High-fat diet-induced brain region-specific phenotypic spectrum of CNS resident microglia

Diets high in fat (HFD) are known to cause an immune response in the periphery as well as the central nervous system. In peripheral adipose tissue, this immune response is primarily mediated by macrophages that are recruited to the tissue. Similarly, reactivity of microglia, the innate immune cells of the brain, has been shown to occur in the hypothalamus of mice fed a high-fat diet. To characterize the nature of the microglial response to diets high in fat in a temporal fashion, we studied the phenotypic spectrum of hypothalamic microglia of mice fed high-fat diet for 3 days and 8 weeks by assessing their tissue reaction and inflammatory signature. While we observed a significant increase in Iba1+ myeloid cells and a reaction of GFAP+ astrocytes in the hypothalamus after 8 weeks of HFD feeding, we found the hypothalamic myeloid cell reaction to be limited to endogenous microglia and not mediated by infiltrating myeloid cells. Moreover, obese humans were found to present with signs of hypothalamic gliosis and exacerbated microglia dystrophy, suggesting a targeted microglia response to diet in humans as well. Notably, the glial reaction occurring in the mouse hypothalamus was not accompanied by an increase in pro-inflammatory cytokines, but rather by an anti-inflammatory reaction. Gene expression analyses of isolated microglia not only confirmed this observation, but also revealed a downregulation of microglia genes important for sensing signals in the microenvironment. Finally, we demonstrate that long-term exposure of microglia to HFD in vivo does not impair the cell’s ability to respond to additional stimuli, like lipopolysaccharide. Taken together, our findings support the notion that microglia react to diets high in fat in a region-specific manner in rodents as well as in humans; however, this response changes over time as it is not exclusively pro-inflammatory nor does exposure to HFD prime microglia in the hypothalamus. Electronic supplementary material The online version of this article (doi:10.1007/s00401-016-1595-4) contains supplementary material, which is available to authorized users

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.Peer Reviewe

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

Development and Technical Validation of an Immunoassay for the Detection of APP669−711 (Aβ−3−40) in Biological Samples

The ratio of amyloid precursor protein (APP)669–711 (Aβ−3–40)/Aβ1–42 in blood plasma was reported to represent a novel Alzheimer’s disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ−3–40 with no appreciable cross reactivity with Aβ1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ42/Aβ−3–40 and Aβ42/Aβ40 ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ42/Aβ−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set

The genomic and transcriptional landscape of primary central nervous system lymphoma

Primary lymphomas of the central nervous system (PCNSL) are mainly diffuse large B-cell lymphomas (DLBCLs) confined to the central nervous system (CNS). Molecular drivers of PCNSL have not been fully elucidated. Here, we profile and compare the whole-genome and transcriptome landscape of 51 CNS lymphomas (CNSL) to 39 follicular lymphoma and 36 DLBCL cases outside the CNS. We find recurrent mutations in JAK-STAT, NFkB, and B-cell receptor signaling pathways, including hallmark mutations in MYD88 L265P (67%) and CD79B (63%), and CDKN2A deletions (83%). PCNSLs exhibit significantly more focal deletions of HLA-D (6p21) locus as a potential mechanism of immune evasion. Mutational signatures correlating with DNA replication and mitosis are significantly enriched in PCNSL. TERT gene expression is significantly higher in PCNSL compared to activated B-cell (ABC)-DLBCL. Transcriptome analysis clearly distinguishes PCNSL and systemic DLBCL into distinct molecular subtypes. Epstein-Barr virus (EBV)+ CNSL cases lack recurrent mutational hotspots apart from IG and HLA-DRB loci. We show that PCNSL can be clearly distinguished from DLBCL, having distinct expression profiles, IG expression and translocation patterns, as well as specific combinations of genetic alterations

The genomic and transcriptional landscape of primary central nervous system lymphoma

Primary lymphomas of the central nervous system (PCNSL) are mainly diffuse large B-cell lymphomas (DLBCLs) confined to the central nervous system (CNS). Molecular drivers of PCNSL have not been fully elucidated. Here, we profile and compare the whole-genome and transcriptome landscape of 51 CNS lymphomas (CNSL) to 39 follicular lymphoma and 36 DLBCL cases outside the CNS. We find recurrent mutations in JAK-STAT, NFkB, and B-cell receptor signaling pathways, including hallmark mutations in MYD88 L265P (67%) and CD79B (63%), and CDKN2A deletions (83%). PCNSLs exhibit significantly more focal deletions of HLA-D (6p21) locus as a potential mechanism of immune evasion. Mutational signatures correlating with DNA replication and mitosis are significantly enriched in PCNSL. TERT gene expression is significantly higher in PCNSL compared to activated B-cell (ABC)-DLBCL. Transcriptome analysis clearly distinguishes PCNSL and systemic DLBCL into distinct molecular subtypes. Epstein-Barr virus (EBV)+ CNSL cases lack recurrent mutational hotspots apart from IG and HLA-DRB loci. We show that PCNSL can be clearly distinguished from DLBCL, having distinct expression profiles, IG expression and translocation patterns, as well as specific combinations of genetic alterations

Study to determine preanalytical influencing factors on CSF cell stability

Einführung: Die exakte Differenzierung der Zellen im Liquor cerebrospinalis ist im Rahmen der diagnostischen Aufarbeitung insbesondere bei neurologischen Krankheitsbildern und Tumorerkrankungen unerlässlich und setzt einen guten Erhaltungszustand der Zellen voraus. Liquor cerebrospinalis gilt als sensibles Probenmaterial, welches bereits 2 Stunden nach Entnahme deutliche autolytische Veränderungen zeigt. Im Institut für Neuropathologie der Charité – Universitätsmedizin Berlin fiel bei der Beurteilung von jährlich über dreitausend Liquorproben ein sehr variabler Erhaltungszustand der Zellen auf. Die große Variabilität im Erhaltungszustand war dabei nicht ausschließlich durch die Dauer des Probentransports zu erklären. Die manuelle Handhabung der Proben im Labor sowie hohe Außentemperaturen schienen einen negativen Einfluss zu haben. In der vorliegenden Arbeit wurden diese Beobachtungen objektiviert und relevante präanalytische Einflussfaktoren auf den Erhaltungszustand der Zellen identifiziert. Methodik: Insgesamt wurden Liquorproben von 119 Patient*innen eingeschlossen und hinsichtlich des Erhaltungszustandes der darin enthaltenen Zellen beurteilt. Dafür wurden Zytospinpräparate angefertigt, nach Pappenheim (May-Grünwald-Giemsa) gefärbt und am Mikroskop beurteilt. Untersucht wurde der Einfluss der Zeit zwischen Probenabnahme und Anfertigung der Zytospinpräparate, der Einfluss des Transportbehältnisses und der Umgebungstemperatur, der Einfluss der manuellen Handhabung im Labor sowie des Gehalts an Eiweiß, Glucose und Lactat. Auch die Variabilität innerhalb einer Probe und in der mikroskopischen Beurteilung wurden erfasst. Ergebnisse: Es zeigten sich eine statistisch signifikante inverse Korrelation der Tageshöchsttemperatur zu dem Anteil intakter Zellen. Bei Tageshöchstwerten über 30°C war zusätzlich der Anteil an Zellschatten signifikant gesteigert. Darüber hinaus war ein signifikanter Einfluss der manuellen Handhabung der Proben auf den Erhaltungszustand der Zellen nachweisbar. Ein relevanter Einfluss der übrigen untersuchten Faktoren zeigte sich hingegen nicht. Schlussfolgerungen: Zusammenfassend lassen sich einzelne Empfehlungen ableiten, welche zu einer besseren Beurteilbarkeit von Zytospinpräparaten im Institut für Neuropathologie der Charité – Universitätsmedizin Berlin beitragen können. Das Labor, in welchem die Zytospinpräparate angefertigt werden, sollte jederzeit mit ausreichend Personal besetzt sein, um eine unmittelbare und gewissenhafte Bearbeitung der Liquorproben zu ermöglichen. Auch sollte das Laborpersonal im Umgang mit Liquor gut und regelmäßig geschult werden. Darüber hinaus muss es vermieden werden, den Liquor hohen Umgebungstemperaturen auszusetzen, sodass sich an heißen Tagen eine Kühlung der Proben während des Transports empfiehlt.The exact differentiation of the cells in the cerebrospinal fluid (CSF) is of great relevance within the diagnostic process, especially in the context of neurological diseases and tumor diseases, and thus requires a good state of preservation of the cells. CSF is a sensitive material that shows significant autolytic changes just 2 hours after collection. In the Department of Neuropathology at the Charité - Universitätsmedizin Berlin, the assessment of over three thousand CSF samples per year revealed a great variety of the state of preservation of the cells which could not only be explained by the duration of the sample transportation. The handling of the samples by the technical staff in the laboratory as well as high outside temperatures also seemed to have a negative impact. This led to the questions if these observations could be confirmed and if some preanalytic factors show a higher influence on the condition of the cells than others. Methods: 119 CSF samples were included and assessed with regard to the state preservation of the cells. For this purpose, cytospins were prepared and assessed under the microscope. The influence of delayed cytospin preparation, the transport container and the ambient temperature, as well as the influence of handling by the technical staff as well as the protein, glucose and lactate levels were examined. The variability within one sample and between two assessors was also documented. Results: There was a statistically significant inverse correlation between the maximum daily temperature and the proportion of intact cells. Additionally, the proportion of cell shadows was also significantly increased with outside temperatures above 30 degrees. Moreover, a significant influence of the handling of the samples by the technical staff on the preservation of the cells could be demonstrated. However, there was no further relevant influence of the other examined factors. Conclusions: The results of the present work are helpful to identify factors that can lead to an improved assessment of cytospins in the Department of Neuropathology of the Charité - Universitätsmedizin Berlin. One conclusion is that the laboratory in which the cytospins are prepared should be sufficiently staffed at all times to enable immediate and conscientious processing of the samples. Furthermore, the laboratory staff should also be well and regularly trained in handling CSF samples. In addition, CSF samples should not be exposed to high ambient temperatures. On hot days, cooling of the samples during transportation is advisable