Gewinn mit nachhaltigen Produkten

Mehr Umsatz mit nachhaltigen Produkten ist auch in Zeiten eines harten Verdrängungswettbewerbs im Einzelhandel möglich. Leitsysteme am Point of Sale sind ein wirksames Instrument, um dieses Ziel zu erreichen. So lautet die Quintessenz eines von der Verbraucher Initiative durchgeführten Projekts

In vitro Rekonstruktion der neurovaskulären Einheiten des Gehirns und der Retina auf einem Organ-on-a-chip System zur Modellierung degenerativer Erkrankungen

Die Zahl degenerativer Erkrankungen wie Parkinson oder diabetische Retinopathie steigt weltweit. Trotz der drastischen Zunahme sind diese Erkrankungen bis heute nicht heilbar. Infolgedessen ist die Entwicklung neuer Behandlungsmethoden und potentieller Wirkstoffe zur Therapie der Erkrankungen von enormer Bedeutung. Dabei gestaltet sich die Validierung der Wirksamkeit dieser Wirkstoffe sowie eine direkte Erforschung im Patienten schwierig. Folglich werden Tierversuche herangezogen, die jedoch aufgrund speziesspezifischer Unterschiede häufig nicht auf den Menschen übertragbar und ethisch schwer vertretbar sind. Infolgedessen beschäftigt sich das Tissue Engineering mit der Entwicklung von in vitro Modellsystemen, welche menschliches Gewebe oder Organe so exakt wie möglich abbilden sollen. Besonders im Fokus stehen dabei Organ-on-a-chip Systeme. Dies sind mikrofluidische funktionelle Modellsysteme, die zur Nachbildung von miniaturisierten Organsystemen und Krankheitsbildern eingesetzt werden können. Solche Modelle auf Basis humaner Zellen können zukünftig zur spezifischeren Wirkstofftestung sowie zur Entwicklung neuer Therapiemöglichkeiten beitragen. Das Ziel der vorliegenden Arbeit war die in vitro Rekonstruktion der retinalen neurovaskulären Einheit (rNVU) im Auge sowie der neurovaskulären Einheit (NVU) im Gehirn im Organ on a chip System vasQchip. Der vasQchip besteht aus einem porösen halbrunden Mikrokanal, welcher zur Nachbildung eines Blutgefäßes mit Endothelzellen ausgekleidet und zur Erzeugung eines artifiziellen Blutflusses an eine Mikrofluidik angeschlossen werden kann. Das umliegende Kompartiment ermöglicht die Rekonstruktion von vaskularisierten 3D-Gewebe. Um die physiologischen Besonderheiten der beiden vaskulären Einheiten zu berücksichtigen, war die Etablierung einer dichten Barriere der Blut-Hirn-Schranke (BBB) in der NVU sowie der inneren Blut-Retina-Schranke (iBRB) in der rNVU maßgeblich. Die iBRB und BBB gewährleisten einen geregelten Stoffaustausch zwischen Retina beziehungsweise Gehirn und dem Blutsystem. Der dichte Barrierecharakter wird unter anderem durch Tight Junctions zwischen benachbarten Endothelzellen und durch spezifische Transporter wie den Efflux Pumpen sichergestellt. Zur Etablierung eines dichten iBRB-Endothels im vasQchip wurden geeignete Kultivierungsbedingungen im Mikrokanal untersucht und optimiert. Durch das Anlegen eines Flusses und den daraus resultierenden Scherkräfte konnte ein dichtes iBRB-Endothel im Mikrokanal des vasQchips erzielt werden. Auch weitere Zelltypen der rNVU wie Perizyten und Astrozyten tragen zur Ausbildung und Aufrechterhaltung der iBRB bei. So konnte nach Validierung einer Kokultur der drei Zelltypen im vasQchip eine erhöhte Expression spezifischer Barriere-Marker sowie die Funktionalität einer der wichtigsten Gatekeeper der Barrierefunktion, die Efflux Pumpe P gp, festgestellt werden. Auch erste Ansätze zur dreidimensionalen Kultivierung von Astrozyten in Gerüststrukturen wie natürlichen Hydrogelen auf Kollagenbasis und semi synthetischen Hydrogelen (GelMA), welche die extrazelluläre Matrix (EZM) imitieren, wurden erzielt. Hierbei wurden Hydrogele verwendet, die der Festigkeit von retinalem Gewebe entsprechen. Im letzten Schritt wurde das iBRB Modell inklusive Perizyten und Astrozyten so verändert, dass es das Krankheitsbild der diabetischen Retinopathie abbilden konnte. So konnte ein Funktionsverlust der iBRB durch eine verminderte Expression wichtiger Barriere Marker sowie eine höhere parazelluläre Permeabilität erzielt werden. Durch zusätzliche Behandlung mit einem in der Literatur bewährten potentiellen Wirkstoff konnte die Barrierefunktion der iBRB zwar erhöht, aber nicht zuverlässig regeneriert werden. Im Vergleich zur iBRB/rNVU gibt es zahlreiche BBB/NVU-Modelle, welchen es jedoch häufig an einigen Zelltypen der NVU fehlt. Neben Perizyten, Astrozyten, Mikroglia und Endothelzellen wurde das NVU-Modell im Rahmen dieser Arbeit ebenfalls aus Neuronen aufgebaut. Diese wurden aus humanen induzierten pluripotenten Stammzellen (iPSC) differenziert und hinsichtlich ihrer Morphologie, Expression zelltypischer Marker und Aktivität evaluiert. Nach einer Untersuchung von geeigneten Kultivierungsbedingungen konnte ein Kokultur-Modell der fünf Zelltypen einschließlich Neuronen im vasQchip etabliert und validiert werden. Auch die in der Literatur beschriebenen positiven Einflüsse der Zellen auf die Barrierefunktion im Endothel konnte mithilfe von RT qPCR Analysen und TEER-Messungen sowie einem Funktionalitätstest der Efflux Pumpe BCRP bestätigt werden. Zur Entwicklung eines 3D-Gewebemodells wurden Neuronen zur Ausdifferenzierung in natürliche Hydrogele auf Kollagenbasis und semi synthetische Hydrogele (GelNB/GelS) eingebracht, welche in ihrer Festigkeit dem Hirngewebe ähneln. So wirkte sich zwar die Einbettung der Neuronen auf die Viabilität der Zellen negativ aus, dennoch konnte im natürlichen Hydrogel ein dichtes neuronales Netzwerk erzeugt werden. Diese dreidimensionalen Netzwerke wurden anschließend in das vollständige NVU Modell im vasQchip integriert und fluidisch langzeitkultiviert. Auch mithilfe des 3D Bioprintings wurden erstmals iPSC-differenzierte Neuronen trotz mangelnder Ausrichtung im druckbaren GelNB/GelS auf dem vasQchip extrudiert und fluidisch kultiviert. Im letzten Schritt wurden erste Ansätze zur Abbildung des Krankheitsbilds von Parkinson in Form einer Neuroinflammation erzielt. So konnte eine Aktivierung der Mikroglia sowie ein Funktionsverlust der BBB nachgewiesen werden. Darüber hinaus wurde mithilfe von Wirkstoffen zur Therapie von Parkinson und neuartigen Drug Delivery-Systemen die Eignung des vasQchips als Testmodell für therapeutische Wirkstoffe überprüft. Anhand der vorliegenden Arbeit wurden wichtige Erkenntnisse zur Krankheitsmodellierung und Wirkstofftestung erzielt, die als Fundament zur Entwicklung einsatzfähiger in vitro Organsysteme fungieren. Durch Kombination verschiedener Technologien des Tissue Engineerings wie 3D Bioprinting und Stammzellsystemen können langfristig exakte Nachbildungen von Organen erzielt werden und damit zur Behandlung von degenerativen Erkrankungen beitragen

Higher polymerase activity of a human influenza virus enhances activation of the hemagglutinin-induced Raf/MEK/ERK signal cascade

Influenza viruses replicate within the nucleus of infected cells. Viral genomic RNA, three polymerase subunits (PB2, PB1, and PA), and the nucleoprotein (NP) form ribonucleoprotein complexes (RNPs) that are exported from the nucleus late during the infectious cycle. The virus-induced Raf/MEK/ERK (MAPK) signal cascade is crucial for efficient virus replication. Blockade of this pathway retards RNP export and reduces virus titers. Hemagglutinin (HA) accumulation and its tight association with lipid rafts activate ERK and enhance localization of cytoplasmic RNPs. We studied the induction of MAPK signal cascade by two seasonal human influenza A viruses A/HK/218449/06 (H3N2) and A/HK/218847/06 (H1N1) that differed substantially in their replication efficiency in tissue culture. Infection with H3N2 virus, which replicates efficiently, resulted in higher HA expression and its accumulation on the cell membrane, leading to substantially increased activation of MAPK signaling compared to that caused by H1N1 subtype. More H3N2-HAs were expressed and accumulated on the cell membrane than did H1N1-HAs. Viral polymerase genes, particularly H3N2-PB1 and H3N2-PB2, were observed to contribute to increased viral polymerase activity. Applying plasmid-based reverse genetics to analyze the role of PB1 protein in activating HA-induced MAPK cascade showed that recombinant H1N1 virus possessing the H3N2-PB1 (rgH1N1/H3N2-PB1) induced greater ERK activation, resulting in increased nuclear export of the viral genome and higr virus titers. We conclude that enhanced viral polymerase activity promotes the replication and transcription of viral RNA leading to increased accumulation of HA on the cell surface and thereby resulting in an upregulation of the MAPK cascade and more efficient nuclear RNP-export as well as virus production

Lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed TNF-related apoptosis-inducing ligand

Mononuclear phagocytes have been attributed a crucial role in the host defense toward influenza virus (IV), but their contribution to influenza-induced lung failure is incompletely understood. We demonstrate for the first time that lung-recruited “exudate” macrophages significantly contribute to alveolar epithelial cell (AEC) apoptosis by the release of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a murine model of influenza-induced pneumonia. Using CC-chemokine receptor 2–deficient (CCR2−/−) mice characterized by defective inflammatory macrophage recruitment, and blocking anti-CCR2 antibodies, we show that exudate macrophage accumulation in the lungs of influenza-infected mice is associated with pronounced AEC apoptosis and increased lung leakage and mortality. Among several proapoptotic mediators analyzed, TRAIL messenger RNA was found to be markedly up-regulated in alveolar exudate macrophages as compared with peripheral blood monocytes. Moreover, among the different alveolar-recruited leukocyte subsets, TRAIL protein was predominantly expressed on macrophages. Finally, abrogation of TRAIL signaling in exudate macrophages resulted in significantly reduced AEC apoptosis, attenuated lung leakage, and increased survival upon IV infection. Collectively, these findings demonstrate a key role for exudate macrophages in the induction of alveolar leakage and mortality in IV pneumonia. Epithelial cell apoptosis induced by TRAIL-expressing macrophages is identified as a major underlying mechanism

Shrews as Reservoir Hosts of Borna Disease Virus

Borna disease virus (BDV) is the causative agent of severe T-cell–mediated meningoencephalitis in horses, sheep, and other animal species in central Europe. Here we report the first unequivocal detection of a BDV reservoir species, the bicolored white-toothed shrew, Crocidura leucodon, in an area in Switzerland with endemic Borna disease

A Novel Antiviral Strategy against MERS-CoV and HCoV-229E Using Binase to Target Viral Genome Replication

© 2016, Springer Science+Business Media New York.RNA viruses cause most of the dangerous communicable diseases. Due to their high mutation rates, RNA viruses quickly evade selective pressures and can adapt to a new host. Therefore, new antiviral approaches are urgently needed, which target more than one specific virus variant and which would optimally prevent development of viral resistance. Among the family of coronaviruses (CoV), several human pathogenic strains (HCoV) are known to cause respiratory diseases and are implied in enteric diseases. While most strains contribute to common cold-like illnesses, others lead to severe infections. One of these viruses is the newly emerged (2012), highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) of zoonotic origin. MERS-CoV causes a severe respiratory infection with a high mortality rate of 35 %. There is no specific treatment or infection prevention available. Here, we show that the bacterial ribonuclease Binase is able to inhibit the replication of MERS-CoV and of the low-pathogenic human coronavirus 229E (HCoV-229E) in cell culture. We demonstrate that at non-toxic concentrations, Binase decreased the titers of MERS-CoV and HCoV-229E. On a molecular level, Binase treatment reduced (i) the viral subgenomic RNAs and (ii) the viral nucleocapsidprotein (N) and non-structural protein 13 (nsp13) accumulation. Furthermore, we show that the quantity of the replication/transcription complexes within the infected cells is diminished. Thus, the data obtained might allow further development of new anti-coronaviral approaches affecting viral replication, independent of the specific virus strain

Inhibitory activity of a standardized elderberry liquid extract against clinically-relevant human respiratory bacterial pathogens and influenza A and B viruses

<p>Abstract</p> <p>Background</p> <p>Black elderberries (<it>Sambucus nigra </it>L.) are well known as supportive agents against common cold and influenza. It is further known that bacterial super-infection during an influenza virus (IV) infection can lead to severe pneumonia. We have analyzed a standardized elderberry extract (Rubini, BerryPharma AG) for its antimicrobial and antiviral activity using the microtitre broth micro-dilution assay against three Gram-positive bacteria and one Gram-negative bacteria responsible for infections of the upper respiratory tract, as well as cell culture experiments for two different strains of influenza virus.</p> <p>Methods</p> <p>The antimicrobial activity of the elderberry extract was determined by bacterial growth experiments in liquid cultures using the extract at concentrations of 5%, 10%, 15% and 20%. The inhibitory effects were determined by plating the bacteria on agar plates. In addition, the inhibitory potential of the extract on the propagation of human pathogenic H5N1-type influenza A virus isolated from a patient and an influenza B virus strain was investigated using MTT and focus assays.</p> <p>Results</p> <p>For the first time, it was shown that a standardized elderberry liquid extract possesses antimicrobial activity against both Gram-positive bacteria of <it>Streptococcus pyogenes </it>and group C and G <it>Streptococci</it>, and the Gram-negative bacterium <it>Branhamella catarrhalis </it>in liquid cultures. The liquid extract also displays an inhibitory effect on the propagation of human pathogenic influenza viruses.</p> <p>Conclusion</p> <p>Rubini elderberry liquid extract is active against human pathogenic bacteria as well as influenza viruses. The activities shown suggest that additional and alternative approaches to combat infections might be provided by this natural product.</p

Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

Copyright © 2012, American Society for Microbiology. All Rights ReservedThe influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.This work was supported by the Research Fund for the Control of Infectious Disease (project 09080892) of the Hong Kong Government, the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, the RESPARI Pasteur Network

Macrophage-expressed IFN-β contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia

Influenza viruses (IV) cause pneumonia in humans with progression to lung failure and fatal outcome. Dysregulated release of cytokines including type I interferons (IFNs) has been attributed a crucial role in immune-mediated pulmonary injury during severe IV infection. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage (AM)-expressed IFN-β significantly contributes to IV-induced alveolar epithelial cell (AEC) injury by autocrine induction of the pro-apoptotic factor TNF-related apoptosis- inducing ligand (TRAIL). Of note, TRAIL was highly upregulated in and released from AM of patients with pandemic H1N1 IV-induced acute lung injury. Elucidating the cell-specific underlying signalling pathways revealed that IV infection induced IFN-β release in AM in a protein kinase R- (PKR-) and NF-κB- dependent way. Bone marrow chimeric mice lacking these signalling mediators in resident and lung-recruited AM and mice subjected to alveolar neutralization of IFN-β and TRAIL displayed reduced alveolar epithelial cell apoptosis and attenuated lung injury during severe IV pneumonia. Together, we demonstrate that macrophage-released type I IFNs, apart from their well-known anti-viral properties, contribute to IV-induced AEC damage and lung injury by autocrine induction of the pro-apoptotic factor TRAIL. Our data suggest that therapeutic targeting of the macrophage IFN-β-TRAIL axis might represent a promising strategy to attenuate IV-induced acute lung injury

Ribonuclease from bacillus acts as an antiviral agent against negative- and positive-sense single stranded human respiratory RNA viruses

Copyright © 2017 Raihan Shah Mahmud et al.Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses