalitis, and myelitis in humans, with a focus on neuropathologic findings.

Cerebral malaria is a life-threatening complication in humans, which the pathogenesis is widely analyzed using a murine model of experimental cerebral malaria (ECM). Here, we show abrogation of ECM by sporozoite induced infection of transgenic Plasmodium berghei line overexpressing profilin, while the same parasite is fully virulent in transfusion-mediated blood infection – thus demonstrating the importance of the clinically silent liver-stage infection for the onset of ECM. Infection with transgenic sporozoites did not lead to vascular damages in the brain, even though both parasites triggered comparable cerebral immune cell accumulation. However, infection with the transgenic strain had different systemic activation of Ly6C monocytes and early induction of peripheral cytokines, such as IFN-γ and IL-10. Collectively, our study reveals the essential role of early host interactions in the liver for decisively controlling the subsequent immunity and the occurrence of ECM, highlighting a novel checkpoint in this fatal pathology.

Neurometabolic and neurodegenerative diseases in children (NDDC) differ from those in adults in that most of the former are autosomal-recessively inherited – few have X-linked inheritance – while the latter are often sporadic or autosomal-dominantly inherited. NDDC may be catabolic and/or anabolic conditions, some of which combine maldevelopmental and degenerative features, for instance, peroxisomal biogenesis disorders or congenital disorders of glycosylation. NDDC are often multiorgan disorders, such as lysosomal, peroxisomal, and polyglucosan disorders. This multiorgan involvement may be marked by extracerebral formation of disease-specific neuropathologic findings, especially in lysosomal diseases allowing diagnostic biopsies in easily accessible tissues, e.g., blood lymphocytes, skin, skeletal muscle, and rectum to be investigated by electron microscopy. NDDC comprise nonvacuolar and vacuolar lysosomal, peroxisomal, polyglucosan, amino and organic acid, white-matter disorders, and congenital disorders of glycosylation.