Publications by authors named "Andreas Hocke"

85 Publications

COVID-19 highlights the model dilemma in biomedical research.

Nat Rev Mater 2021 Mar 17:1-3. Epub 2021 Mar 17.

Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC Canada.

Scientists worldwide struggle to identify suitable animal models to study SARS-CoV-2 infections. Interspecies-related differences, such as host specificity, divergent immune responses, or the unavailability of species-specific reagents hamper the research. Human-based models, such as micro-engineered multi-organs-on-chip, may hold the solution.
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http://dx.doi.org/10.1038/s41578-021-00305-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7967778PMC
March 2021

Transcriptomic profiling of SARS-CoV-2 infected human cell lines identifies HSP90 as target for COVID-19 therapy.

iScience 2021 Mar 6;24(3):102151. Epub 2021 Feb 6.

Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Hannoversche Str 28, 10115 Berlin, Germany.

Detailed knowledge of the molecular biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is crucial for understanding of viral replication, host responses, and disease progression. Here, we report gene expression profiles of three SARS-CoV- and SARS-CoV-2-infected human cell lines. SARS-CoV-2 elicited an approximately two-fold higher stimulation of the innate immune response compared to SARS-CoV in the human epithelial cell line Calu-3, including induction of miRNA-155. Single-cell RNA sequencing of infected cells showed that genes induced by virus infections were broadly upregulated, whereas interferon beta/lambda genes, a pro-inflammatory cytokines such as IL-6, were expressed only in small subsets of infected cells. Temporal analysis suggested that transcriptional activities of interferon regulatory factors precede those of nuclear factor κB. Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 activity resulted in a reduction of viral replication and pro-inflammatory cytokine expression in primary human airway epithelial cells.
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http://dx.doi.org/10.1016/j.isci.2021.102151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866843PMC
March 2021

A Therapeutic Non-self-reactive SARS-CoV-2 Antibody Protects from Lung Pathology in a COVID-19 Hamster Model.

Cell 2020 11 23;183(4):1058-1069.e19. Epub 2020 Sep 23.

German Center for Neurodegenerative Diseases (DZNE) Berlin, 10117 Berlin, Germany; Helmholtz Innovation Lab BaoBab (Brain Antibody-omics and B-cell Lab), 10117 Berlin, Germany; Department of Neurology and Experimental Neurology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, 10117 Berlin, Germany. Electronic address:

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from 10 COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb, CV07-209, neutralized authentic SARS-CoV-2 with an IC value of 3.1 ng/mL. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2-neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss, and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.
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http://dx.doi.org/10.1016/j.cell.2020.09.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510528PMC
November 2020

Reversion of pneumolysin induced executioner caspase activation redirects cells to survival.

J Infect Dis 2020 Oct 12. Epub 2020 Oct 12.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Internal Medicine/Infectious Diseases and Respiratory Medicine, Charitéplatz, Berlin, Germany.

Apoptosis is an indispensable mechanism for eliminating infected cells and activation of executioner caspases is considered as a point of no return. Streptococcus pneumoniae, the most common bacterial pathogen causing community-acquired pneumonia, induces apoptosis via its pore forming toxin pneumolysin, leading to rapid influxes of mitochondrial calcium [Ca 2+]m as well as fragmentation, loss of motility and membrane potential, which is accompanied by caspase-3/7 activation. Using machine-learning and quantitative live-cell microscopy, we identified a significant number of alveolar epithelial cells surviving such executioner caspase activation after pneumolysin attack. Precise single cell analysis revealed the [Ca 2+]m amplitude and efflux rate as decisive parameters for survival and death, which was verified by pharmacological inhibition of [Ca 2+]m efflux shifting the surviving cells towards the dying fraction. Taken together, we identified the regulation of [Ca 2+]m as critical for controlling the cellular fate under pneumolysin attack, which might be useful for therapeutic intervention during pneumococcal infection.
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http://dx.doi.org/10.1093/infdis/jiaa639DOI Listing
October 2020

A SARS-CoV-2 neutralizing antibody protects from lung pathology in a COVID-19 hamster model.

bioRxiv 2020 Aug 16. Epub 2020 Aug 16.

German Center for Neurodegenerative Diseases (DZNE) Berlin, Berlin, Germany.

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 A revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy.
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http://dx.doi.org/10.1101/2020.08.15.252320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430590PMC
August 2020

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Cell 2020 09 5;182(6):1419-1440.e23. Epub 2020 Aug 5.

Department of Infectious Diseases and Respiratory Medicine, Charité, Universitätsmedizin Berlin, Berlin, Germany; German Center for Lung Research (DZL).

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRCD11c inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
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http://dx.doi.org/10.1016/j.cell.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405822PMC
September 2020

SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19.

Nature 2020 11 29;587(7833):270-274. Epub 2020 Jul 29.

Si-M/'Der Simulierte Mensch', Technische Universität Berlin and Charité-Universitätsmedizin Berlin, Berlin, Germany.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the rapidly unfolding coronavirus disease 2019 (COVID-19) pandemic. Clinical manifestations of COVID-19 vary, ranging from asymptomatic infection to respiratory failure. The mechanisms that determine such variable outcomes remain unresolved. Here we investigated CD4 T cells that are reactive against the spike glycoprotein of SARS-CoV-2 in the peripheral blood of patients with COVID-19 and SARS-CoV-2-unexposed healthy donors. We detected spike-reactive CD4 T cells not only in 83% of patients with COVID-19 but also in 35% of healthy donors. Spike-reactive CD4 T cells in healthy donors were primarily active against C-terminal epitopes in the spike protein, which show a higher homology to spike glycoproteins of human endemic coronaviruses, compared with N-terminal epitopes. Spike-protein-reactive T cell lines generated from SARS-CoV-2-naive healthy donors responded similarly to the C-terminal region of the spike proteins of the human endemic coronaviruses 229E and OC43, as well as that of SARS-CoV-2. This results indicate that spike-protein cross-reactive T cells are present, which were probably generated during previous encounters with endemic coronaviruses. The effect of pre-existing SARS-CoV-2 cross-reactive T cells on clinical outcomes remains to be determined in larger cohorts. However, the presence of spike-protein cross-reactive T cells in a considerable fraction of the general population may affect the dynamics of the current pandemic, and has important implications for the design and analysis of upcoming trials investigating COVID-19 vaccines.
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http://dx.doi.org/10.1038/s41586-020-2598-9DOI Listing
November 2020

Ultra-High-Throughput Clinical Proteomics Reveals Classifiers of COVID-19 Infection.

Cell Syst 2020 07 2;11(1):11-24.e4. Epub 2020 Jun 2.

The Francis Crick Institute, Molecular Biology of Metabolism Laboratory, London NW11AT, UK; Charité Universitätsmedizin, Department of Biochemistry, 10117 Berlin, Germany. Electronic address:

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies. We use our platform on samples collected from a cohort of early hospitalized cases of the SARS-CoV-2 pandemic and identify 27 potential biomarkers that are differentially expressed depending on the WHO severity grade of COVID-19. They include complement factors, the coagulation system, inflammation modulators, and pro-inflammatory factors upstream and downstream of interleukin 6. All protocols and software for implementing our approach are freely available. In total, this work supports the development of routine proteomic assays to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets.
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http://dx.doi.org/10.1016/j.cels.2020.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264033PMC
July 2020

COVID-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis.

Nat Biotechnol 2020 08 26;38(8):970-979. Epub 2020 Jun 26.

Center for Digital Health, Berlin Institute of Health (BIH) and Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.

To investigate the immune response and mechanisms associated with severe coronavirus disease 2019 (COVID-19), we performed single-cell RNA sequencing on nasopharyngeal and bronchial samples from 19 clinically well-characterized patients with moderate or critical disease and from five healthy controls. We identified airway epithelial cell types and states vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In patients with COVID-19, epithelial cells showed an average three-fold increase in expression of the SARS-CoV-2 entry receptor ACE2, which correlated with interferon signals by immune cells. Compared to moderate cases, critical cases exhibited stronger interactions between epithelial and immune cells, as indicated by ligand-receptor expression profiles, and activated immune cells, including inflammatory macrophages expressing CCL2, CCL3, CCL20, CXCL1, CXCL3, CXCL10, IL8, IL1B and TNF. The transcriptional differences in critical cases compared to moderate cases likely contribute to clinical observations of heightened inflammatory tissue damage, lung injury and respiratory failure. Our data suggest that pharmacologic inhibition of the CCR1 and/or CCR5 pathways might suppress immune hyperactivation in critical COVID-19.
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http://dx.doi.org/10.1038/s41587-020-0602-4DOI Listing
August 2020

Studying the pathophysiology of coronavirus disease 2019: a protocol for the Berlin prospective COVID-19 patient cohort (Pa-COVID-19).

Infection 2020 Aug 13;48(4):619-626. Epub 2020 Jun 13.

Department of Infectious Diseases and Respiratory Medicine, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, and Berlin Institute of Health, Campus Virchow Klinikum, Augustenburgerplatz 1, 13353, Berlin, Germany.

Purpose: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide causing a global health emergency. Pa-COVID-19 aims to provide comprehensive data on clinical course, pathophysiology, immunology and outcome of COVID-19, to identify prognostic biomarkers, clinical scores, and therapeutic targets for improved clinical management and preventive interventions.

Methods: Pa-COVID-19 is a prospective observational cohort study of patients with confirmed SARS-CoV-2 infection treated at Charité - Universitätsmedizin Berlin. We collect data on epidemiology, demography, medical history, symptoms, clinical course, and pathogen testing and treatment. Systematic, serial blood sampling will allow deep molecular and immunological phenotyping, transcriptomic profiling, and comprehensive biobanking. Longitudinal data and sample collection during hospitalization will be supplemented by long-term follow-up.

Results: Outcome measures include the WHO clinical ordinal scale on day 15 and clinical, functional, and health-related quality-of-life assessments at discharge and during follow-up. We developed a scalable dataset to (i) suit national standards of care, (ii) facilitate comprehensive data collection in medical care facilities with varying resources, and (iii) allow for rapid implementation of interventional trials based on the standardized study design and data collection. We propose this scalable protocol as blueprint for harmonized data collection and deep phenotyping in COVID-19 in Germany.

Conclusion: We established a basic platform for harmonized, scalable data collection, pathophysiological analysis, and deep phenotyping of COVID-19, which enables rapid generation of evidence for improved medical care and identification of candidate therapeutic and preventive strategies. The electronic database accredited for interventional trials allows fast trial implementation for candidate therapeutic agents.

Trial Registration: Registered at the German registry for clinical studies (DRKS00021688).
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http://dx.doi.org/10.1007/s15010-020-01464-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293426PMC
August 2020

Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry.

Nat Nanotechnol 2020 05 30;15(5):373-379. Epub 2020 Mar 30.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies and peptides or that address late steps of the viral replication cycle.
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http://dx.doi.org/10.1038/s41565-020-0660-2DOI Listing
May 2020

Multi-target immunofluorescence by separation of antibody cross-labelling via spectral-FLIM-FRET.

Sci Rep 2020 03 2;10(1):3820. Epub 2020 Mar 2.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Internal Medicine/Infectious Diseases and Respiratory Medicine, Charitéplatz 1, 10117, Berlin, Germany.

In biomedical research, indirect immunofluorescence labelling by use of primary and secondary antibodies is central for revealing the spatial distribution of multiple cellular antigens. However, labelling is regularly restricted to few antigens since species variation of primary and corresponding secondary antibodies is limited bearing the risk of unspecific cross-labelling. Here, we introduce a novel microscopic procedure for leveraging undesirable cross-labelling effects among secondary antibodies thereby increasing the number of fluorophore channels. Under cross-labelling conditions, commonly used fluorophores change chemical-physical properties by 'Förster resonance energy transfer' leading to defined changes in spectral emission and lifetime decay. By use of spectral fluorescence lifetime imaging and pattern-matching, we demonstrate precise separation of cross-labelled cellular antigens where conventional imaging completely fails. Consequently, this undesired effect serves for an innovative imaging procedure to separate critical antigens where antibody species variation is limited and allows for multi-target labelling by attribution of new fluorophore cross-labelling channels.
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http://dx.doi.org/10.1038/s41598-020-60877-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052234PMC
March 2020

Photon arrival time tagging with many channels, sub-nanosecond deadtime, very high throughput, and fiber optic remote synchronization.

Rev Sci Instrum 2020 Jan;91(1):013108

Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Internal Medicine/Infectious Diseases and Respiratory Medicine, Charitéplatz 1, D-10117 Berlin, Germany.

Time-Correlated Single Photon Counting (TCSPC) and time tagging of individual photon detections are powerful tools in many quantum optical experiments and other areas of applied physics. Using TCSPC, e.g., for the purpose of fluorescence lifetime measurements, is often limited in speed due to dead-time losses and pileup. We show that this limitation can be lifted by reducing the dead-time of the timing electronics to the absolute minimum imposed by the speed of the detector signals while maintaining high temporal resolution. A complementing approach to speedy data acquisition is parallelization by means of simultaneous readout of many detector channels. This puts high demands on the data throughput of the TCSPC system, especially in time tagging of individual photon arrivals. Here, we present a new design approach, supporting up to 16 input channels, an extremely short dead-time of 650 ps, very high time tagging throughput, and a timing resolution of 80 ps. In order to facilitate remote synchronization of multiple such instruments with highest precision, the new TCSPC electronics provide an interface for White Rabbit fiber optic networks. Beside fundamental research in the field of astronomy, such remote synchronization tasks arise routinely in quantum communication networks with node to node distances on the order of tens of kilometers. In addition to showing design features and benchmark results of new TCSPC electronics, we present application results from spectrally resolved and high-speed fluorescence lifetime imaging in medical research. We furthermore show how pulse-pileup occurring in the detector signals at high photon flux can be corrected for and how this data acquisition scheme performs in terms of accuracy and efficiency.
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http://dx.doi.org/10.1063/1.5121412DOI Listing
January 2020

Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities.

Emerg Microbes Infect 2019 ;8(1):1763-1776

Institute of Virology Muenster, Westfaelische Wilhelms-University, Muenster, Germany.

Influenza is an acute respiratory infection causing high morbidity and mortality in annual outbreaks worldwide. Antiviral drugs are limited and pose the risk of resistance development, calling for new treatment options. IFN-α subtypes are immune-stimulatory cytokines with strong antiviral activities against IAV and However, the clinical use of IFN-α2, the only licensed subtype of this multi-gene family, could not prevent or limit IAV infections in humans. However, the other subtypes were not investigated.Therefore, this study evaluated the induction and antiviral potential of all human IFN-α subtypes during H3N2 IAV infection in human lung explants. We found that subtypes with weak antiviral activities were preferentially induced during IAV infection in human lungs. Intriguingly, non-induced subtypes α16, α5 and α4 suppressed viral replication up to 230-fold more efficiently than α2. Furthermore, our results demonstrate that subtypes with stronger antiviral activities induce higher expression of IAV-specific restriction factors and that MxA expression is a determinant of the subtype-specific antiviral activity towards H3N2 IAV. These results corroborate that IFN-α subtypes exhibit differential antiviral activities and emphasize that subtypes α16, α5 and α4 should be further investigated for the prevention and treatment of severe infections with seasonal H3N2 IAV.
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http://dx.doi.org/10.1080/22221751.2019.1698271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913622PMC
April 2020

Stable integration of the Mrx1-roGFP2 biosensor to monitor dynamic changes of the mycothiol redox potential in Corynebacterium glutamicum.

Redox Biol 2019 01 17;20:514-525. Epub 2018 Nov 17.

Freie Universität Berlin, Institute for Biology-Microbiology, D-14195 Berlin, Germany. Electronic address:

Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (E) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial E throughout the growth curve with basal E levels of ~- 296 mV. Consistent with its HO resistant phenotype, C. glutamicum responds only weakly to 40 mM HO, but is rapidly oxidized by low doses of NaOCl. We further monitored basal E changes and the HO response in various mutants which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal E was observed in the sigH mutant. The catalase KatA was confirmed as major HO detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on E and HO detoxification. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic E changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level E and the role of OxyR and KatA for efficient HO detoxification under oxidative stress.
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http://dx.doi.org/10.1016/j.redox.2018.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258114PMC
January 2019

3D organ models-Revolution in pharmacological research?

Pharmacol Res 2019 01 3;139:446-451. Epub 2018 Nov 3.

Freie Universität Berlin, Institute for Pharmacy, Pharmacology & Toxicology, Königin-Luise-Str. 2-4, Berlin, 14195, Germany. Electronic address:

3D organ models have gained increasing attention as novel preclinical test systems and alternatives to animal testing. Over the years, many excellent in vitro tissue models have been developed. In parallel, microfluidic organ-on-a-chip tissue cultures have gained increasing interest for their ability to house several organ models on a single device and interlink these within a human-like environment. In contrast to these advancements, the development of human disease models is still in its infancy. Although major advances have recently been made, efforts still need to be intensified. Human disease models have proven valuable for their ability to closely mimic disease patterns in vitro, permitting the study of pathophysiological features and new treatment options. Although animal studies remain the gold standard for preclinical testing, they have major drawbacks such as high cost and ongoing controversy over their predictive value for several human conditions. Moreover, there is growing political and social pressure to develop alternatives to animal models, clearly promoting the search for valid, cost-efficient and easy-to-handle systems lacking interspecies-related differences. In this review, we discuss the current state of the art regarding 3D organ as well as the opportunities, limitations and future implications of their use.
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http://dx.doi.org/10.1016/j.phrs.2018.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7129286PMC
January 2019

Generation of a 3D Liver Model Comprising Human Extracellular Matrix in an Alginate/Gelatin-Based Bioink by Extrusion Bioprinting for Infection and Transduction Studies.

Int J Mol Sci 2018 Oct 12;19(10). Epub 2018 Oct 12.

Institute of Biotechnology, Department of Applied Biochemistry, Technische Universität Berlin, 13355 Berlin, Germany.

Bioprinting is a novel technology that may help to overcome limitations associated with two-dimensional (2D) cell cultures and animal experiments, as it allows the production of three-dimensional (3D) tissue models composed of human cells. The present study describes the optimization of a bioink composed of alginate, gelatin and human extracellular matrix (hECM) to print human HepaRG liver cells with a pneumatic extrusion printer. The resulting tissue model was tested for its suitability for the study of transduction by an adeno-associated virus (AAV) vector and infection with human adenovirus 5 (hAdV5). We found supplementation of the basic alginate/gelatin bioink with 0.5 and 1 mg/mL hECM provides desirable properties for the printing process, the stability of the printed constructs, and the viability and metabolic functions of the printed HepaRG cells. The tissue models were efficiently transduced by AAV vectors of serotype 6, which successfully silenced an endogenous target (cyclophilin B) by means of RNA interference. Furthermore, the printed 3D model supported efficient adenoviral replication making it suitable to study virus biology and develop new antiviral compounds. We consider the approach described here paradigmatic for the development of 3D tissue models for studies including viral vectors and infectious viruses.
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http://dx.doi.org/10.3390/ijms19103129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213460PMC
October 2018

Optimization of cell-laden bioinks for 3D bioprinting and efficient infection with influenza A virus.

Sci Rep 2018 09 17;8(1):13877. Epub 2018 Sep 17.

Institute of Biotechnology, Department of Applied Biochemistry, Technische Universität Berlin, 13355, Berlin, Germany.

Bioprinting is a new technology, which arranges cells with high spatial resolution, but its potential to create models for viral infection studies has not yet been fully realized. The present study describes the optimization of a bioink composition for extrusion printing. The bioinks were biophysically characterized by rheological and electron micrographic measurements. Hydrogels consisting of alginate, gelatin and Matrigel were used to provide a scaffold for a 3D arrangement of human alveolar A549 cells. A blend containing 20% Matrigel provided the optimal conditions for spatial distribution and viability of the printed cells. Infection of the 3D model with a seasonal influenza A strain resulted in widespread distribution of the virus and a clustered infection pattern that is also observed in the natural lung but not in two-dimensional (2D) cell culture, which demonstrates the advantage of 3D printed constructs over conventional culture conditions. The bioink supported viral replication and proinflammatory interferon release of the infected cells. We consider our strategy to be paradigmatic for the generation of humanized 3D tissue models by bioprinting to study infections and develop new antiviral strategies.
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http://dx.doi.org/10.1038/s41598-018-31880-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141611PMC
September 2018

A novel European H5N8 influenza A virus has increased virulence in ducks but low zoonotic potential.

Emerg Microbes Infect 2018 Jul 19;7(1):132. Epub 2018 Jul 19.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.

We investigated in a unique setup of animal models and a human lung explant culture biological properties, including zoonotic potential, of a representative 2016 highly pathogenic avian influenza virus (HPAIV) H5N8, clade 2.3.4.4 group B (H5N8B), that spread rapidly in a huge and ongoing outbreak series in Europe and caused high mortality in waterfowl and domestic birds. HPAIV H5N8B showed increased virulence with rapid onset of severe disease and mortality in Pekin ducks due to pronounced neuro- and hepatotropism. Cross-species infection was evaluated in mice, ferrets, and in a human lung explant culture model. While the H5N8B isolate was highly virulent for Balb/c mice, virulence and transmissibility were grossly reduced in ferrets, which was mirrored by marginal replication in human lung cultures infected ex vivo. Our data indicate that the 2016 HPAIV H5N8B is avian-adapted with augmented virulence for waterfowl, but has low zoonotic potential. The here tested combination of animal studies with the inoculation of human explants provides a promising future workflow to evaluate zoonotic potential, mammalian replication competence and avian virulence of HPAIV.
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http://dx.doi.org/10.1038/s41426-018-0130-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053424PMC
July 2018

Long-Term Culture of Distal Airway Epithelial Cells Allows Differentiation Towards Alveolar Epithelial Cells Suited for Influenza Virus Studies.

EBioMedicine 2018 Jul 22;33:230-241. Epub 2018 Jun 22.

Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany. Electronic address:

As the target organ for numerous pathogens, the lung epithelium exerts critical functions in health and disease. However, research in this area has been hampered by the quiescence of the alveolar epithelium under standard culture conditions. Here, we used human distal airway epithelial cells (DAECs) to generate alveolar epithelial cells. Long-term, robust growth of human DAECs was achieved using co-culture with feeder cells and supplementation with epidermal growth factor (EGF), Rho-associated protein kinase inhibitor Y27632, and the Notch pathway inhibitor dibenzazepine (DBZ). Removal of feeders and priming with DBZ and a cocktail of lung maturation factors prevented the spontaneous differentiation into airway club cells and instead induced differentiation to alveolar epithelial cells. We successfully transferred this approach to chicken distal airway cells, thus generating a zoonotic infection model that enables studies on influenza A virus replication. These cells are also amenable for gene knockdown using RNAi technology, indicating the suitability of the model for mechanistic studies into lung function and disease.
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http://dx.doi.org/10.1016/j.ebiom.2018.05.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085545PMC
July 2018

Protein arginine methyltransferase 5 mediates enolase-1 cell surface trafficking in human lung adenocarcinoma cells.

Biochim Biophys Acta Mol Basis Dis 2018 May 2;1864(5 Pt A):1816-1827. Epub 2018 Mar 2.

Department of Biochemistry, Faculty of Medicine, Universities of Giessen and Marburg Lung Center, Friedrichstrasse 24, 35392 Giessen, Germany; Member of the German Center for Lung Research, Giessen, Germany.

Objectives: Enolase-1-dependent cell surface proteolysis plays an important role in cell invasion. Although enolase-1 (Eno-1), a glycolytic enzyme, has been found on the surface of various cells, the mechanism responsible for its exteriorization remains elusive. Here, we investigated the involvement of post-translational modifications (PTMs) of Eno-1 in its lipopolysaccharide (LPS)-triggered trafficking to the cell surface.

Results: We found that stimulation of human lung adenocarcinoma cells with LPS triggered the monomethylation of arginine 50 (R50me) within Eno-1. The Eno-1R50me was confirmed by its interaction with the tudor domain (TD) from TD-containing 3 (TDRD3) protein recognizing methylarginines. Substitution of R50 with lysine (R50K) reduced Eno-1 association with epithelial caveolar domains, thereby diminishing its exteriorization. Similar effects were observed when pharmacological inhibitors of arginine methyltransferases were applied. Protein arginine methyltransferase 5 (PRMT5) was identified to be responsible for Eno-1 methylation. Overexpression of PRMT5 and caveolin-1 enhanced levels of membrane-bound extracellular Eno-1 and, conversely, pharmacological inhibition of PRMT5 attenuated Eno-1 cell-surface localization. Importantly, Eno-1R50me was essential for cancer cell motility since the replacement of Eno-1 R50 by lysine or the suppression of PRMT 5 activity diminished Eno-1-triggered cell invasion.

Conclusions: LPS-triggered Eno-1R50me enhances Eno-1 cell surface levels and thus potentiates the invasive properties of cancer cells. Strategies to target Eno-1R50me may offer novel therapeutic approaches to attenuate tumor metastasis in cancer patients.
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http://dx.doi.org/10.1016/j.bbadis.2018.02.021DOI Listing
May 2018

Digital Image Analyses on Whole-Lung Slides in Mouse Models of Acute Pneumonia.

Am J Respir Cell Mol Biol 2018 04;58(4):440-448

1 Department of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany.

Descriptive histopathology of mouse models of pneumonia is essential in assessing the outcome of infections, molecular manipulations, or therapies in the context of whole lungs. Quantitative comparisons between experimental groups, however, have been limited to laborious stereology or ill-defined scoring systems that depend on the subjectivity of a more or less experienced observer. Here, we introduce self-learning digital image analyses that allow us to transform optical information from whole mouse lung sections into statistically testable data. A pattern-recognition-based software and a nuclear count algorithm were adopted to quantify user-defined pathologies from whole slide scans of lungs infected with Streptococcus pneumoniae or influenza A virus compared with PBS-challenged lungs. The readout parameters "relative area affected" and "nuclear counts per area" are proposed as relevant criteria for the quantification of lesions from hematoxylin and eosin-stained sections, also allowing for the generation of a heat map of, for example, immune cell infiltrates with anatomical assignments across entire lung sections. Moreover, when combined with immunohistochemical labeling of marker proteins, both approaches are useful for the identification and counting of, for example, immune cell populations, as validated here by direct comparisons with flow cytometry data. The solutions can easily and flexibly be adjusted to specificities of different models or pathogens. Automated digital analyses of whole mouse lung sections may set a new standard for the user-defined, high-throughput comparative quantification of histological and immunohistochemical images. Still, our algorithms established here are only a start, and need to be tested in additional studies and other applications in the future.
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http://dx.doi.org/10.1165/rcmb.2017-0337MADOI Listing
April 2018

A comparative analysis of human bone marrow-resident and peripheral memory B cells.

J Allergy Clin Immunol 2018 05 31;141(5):1911-1913.e7. Epub 2018 Jan 31.

Experimental and Clinical Research Center, Berlin, Germany; Department of Hematology, Oncology and Tumor Immunology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany; Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany; Berlin Institute of Health, Berlin, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2017.12.983DOI Listing
May 2018

Pneumolysin induced mitochondrial dysfunction leads to release of mitochondrial DNA.

Sci Rep 2018 01 9;8(1):182. Epub 2018 Jan 9.

Department of Internal Medicine/Infectious Diseases and Respiratory Medicine, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.

Streptococcus pneumoniae (S.pn.) is the most common bacterial pathogen causing community acquired pneumonia. The pore-forming toxin pneumolysin (PLY) is the major virulence factor of S.pn. and supposed to affect alveolar epithelial cells thereby activating the immune system by liberation of danger-associated molecular patterns (DAMP). To test this hypothesis, we established a novel live-cell imaging based assay to analyse mitochondrial function and associated release of mitochondrial DNA (mtDNA) as DAMP in real-time. We first revealed that bacterially released PLY caused significant changes of the cellular ATP homeostasis and led to morphologic alterations of mitochondria in human alveolar epithelial cells in vitro and, by use of spectral live-tissue imaging, in human alveoli. This was accompanied by strong mitochondrial calcium influx and loss of mitochondrial membrane potential resulting in opening of the mitochondrial permeability transition pore and mtDNA release without activation of intrinsic apoptosis. Moreover, our data indicate cellular mtDNA liberation via microvesicles, which may contribute to S.pn. related pro-inflammatory immune activation in the human alveolar compartment.
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http://dx.doi.org/10.1038/s41598-017-18468-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760655PMC
January 2018

Sphingosine Kinase 1 Regulates Inflammation and Contributes to Acute Lung Injury in Pneumococcal Pneumonia via the Sphingosine-1-Phosphate Receptor 2.

Crit Care Med 2018 03;46(3):e258-e267

Department of Infectious Diseases and Pulmonary Medicine, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Objectives: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia.

Design: Controlled, in vitro, ex vivo, and in vivo laboratory study.

Subjects: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells.

Interventions: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin.

Measurements And Main Results: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase.

Conclusions: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.
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http://dx.doi.org/10.1097/CCM.0000000000002916DOI Listing
March 2018

Spectrum of pathogen- and model-specific histopathologies in mouse models of acute pneumonia.

PLoS One 2017 20;12(11):e0188251. Epub 2017 Nov 20.

Department of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany.

Pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. Murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. Despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. Here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by Streptococcus (S.) pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Legionella pneumophila, Escherichia coli, Middle East respiratory syndrome (MERS) coronavirus, influenza A virus (IAV) and superinfection of IAV-incuced pneumonia with S. pneumoniae. Systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. We therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogen- and model-specific patterns of pneumonia. Other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. The substantial differences between the model-specific pathologies underscore the necessity of pathogen- and model-adapted criteria for the comparative quantification of experimental outcomes. These criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188251PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695780PMC
December 2017

Human Pulmonary 3D Models For Translational Research.

Biotechnol J 2018 Jan 20;13(1). Epub 2017 Sep 20.

Dept. of Internal Medicine/Infectious and Respiratory Diseases, Charité - Universitätsmedizin Berlin, Charitèplatz 1, Berlin 10117, Germany.

Lung diseases belong to the major causes of death worldwide. Recent innovative methodological developments now allow more and more for the use of primary human tissue and cells to model such diseases. In this regard, the review covers bronchial air-liquid interface cultures, precision cut lung slices as well as ex vivo cultures of explanted peripheral lung tissue and de-/re-cellularization models. Diseases such as asthma or infections are discussed and an outlook on further areas for development is given. Overall, the progress in ex vivo modeling by using primary human material could make translational research activities more efficient by simultaneously fostering the mechanistic understanding of human lung diseases while reducing animal usage in biomedical research.
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http://dx.doi.org/10.1002/biot.201700341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161817PMC
January 2018

Tyk2 as a target for immune regulation in human viral/bacterial pneumonia.

Eur Respir J 2017 07 13;50(1). Epub 2017 Jul 13.

Dept of Internal Medicine/Infectious Diseases and Respiratory Medicine, Charité - Universitätsmedizin Berlin, Berlin, Germany

The severity and lethality of influenza A virus (IAV) infections is frequently aggravated by secondary bacterial pneumonia. However, the mechanisms in human lung tissue that provoke this increase in fatality are unknown and therapeutic immune modulatory options are lacking.We established a human lung co-infection model to investigate innate immune related mechanisms contributing to the susceptibility of secondary pneumococcal pneumonia.We revealed that type I and III interferon (IFN) inhibits -induced interleukin (IL)-1β release. The lack of IL-1β resulted in the repression of bacterially induced granulocyte-macrophage colony-stimulating factor (GM-CSF) liberation. Specific inhibition of IFN receptor I and III-associated tyrosine kinase 2 (Tyk2) completely restored the -induced IL-1β-GM-CSF axis, leading to a reduction of bacterial growth. A preceding IAV infection of the human alveolus leads to a type I and III IFN-dependent blockade of the early cytokines IL-1β and GM-CSF, which are key for orchestrating an adequate innate immune response against bacteria. Their virally induced suppression may result in impaired bacterial clearance and alveolar repair.Pharmacological inhibition of Tyk2 might be a new treatment option to sustain beneficial endogenous GM-CSF levels in IAV-associated secondary bacterial pneumonia.
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http://dx.doi.org/10.1183/13993003.01953-2016DOI Listing
July 2017