"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.

The neurobiology of SARS-CoV-2 infection.

Worldwide, over 694 million people have been infected with SARS-CoV-2, with an estimated 55-60% of those infected developing COVID-19. Since the beginning of the pandemic in December 2019, different variants of concern have appeared and continue to occur. With the emergence of different variants, an increasing rate of vaccination and previous infections, the acute neurological symptomatology of COVID-19 changed. Moreover, 10-45% of individuals with a history of SARS-CoV-2 infection experience symptoms even 3 months after disease onset, a condition that has been defined as 'post-COVID-19' by the World Health Organization and that occurs independently of the virus variant. The pathomechanisms of COVID-19-related neurological complaints have become clearer during the past 3 years. To date, there is no overt - that is, truly convincing - evidence for SARS-CoV-2 particles in the brain. In this Review, we put special emphasis on discussing the  methodological difficulties of viral detection in CNS tissue and discuss immune-based (systemic and central) effects contributing to COVID-19-related CNS affection. We sequentially review the reported changes to CNS cells in COVID-19, starting with the blood-brain barrier and blood-cerebrospinal fluid barrier - as systemic factors from the periphery appear to primarily influence barriers and conduits - before we describe changes in brain parenchymal cells, including microglia, astrocytes, neurons and oligodendrocytes as well as cerebral lymphocytes. These findings are critical to understanding CNS affection in acute COVID-19 and post-COVID-19 in order to translate these findings into treatment options, which are still very limited.

K2P2.1 is a regulator of inflammatory cell responses in idiopathic inflammatory myopathies.

K2P2.1 (TREK1), a two-pore domain potassium channel, has emerged as regulator of leukocyte transmigration into the central nervous system. In the context of skeletal muscle, immune cell infiltration constitutes the pathogenic hallmark of idiopathic inflammatory myopathies (IIMs). However, the underlying mechanisms remain to be elucidated. In this study, we investigated the role of K2P2.1 in the autoimmune response of IIMs. We detected K2P2.1 expression in primary skeletal muscle and endothelial cells of murine and human origin. We observed an increased pro-inflammatory cell response, adhesion and transmigration by pharmacological blockade or genetic deletion of K2P2.1 in vitro and in in vivo myositis mouse models. Of note, our findings were not restricted to endothelial cells as skeletal muscle cells with impaired K2P2.1 function also demonstrated a strong pro-inflammatory response. Conversely, these features were abrogated by activation of K2P2.1 and improved the disease course of a myositis mouse model. In humans, K2P2.1 expression was diminished in IIM patients compared to non-diseased controls arguing for the translatability of our findings. In summary, K2P2.1 may regulate the inflammatory response of skeletal muscle. Further research is required to understand whether K2P2.1 could serve as novel therapeutic target.

Distinct tissue niches direct lung immunopathology via CCL18 and CCL21 in severe COVID-19.

Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling.

Adult intracranial ependymoma-relevance of DNA methylation profiling for diagnosis, prognosis, and treatment.

BACKGROUND: A methylation-based classification of ependymoma has recently found broad application. However, the diagnostic advantage and implications for treatment decisions remain unclear. Here, we retrospectively evaluate the impact of surgery and radiotherapy on outcome after molecular reclassification of adult intracranial ependymomas. METHODS: Tumors diagnosed as intracranial ependymomas from 170 adult patients collected from 8 diagnostic institutions were subjected to DNA methylation profiling. Molecular classes, patient characteristics, and treatment were correlated with progression-free survival (PFS). RESULTS: The classifier indicated an ependymal tumor in 73.5%, a different tumor entity in 10.6%, and non-classifiable tumors in 15.9% of cases, respectively. The most prevalent molecular classes were posterior fossa ependymoma group B (EPN-PFB, 32.9%), posterior fossa subependymoma (PF-SE, 25.9%), and supratentorial ZFTA fusion-positive ependymoma (EPN-ZFTA, 11.2%). With a median follow-up of 60.0 months, the 5- and 10-year-PFS rates were 64.5% and 41.8% for EPN-PFB, 67.4% and 45.2% for PF-SE, and 60.3% and 60.3% for EPN-ZFTA. In EPN-PFB, but not in other molecular classes, gross total resection (GTR) (P = .009) and postoperative radiotherapy (P = .007) were significantly associated with improved PFS in multivariable analysis. Histological tumor grading (WHO 2 vs. 3) was not a predictor of the prognosis within molecularly defined ependymoma classes. CONCLUSIONS: DNA methylation profiling improves diagnostic accuracy and risk stratification in adult intracranial ependymoma. The molecular class of PF-SE is unexpectedly prevalent among adult tumors with ependymoma histology and relapsed as frequently as EPN-PFB, despite the supposed benign nature. GTR and radiotherapy may represent key factors in determining the outcome of EPN-PFB patients.

[Neuropathology II: diseases of the central and peripheral nervous systems : Outlook on new techniques in electron microscopy]. / Neuropathologie II: Erkrankungen des zentralen und peripheren Nervensystems : Ausblick auf neue Techniken in der Elektronenmikroskopie.

In the diagnosis of diseases of the central and peripheral nervous systems, the use of electron microscopic analyses has become rare these days. However, there are questions in which the method is helpful in confirming the etiopathogenesis of the disease. Hereditary neurodegenerative and metabolic diseases, such as the lysosomal storage disease neuronal ceroid lipofuscinosis, are associated with pathognomonic storage products not only in the central nervous system (CNS) but also in extracerebral tissues such as sweat glands and lymphocytes. These tissues are easily accessible and thus function as "windows to the CNS". In addition, there are new methods that overcome limitations of conventional electron microscopy and may improve ultrastructural diagnostics. This is particularly important for the correct classification of viral particles such as SARS-CoV­2, leading to a better understanding of COVID19-associated diseases in the CNS and peripheral nervous system.

Microglia regulate central nervous system myelin growth and integrity.

Myelin is required for the function of neuronal axons in the central nervous system, but the mechanisms that support myelin health are unclear. Although macrophages in the central nervous system have been implicated in myelin health1, it is unknown which macrophage populations are involved and which aspects they influence. Here we show that resident microglia are crucial for the maintenance of myelin health in adulthood in both mice and humans. We demonstrate that microglia are dispensable for developmental myelin ensheathment. However, they are required for subsequent regulation of myelin growth and associated cognitive function, and for preservation of myelin integrity by preventing its degeneration. We show that loss of myelin health due to the absence of microglia is associated with the appearance of a myelinating oligodendrocyte state with altered lipid metabolism. Moreover, this mechanism is regulated through disruption of the TGFß1-TGFßR1 axis. Our findings highlight microglia as promising therapeutic targets for conditions in which myelin growth and integrity are dysregulated, such as in ageing and neurodegenerative disease2,3.

Analysing cerebrospinal fluid with explainable deep learning: From diagnostics to insights.

AIM: Analysis of cerebrospinal fluid (CSF) is essential for diagnostic workup of patients with neurological diseases and includes differential cell typing. The current gold standard is based on microscopic examination by specialised technicians and neuropathologists, which is time-consuming, labour-intensive and subjective. METHODS: We, therefore, developed an image analysis approach based on expert annotations of 123,181 digitised CSF objects from 78 patients corresponding to 15 clinically relevant categories and trained a multiclass convolutional neural network (CNN). RESULTS: The CNN classified the 15 categories with high accuracy (mean AUC 97.3%). By using explainable artificial intelligence (XAI), we demonstrate that the CNN identified meaningful cellular substructures in CSF cells recapitulating human pattern recognition. Based on the evaluation of 511 cells selected from 12 different CSF samples, we validated the CNN by comparing it with seven board-certified neuropathologists blinded for clinical information. Inter-rater agreement between the CNN and the ground truth was non-inferior (Krippendorff's alpha 0.79) compared with the agreement of seven human raters and the ground truth (mean Krippendorff's alpha 0.72, range 0.56-0.81). The CNN assigned the correct diagnostic label (inflammatory, haemorrhagic or neoplastic) in 10 out of 11 clinical samples, compared with 7-11 out of 11 by human raters. CONCLUSIONS: Our approach provides the basis to overcome current limitations in automated cell classification for routine diagnostics and demonstrates how a visual explanation framework can connect machine decision-making with cell properties and thus provide a novel versatile and quantitative method for investigating CSF manifestations of various neurological diseases.

[Neuropathology I: muscular diseases]. / Neuropathologie I: Muskuläre Erkrankungen.

Muscle diseases include hereditary and acquired diseases with clinical manifestation in both childhood and adulthood. The different muscle diseases may have ultrastructural alterations that help us further understand the pathology of the disease. Specific changes in sarcomere structure help to classify a congenital myopathy. The detection of cellular aggregates supports the classification of myositis. Pathologically altered mitochondria, on the other hand, can occur both in genetic mitochondriopathies but also secondarily in acquired muscle diseases like myositis. Ultrastructural analysis of the myocardium is also helpful in the diagnosis of hereditary cardiomyopathies in childhood. This review article highlights the ultrastructural features of different muscle diseases and pathognomonic findings in specific disease groups.

DNA methylation-based classification of sinonasal tumors.

The diagnosis of sinonasal tumors is challenging due to a heterogeneous spectrum of various differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (SNUCs). In this study, we apply a machine learning algorithm based on DNA methylation patterns to classify sinonasal tumors with clinical-grade reliability. We further show that sinonasal tumors with SNUC morphology are not as undifferentiated as their current terminology suggests but rather reassigned to four distinct molecular classes defined by epigenetic, mutational and proteomic profiles. This includes two classes with neuroendocrine differentiation, characterized by IDH2 or SMARCA4/ARID1A mutations with an overall favorable clinical course, one class composed of highly aggressive SMARCB1-deficient carcinomas and another class with tumors that represent potentially previously misclassified adenoid cystic carcinomas. Our findings can aid in improving the diagnostic classification of sinonasal tumors and could help to change the current perception of SNUCs.

Assessing and improving the validity of COVID-19 autopsy studies - A multicentre approach to establish essential standards for immunohistochemical and ultrastructural analyses.

BACKGROUND: Autopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain about whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, such as overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. METHODS: We assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 proteins in cultured cell lines and COVID-19 autopsy tissues. In a multicentre study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 IHC. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. FINDINGS: Publications show high variability in detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. We show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (N= 35; r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and provide recommendations for optimized sampling and analysis. 135 of 144 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM sections as a reference and for training purposes. INTERPRETATION: Since detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2. FUNDING: German Federal Ministry of Health, German Federal Ministry of Education and Research, Berlin University Alliance, German Research Foundation, German Center for Infectious Research.

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.

Congenital myopathies: The current status.

Within the history of neuromuscular diseases (NMD), congenital myopathies (CM) represent a relatively new category introduced in the mid-nineteen hundreds upon advent and subsequent application of enzyme histochemistry and electron microscopy by establishing the three major CM, central core disease, nemaline myopathy, and centronuclear myopathy which later pluralized each when the molecular era began at the end of last century. Quickly, during the following 5 decades, many new CM entities were described, based on muscle biopsies and their CM-characteristic myopathology, the former a prerequisite to recognizing an individual CM, the latter of the nosological hallmark of the individual CM. When the molecular era ushered in immunohistochemistry the spectrum and nosography of CM altered in that some CM became allelic to other cohorts of NMD, e.g., congenital muscular dystrophies, other muscular dystrophies, distal myopathies based on different or identical mutations in the same gene. The nosological spectrum of a defective gene also enlarged by recognizing several entities with mutations in the same gene, and same or similar nosological conditions originated from mutations in different genes. Lately, however, CM were reported which lacked any individual myopathological hallmarks, but were clearly based on molecular defects, a fair number of them being newly identified ones. Few CM still remain without any molecular clarification. This nosographic development rendered the original definition of such new CM questionable and brought uncertainty to their classification and nomenclature.

Skeletal muscle provides the immunological micro-milieu for specific plasma cells in anti-synthetase syndrome-associated myositis.

Anti-synthetase syndrome (ASyS)-associated myositis is a major subgroup of the idiopathic inflammatory myopathies (IIM) and is characterized by disease chronicity with musculoskeletal, dermatological and pulmonary manifestations. One of eight autoantibodies against the aminoacyl-transferase RNA synthetases (ARS) is detectable in the serum of affected patients. However, disease-specific therapeutic approaches have not yet been established.To obtain a deeper understanding of the underlying pathogenesis and to identify putative therapeutic targets, we comparatively investigated the most common forms of ASyS associated with anti-PL-7, anti-PL-12 and anti-Jo-1. Our cohort consisted of 80 ASyS patients as well as healthy controls (n = 40), diseased controls (n = 40) and non-diseased controls (n = 20). We detected a reduced extent of necrosis and regeneration in muscle biopsies from PL-12+ patients compared to Jo-1+ patients, while PL-7+ patients had higher capillary dropout in biopsies of skeletal muscle. Aside from these subtle alterations, no significant differences between ASyS subgroups were observed. Interestingly, a tissue-specific subpopulation of CD138+ plasma cells and CXCL12+/CXCL13+CD20+ B cells common to ASyS myositis were identified. These cells were localized in the endomysium associated with alkaline phosphatase+ activated mesenchymal fibroblasts and CD68+MHC-II+CD169+ macrophages. An MHC-I+ and MHC-II+ MxA negative type II interferon-driven milieu of myofiber activation, topographically restricted to the perifascicular area and the adjacent perimysium, as well as perimysial clusters of T follicular helper cells defined an extra-medullary immunological niche for plasma cells and activated B cells. Consistent with this, proteomic analyses of muscle tissues from ASyS patients demonstrated alterations in antigen processing and presentation. In-depth immunological analyses of peripheral blood supported a B-cell/plasma-cell-driven pathology with a shift towards immature B cells, an increase of B-cell-related cytokines and chemokines, and activation of the complement system. We hypothesize that a B-cell-driven pathology with the presence and persistence of a specific subtype of plasma cells in the skeletal muscle is crucially involved in the self-perpetuating chronicity of ASyS myositis. This work provides the conceptual framework for the application of plasma-cell-targeting therapies in ASyS myositis.

The ultrastructural heterogeneity of lung surfactant revealed by serial section electron tomography: insights into the 3-D architecture of human tubular myelin.

Weibel's hypothetical three-dimensional (3-D) model in 1966 provided first ultrastructural details into tubular myelin (TM), a unique, complex surfactant subtype found in the hypophase of the alveolar lining layer. Although initial descriptions by electron microscopy (EM) were already published in the 1950s, a uniform morphological differentiation from other intra-alveolar surfactant subtypes is still missing and potential structure-function relationships remain enigmatic. Technical developments in volume EM methods now allow a more detailed reinvestigation, to address unanswered ultrastructural questions, we analyzed ultrathin sections of humanized SP-A1/SP-A2 coexpressing mouse and human lung samples by conventional transmission EM. We combined these two-dimensional (2-D) information with 3-D analysis of single- and dual-axis electron tomography of serial sections for high z-resolution (in a range of a few nanometers) and extended volumes of up to 1 µm total z-information, this study reveals that TM constitutes a heterogeneous surfactant organization mainly comprised of distorted parallel membrane planes with local intersections, which are distributed all over the TM substructure. These intersecting membrane planes form, among other various polygons, the well-known 2-D "lattice", respectively 3-D quadratic tubules, which in many analyzed spots of human alveoli appear to be less abundant than also observed nonconcentric 3-D lamellae, the additional application of serial section electron tomography to conventional transmission EM demonstrates a high heterogeneity of TM membrane networks, which indicates dynamic transformations between its substructures. Our method provides an ideal basis for further in and ex vivo structural analyses of surfactant under various conditions at nanometer scale.

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). ​.