Neuropathological changes in severe COVID-19

Einleitung Eine Infektion mit dem neuartigen Erreger SARS-CoV-2 verursacht COVID-19 und geht in mehr als einem Drittel der Fälle mit einem breiten Spektrum neurologischer Symptome einher, deren Ätiopathogenesen bisher nicht vollumfänglich geklärt sind. Riech- und Geschmacksstörungen, insbesondere die Anosmie und Ageusie, sind für einen Teil der Patienten*innen sogar die führenden Symptome einer SARS-CoV-2-Infektion. Die Rezeptionszone des olfaktorischen Systems wird als Riechschleimhaut bezeichnet und bedeckt die obere Nasenmuschel direkt unterhalb der Siebplatte. In Anbetracht des direkten Kontaktes von neuralen/neuronalen Zellen mit der Umwelt stellt die Riechschleimhaut eine Schnittstelle und somit eine potentielle Eintrittspforte von SARS-CoV-2 in das ZNS dar. Methodik In der vorliegenden Arbeit erfolgte eine systematische postmortem Untersuchung des ZNS sowie peripherer Gewebeproben aus dem Respirationstrakt von Personen, die infolge einer SARS-CoV-2-Infektion verstorben sind. Mittels der Korrelation klinischer Daten und (neuro-)pathologischer Untersuchungen wurden SARS-CoV-2-spezifische Veränderungen des ZNS ermittelt. Zur Quantifizierung pro- und antiinflammatorischer Zytokine im Liquor der Verstorbenen wurde ein humaner Zytokin Array (Bio-techne) durchgeführt. Der Nachweis von SARS-CoV-2-RNA sowie subgenomischer (sg)RNA, als Surrogat aktiver Virusreplikation, erfolgte mittels quantitativer Echtzeit-Polymerase-Kettenreaktion (qPCR). Zur Charakterisierung des Tropismus von SARS-CoV-2 wurde die RNAscope® in situ Hybridisierung (ISH), Immunhistochemie (IHC) und Elektronenmikroskopie genutzt. Ergebnisse Akute thromboembolische ischämische Infarkte (n=13/86; 15%) und frische Blutungen (n=13/86; 15%) sowie eine starke angeborene Immunantwort, vermittelt durch HLA-DR+ Mikroglia mit korrelierender Erhöhung proinflammatorischer Mediatoren wie IL-6, IL-18, CCL2 und sICAM-1 im Liquor, sind die führenden Veränderungen im ZNS. Die höchste Viruslast ließ sich mittels qPCR in der Riechschleimhaut detektieren. SgRNA fand sich am häufigsten in der Riechschleimhaut (n=18/57) und Uvula (n=8/45); es ergab sich kein Nachweis von sgRNA im ZNS. Die Viruslast korreliert invers mit der Erkrankungsdauer der Patienten*innen (r=-0,5; P=0,006). Im Riechepithel - hier in Kolokalisation mit neuralen/neuronalen Zellen - sowie in den cerebralen Gefäßendothelzellen fand sich eine distinkte Immunreaktivität für SARS-CoV Spike-Protein. Mittels RNAscope® ISH gelang es SARS-CoV-2 RNA innerhalb des Riechepithels darzustellen. Ultrastrukturell konnten wir elektronenmikroskopische Aufnahmen intakter Coronaviruspartikel in der Riechschleimhaut anfertigen. Schlussfolgerung Die Ergebnisse weisen darauf hin, dass SARS-CoV-2 die Nervenzellen der Riechschleimhaut als Eintrittspforte in das Gehirn benutzen kann. Eine SARS-CoV-2-Infektion führt im ZNS zu einer Immunantwort mit Aktivierung von HLA-DR+ Mikroglia und erhöhten Spiegeln von Entzündungsmediatoren.Introduction The newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus disease 2019 (COVID-19) which is accompanied by a broad spectrum of neurological manifestations in more than one-third of COVID-19 cases. ­­­For the latter, olfactory and gustatory disturbances such as anosmia and ageusia, are often leading symptoms of SARS-CoV-2 infection. The reception area of the human olfactory system is known as the olfactory mucosa and covers the upper turbinate directly underneath the cribriform plate. Given the close proximity of neural/neuronal cells and the environment the olfactory mucosa as a neural-mucosal interface may represent a potential port of CNS entry for SARS-CoV-2. Methods Here, we systematically investigated postmortem tissue of the CNS and respiratory tract from individuals with COVID-19. Based on the correlation of clinical data and (neuro-)pathological examinations, SARS-CoV-2-specific morphological changes were determined. To quantify pro- and anti-inflammatory cytokines in the cerebrospinal fluid (CSF) of the deceased, a human cytokine array (Bio-techne) was performed. Detection of SARS-CoV-2 RNA, including subgenomic (sg)RNA as a surrogate of active virus replication, was performed using quantitative real-time PCR (qPCR). By various means, such as RNAscope® in situ hybridization (ISH), immunohistochemistry and electron microscopy, we further characterized the CNS tropism of SARS-CoV-2 and the consequences thereof. Results Acute thromboembolic ischemic infarcts (n=13/86; 15%) and a strong innate immune response, mediated by HLA-DR+ microglia with a linked increase in proinflammatory mediators such as IL-6, IL-18, CCL2 and sICAM-1 in the CSF, are leading alterations in the CNS. The highest levels of viral RNA for SARS-CoV-2 were found within the olfactory mucosa. SgRNA was most frequently found in the olfactory mucosa (n=18/57) and uvula (n=8/45), although there was no evidence of sgRNA in the CNS. An inverse correlation between the duration of illness and viral load could be determined (r=-0,5; P=0,006). Besides, a distinct immunoreactivity for SARS-CoV Spike (S) protein was found in the olfactory epithelium – here co-localizing with neural/neuronal cells - and within cerebral endothelial cells. Using RNAscope® ISH, SARS-CoV-2 RNA was successfully detected within the olfactory epithelium. We were also able to illustrate intact coronavirus particles in the olfactory mucosa ultrastructurally. Conclusion The results indicate that SARS-CoV-2 can enter the nervous system by crossing the neural-mucosal interface in the olfactory mucosa. SARS-CoV-2 infection results in an innate immune response with activation of HLA-DR+ microglia and increased levels of inflammatory mediators in the CNS

COVID-19: a fatal case of acute liver failure associated with SARS-CoV-2 infection in pre-existing liver cirrhosis

Background: The detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2) is challenging, particularly in post-mortem human tissues. However, there is increasing evidence for viral SARS-CoV-2 manifestation in non-respiratory tissues. In this context, it is a current matter of debate, whether SARS-CoV-2 shows hepatotropism. Case presentation: Here, we report a case of an 88-year-old women with massive SARS-CoV-2 viremia, severe jaundice and clinical signs of an acute hepatitis, who died within a few days from an acute liver failure without showing any clinical signs of pneumonia. Autopsy revealed a severe chronic and acute liver damage with bile duct infestation by SARS-CoV-2 that was accompanied by higher expressions of angiotensin-converting enzyme-2 (ACE2), Cathepsin L and transmembrane serine protease 2 (TMPRSS2). Conclusion: Our findings indicate an enhanced biliary susceptibility to viral infection with SARS-CoV-2, that might have resulted from pre-existing severe liver damage. Furthermore, our findings emphasize the differential diagnosis of coronavirus disease 2019 (COVID-19)-associated liver failure in the clinical setting of an inexplicable jaundice

Human small intestinal infection by SARS-CoV-2 is characterized by a mucosal infiltration with activated CD8+ T cells

The SARS-CoV-2 pandemic has so far claimed over three and a half million lives worldwide. Though the SARS-CoV-2 mediated disease COVID-19 has first been characterized by an infection of the upper airways and the lung, recent evidence suggests a complex disease including gastrointestinal symptoms. Even if a direct viral tropism of intestinal cells has recently been demonstrated, it remains unclear, whether gastrointestinal symptoms are caused by direct infection of the gastrointestinal tract by SARS-CoV-2 or whether they are a consequence of a systemic immune activation and subsequent modulation of the mucosal immune system. To better understand the cause of intestinal symptoms we analyzed biopsies of the small intestine from SARS-CoV-2 infected individuals. Applying qRT-PCR and immunohistochemistry, we detected SARS-CoV-2 RNA and nucleocapsid protein in duodenal mucosa. In addition, applying imaging mass cytometry and immunohistochemistry, we identified histomorphological changes of the epithelium, which were characterized by an accumulation of activated intraepithelial CD8(+) T cells as well as epithelial apoptosis and subsequent regenerative proliferation in the small intestine of COVID-19 patients. In summary, our findings indicate that intraepithelial CD8(+) T cells are activated upon infection of intestinal epithelial cells with SARS-CoV-2, providing one possible explanation for gastrointestinal symptoms associated with COVID-19

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.Peer Reviewe

Quantitative analysis of the natural history of prolidase deficiency: description of 17 families and systematic review of published cases.

PURPOSE: Prolidase deficiency is a rare inborn error of metabolism causing ulcers and other skin disorders, splenomegaly, developmental delay, and recurrent infections. Most of the literature is constituted of isolated case reports. We aim to provide a quantitative description of the natural history of the condition by describing 19 affected individuals and reviewing the literature. METHODS: Nineteen patients were phenotyped per local institutional procedures. A systematic review following PRISMA criteria identified 132 articles describing 161 patients. Main outcome analyses were performed for manifestation frequency, diagnostic delay, overall survival, symptom-free survival, and ulcer-free survival. RESULTS: Our cohort presented a wide variability of severity. Autoimmune disorders were found in 6/19, including Crohn disease, systemic lupus erythematosus, and arthritis. Another immune finding was hemophagocytic lymphohistiocytosis (HLH). Half of published patients were symptomatic by age 4 and had a delayed diagnosis (mean delay 11.6 years). Ulcers were present initially in only 30% of cases, with a median age of onset at 12 years old. CONCLUSION: Prolidase deficiency has a broad range of manifestations. Symptoms at onset may be nonspecific, likely contributing to the diagnostic delay. Testing for this disorder should be considered in any child with unexplained autoimmunity, lower extremity ulcers, splenomegaly, or HLH

Impact of Hydrological Modellers’ Decisions and Attitude on the Performance of a Calibrated Conceptual Catchment Model: Results from a ‘Modelling Contest’

In this study, 17 hydrologists with different experience in hydrological modelling applied the same conceptual catchment model (HBV) to a Greek catchment, using identical data and model code. Calibration was performed manually. Subsequently, the modellers were asked for their experience, their calibration strategy, and whether they enjoyed the exercise. The exercise revealed that there is considerable modellers’ uncertainty even among the experienced modellers. It seemed to be equally important whether the modellers followed a good calibration strategy, and whether they enjoyed modelling. The exercise confirmed previous studies about the benefit of model ensembles: Different combinations of the simulation results (median, mean) outperformed the individual model simulations, while filtering the simulations even improved the quality of the model ensembles. Modellers’ experience, decisions, and attitude, therefore, have an impact on the hydrological model application and should be considered as part of hydrological modelling uncertainty

Using EM data to understand COVID-19 pathophysiology – Authors' reply

Peer Reviewe

Human small intestinal infection by SARS-CoV-2 is characterized by a mucosal infiltration with activated CD8+ T cells

Abstract The SARS-CoV-2 pandemic has so far claimed over three and a half million lives worldwide. Though the SARS-CoV-2 mediated disease COVID-19 has first been characterized by an infection of the upper airways and the lung, recent evidence suggests a complex disease including gastrointestinal symptoms. Even if a direct viral tropism of intestinal cells has recently been demonstrated, it remains unclear, whether gastrointestinal symptoms are caused by direct infection of the gastrointestinal tract by SARS-CoV-2 or whether they are a consequence of a systemic immune activation and subsequent modulation of the mucosal immune system. To better understand the cause of intestinal symptoms we analyzed biopsies of the small intestine from SARS-CoV-2 infected individuals. Applying qRT-PCR and immunohistochemistry, we detected SARS-CoV-2 RNA and nucleocapsid protein in duodenal mucosa. In addition, applying imaging mass cytometry and immunohistochemistry, we identified histomorphological changes of the epithelium, which were characterized by an accumulation of activated intraepithelial CD8 + T cells as well as epithelial apoptosis and subsequent regenerative proliferation in the small intestine of COVID-19 patients. In summary, our findings indicate that intraepithelial CD8 + T cells are activated upon infection of intestinal epithelial cells with SARS-CoV-2, providing one possible explanation for gastrointestinal symptoms associated with COVID-19

Dynamics of Core Planar Polarity Protein Turnover and Stable Assembly into Discrete Membrane Subdomains

The core planar polarity proteins localize asymmetrically to the adherens junctions of epithelial cells, where they have been hypothesized to assemble into intercellular complexes. Here, we show that the core proteins are preferentially distributed to discrete membrane subdomains (“puncta”), where they form asymmetric contacts between neighboring cells. Using an antibody internalization assay and fluorescence recovery after photobleaching in prepupal and pupal wings, we have investigated the turnover of two key core proteins, Flamingo and Frizzled, and find that the localization of both within puncta is highly stable. Furthermore, the transmembrane core proteins, Flamingo, Frizzled, and Strabismus, are necessary for stable localization of core proteins to junctions, whereas the cytoplasmic core proteins are required for their concentration into puncta. Thus, we define the distinct roles of specific core proteins in the formation of asymmetric contacts between cells, which is a key event in the generation of coordinated cellular asymmetry

B cell depletion and signs of sepsis-acquired immunodeficiency in bone marrow and spleen of COVID-19 deceased

Objectives: In coronavirus disease 2019 (COVID-19), the adaptive immune response is of considerable importance, and detailed cellular immune reactions in the hematological system of patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are yet to be clarified. Methods: This study reports the morphological characterization of both bone marrow and spleen in 11 COVID-19 decedents with respect to findings in the peripheral blood and pulmonary SARS-CoV-2 burden. Results: In the bone marrow, activation and left shift were found in at least 55% of patients, which was mirrored by peripheral anaemia, granulocytic immaturity and multiple thromboembolic events. Signs of sepsis-acquired immunodeficiency were found in the setting of an abscess-forming superinfection of viral COVID-19 pneumonia. Furthermore, a severe B cell loss was observed in the bone marrow and/or spleen in 64% of COVID-19 patients. This was reflected by lymphocytopenia in the peripheral blood. As compared to B cell preservation, B cell loss was associated with a higher pulmonary SARS-CoV-2 burden and only a marginal decrease of of T cell counts. Conclusions: The results of this study suggest the presence of sepsis-related immunodeficiency in severe COVID-19 pneumonia with superinfection. Furthermore, our findings indicate that lymphocytopenia in COVID-19 is accompanied by B cell depletion in hematopoietic tissue, which might impede the durability of the humoral immune response to SARS-CoV-2. (C) 2021 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases