TY - JOUR A1 - Shityakov, Sergey A1 - Salvador, Ellaine A1 - Pastorin, Giorgia A1 - Förster, Carola T1 - Blood-brain barrier transport studies, aggregation, and molecular dynamics simulation of multiwalled carbon nanotube functionalized with fluorescein isothiocyanate JF - International Journal of Nanomedicine N2 - In this study, the ability of a multiwalled carbon nanotube functionalized with fluorescein isothiocyanate (MWCNT-FITC) was assessed as a prospective central nervous system-targeting drug delivery system to permeate the blood-brain barrier. The results indicated that the MWCNT-FITC conjugate is able to penetrate microvascular cerebral endothelial monolayers; its concentrations in the Transwell® system were fully equilibrated after 48 hours. Cell viability test, together with phase-contrast and fluorescence microscopies, did not detect any signs of MWCNT-FITC toxicity on the cerebral endothelial cells. These microscopic techniques also revealed presumably the intracellular localization of fluorescent MWCNT-FITCs apart from their massive nonfluorescent accumulation on the cellular surface due to nanotube lipophilic properties. In addition, the 1,000 ps molecular dynamics simulation in vacuo discovered the phenomenon of carbon nanotube aggregation driven by van der Waals forces via MWCN-TFITC rapid dissociation as an intermediate phase. KW - endothelial cells KW - cytotoxicity KW - blood-brain barrier KW - fluorescein isothiocyanate KW - aggregation KW - molecular dynamics KW - fluorescence microscopy KW - Transwell® system KW - multiwalled carbon nanotube KW - mice Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149233 VL - 10 ER - TY - THES A1 - Seystahl, Katharina Gertrud T1 - Einfluss von Dasatinib auf die Expansion, Zytotoxizität und Zytokinproduktion von humanen Natürlichen Killer-Zellen T1 - The impact of dasatinib on expansion, cytotoxicity and cytokine production of human Natural Killer cells N2 - NK-Zellen spielen eine wichtige Rolle im menschlichen Immunsystem, insbesondere durch die Zerstörung von virusinfizierten Zellen und Tumorzellen sowie durch die Produktion von Zytokinen. Eine gezielte Modulation der Effektorfunktionen von NK-Zellen kann den Weg für neue Therapiestrategien gegenüber malignen Erkrankungen oder auch Autoimmunerkrankungen bahnen. Dasatinib ist ein potenter Inhibitor einer Vielzahl von Kinasen, die an der Regulation von NK-Zelleffektorfunktionen beteiligt sind und für die bereits eine Inhibition von T-Zelleffektorfunktionen gezeigt werden konnte [Schade et al. 2008; Weichsel et al. 2008]. Ein besseres Verständnis der immunmodulatorischen Eigenschaften von Dasatinib kann nicht nur neue Einsatzbereiche identifizieren, sondern auch die bereits bewährte Therapie der CML optimieren. Daher wurde im Rahmen dieser Arbeit der Einfluss von Dasatinib auf die Expansion, Zytotoxizität und Zytokinproduktion von humanen NK-Zellen analysiert. Dazu wurden aus peripheren Blutlymphozyten gesunder Spender polyklonale NK-Zellen in Kokultur mit bestrahlten RPMI 8866-Zellen mit und ohne Dasatinib expandiert und NK-Zelleffektorfunktionen mit Durchfluszytometrie-basierten Experimenten untersucht. Im Detail wurde die Zytotoxizität nach dem Prinzip des FATAL-Experiments [Sheehy et al. 2001], die Degranulationsaktivität über die Expression von CD107a/b, die Produktion von TNF-α bzw. IFN-γ mit einer intrazellulären Färbung und die Apoptose- und Zelltodanalyse über Annexin-V und 7-AAD gemessen. Die Daten dieser Arbeit zeigen, dass Dasatinib die Haupteffektorfunktionen von NK-Zellen gesunder Blutspender in vitro reguliert: Die Expansionskapazität von NK-Zellen wird dosisabhängig und bei 50 nM Dasatinib vollständig inhibiert, ohne dass dies durch ein Absterben der NK-Zellen bedingt ist. Die Zytotoxizität von NK-Zellen, die unter 10 nM Dasatinib expandiert sind, ist nach Entfernen des Medikamentes restauriert, und die Degranulationskapazität und die Zytokinproduktion sind gesteigert. Bei unbehandelt expandierten NK-Zellen führt die direkte Anwesenheit von Dasatinib zu einer dosisabhängigen Hemmung der Zytotoxizität gegenüber K562-Zellen. Darüber hinaus inhibiert Dasatinib dosisabhängig die Degranulation und Zytokinproduktion von NK-Zellen bei einer Stimulation mit K562-Zellen nicht aber bei einer Stimulation mit PMA/Ca2+Ionophor. Eine indirekte Veränderung des Lyseverhaltens der NK-Zellen durch Effekte von Dasatinib auf die K562-Zellen zeigt sich nicht nach 4h, aber nach 24h im Sinne einer erhöhten Spontanlyse, aber geringeren spezifischen Lyse. Eine 24h-Vorbehandlung von K562-Zellen mit Dasatinib führt außerdem zu einer verminderten Degranulationsaktivität und Zytokinproduktion von unbehandelten NK-Zellen. Die Hemmung der NK-Zelleffektorfunktionen bei direkter Anwesenheit von Dasatinib und deren Restauration respektive Steigerung nach Entfernen des Medikaments ist am ehesten auf eine reversible Inhibition von Src-Kinase-abhängigen Prozessen der intrazellulären Signalübertragung zurückzuführen. Eine kompromittierte NK-Zellfunktion könnte während einer Behandlung mit Dasatinib zu einer Verminderung der Infektabwehr und der immunologischen Tumorüberwachung führen. Möglicherweise lassen sich jedoch die unerwünschten Wirkungen durch ein verändertes Dosisregime, wie eine Hochdosispulstherapie, bei guter Therapieeffizienz minimieren. Eine supprimierte Aktivität der NK-Zellen durch Dasatinib könnte dagegen bei der Therapie von NK-Zelllymphomen oder auch von Autoimmunerkrankungen eine neue Behandlungsoption darstellen. Aufgrund der bereits bekannten inhibitorischen Wirkung auf T-Zellfunktionen gibt es dabei möglicherweise Synergien in der immunsuppressiven Wirkung. Das immunmodulatorische Potential von Dasatinib birgt daher große Chancen sowohl im Einsatz als Immunsuppressivum, als auch in der Optimierung der bereits bewährten Therapie der CML. N2 - NK cells play an important role in the human immune system, especially by the lysis of virally infected cells and tumor cells, but also by the production of cytokines. Modulating NK cell effector functions may help to identify new strategies in the therapy of cancer or autoimmune diseases. Dasatinib is a potent inhibitor of multiple kinases regulating NK cell effector functions and the drug was already shown to inhibit T cell effector functions. A better understanding of the modulatory effect of dasatinib on the immune system may not only identify new applications of the drug but may also help to improve the current therapy of chronic myelogenous leukemia. Thus, the impact of dasatinib on the expansion, cytotoxicity and cytokine production of human NK cells was analyzed in this thesis. NK cells from healthy human blood donors were expanded by co-culturing irradiated RPMI 8866 cells with and without dasatinib. NK cell effector functions have been examined by flow cytometry. Cytotoxicity was analyzed by a FATAL-based experiment, degranulation activity by CD107a/b expression, TNF-α and IFN-γ production by intracellular staining and viability by Annexin V and 7-AAD. The data of this work show that dasatinib regulates the main NK cell effector functions of healthy blood donors in vitro. Expansion of NK cells is dose-dependently inhibited, including a complete inhibition at 50 nM dasatinib, which is not due to a decreased viability of NK cells. After removing the drug, cytotoxicity of NK cells being expanded at 10 nM dasatinib is restored while degranulation and cytokine production are increased. When no drug is present during expansion, dasatinib inhibits NK cell cytotoxicity against K562 cells in a dose-dependent manner. Furthermore, dasatinib leads to a dose-dependent inhibition of degranulation and cytokine production of NK cells after stimulation by K562 cells but not after stimulation by PMA/Ca2+Ionophor. Indirect effects of dasatinib on NK cell cytotoxic activity by an impairment of K562 cells is not detected after 4h, but after 24h showing an increased spontaneous lysis but a decreased specific lysis. 24h-pretreating of K562 cells with dasatinib decreases degranulation and cytokine production of untreated NK cells. The inhibition of NK cell effector functions by direct presence of dasatinib and their restoration or enhancement after removing the drug are most likely due to a reversible inhibition of SRC-kinase dependent processes during intracellular signal transduction. An impaired NK cell function during the treatment with dasatinib might alter immune defense or tumor immunosurveillance. However, adverse effects could also be reduced by a high-dose pulse therapy with an equal anti-tumor efficacy. Suppressing NK cell activity by dasatinib might also be helpful in the therapy of NK cell lymphomas or autoimmune diseases. As an inhibitory effect was already shown on T cell functions, there might be a synergistic action regarding the immunosuppressive effect. The potential of dasatinib is promising not only as an immunosuppressant but also by improving the current therapy of chronic myelogenous leukemia. KW - Natürliche Killerzelle KW - Protein-Tyrosin-Kinasen KW - Cytotoxizität KW - dasatinib KW - NK cells KW - dasatinib KW - cytotoxicity Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-51843 ER - TY - JOUR A1 - Rasche, Leo A1 - Duell, Johannes A1 - Morgner, Charlotte A1 - Chatterjee, Manik A1 - Hensel, Frank A1 - Rosenwald, Andreas A1 - Einsele, Hermann A1 - Topp, Max S. A1 - Brändlein, Stephanie T1 - The Natural Human IgM Antibody PAT-SM6 Induces Apoptosis in Primary Human Multiple Myeloma Cells by Targeting Heat Shock Protein GRP78 JF - PLoS ONE N2 - In contrast to other haematological malignancies, targeted immunotherapy has not entered standard treatment regimens for de novo or relapsed multiple myeloma (MM) yet. While a number of IgG-formatted monoclonal antibodies are currently being evaluated in clinical trials in MM, our study aimed to investigate whether the fully human IgM monoclonal antibody PAT-SM6 that targets a tumour-specific variant of the heat shock protein GRP78 might be an attractive candidate for future immunotherapeutic approaches. We here show that GRP78 is stably and consistently expressed on the surface on tumour cells from patients with de novo, but also relapsed MM and that binding of PAT-SM6 to MM cells can specifically exert cytotoxic effects on malignant plasma cells, whereas non-malignant cells are not targeted. We demonstrate that the induction of apoptosis and, to a lesser extent, complement dependent cytotoxicity is the main mode of action of PAT-SM6, whereas antibody dependent cellular cytotoxicity does not appear to contribute to the cytotoxic properties of this antibody. Given the favourable safety profile of PAT-SM6 in monkeys, but also in a recent phase I trial in patients with malignant melanoma, our results form the basis for a planned phase I study in patients with relapsed MM. KW - cytotoxicity KW - apoptosis KW - immunohistochemistry techniques KW - enzyme-linked immunoassays KW - multiple myeloma KW - cell staining KW - cell binding KW - complement system Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130125 VL - 8 IS - 5 ER - TY - THES A1 - Glückermann, Susanne Karola T1 - Zytotoxizitätsprüfung verschiedener Silbertitanlegierungen auf Titanbasis mittels humaner Osteoblasten T1 - Cytotoxicity of silver-titanium-alloy on titan grade 2 with human osteoblasts N2 - In der vorliegenden Arbeit wurde die Zytotoxizität verschiedener Schichten bestehend aus Silber-Titanlegierungen auf Titanbasis mittels der humanen Osteoblastenzelllinie hFOB 1.19 geprüft. Es sollte der Einfluß des Silberanteils in der Beschichtung auf die Zellen getestet werden. Es wurde die Wirkung auf die Proliferations- und Differenzierungsleistung der Zellen mit standardisierten Untersuchungsmethoden getestet. Des weiteren wurde die Aktivität der alkalischen Phosphatase und die Biomassebestimmung vorgenommen.Als Kontrolluntersuchung wurde der gleiche Versuch mit der bronchialen Epithelzelllinie HBE 16 durchgeführt. Die Zellkultivierung erfolgte über einen 14-tägigen Zeitraum. Als Referenzoberfläche wurde konventionelles Zellkultur-Polystyrol verwendet. Die Zellvitalität wurde mit Hilfe des WST-1-Tests, der Differenzierungstatus anhand der Aktivität der alkalischen Phosphatase und der Proteingehalt mittels der Proteinbestimmung nach Bradford erfaßt. Es zeigten sich bei allen Messungen starke Schwankungen der Zellzahl, Zellvitalität, der spezifischen Aktivität der alkalischen Phosphatase und des Proteingehalts auf den Oberflächen. Eine Proportionalität zwischen den verschiedenen Silberkonzentrationen und den Proliferationszahlen war nicht zu beobachten. Mit dem Wissen über die hervorragende Biokompatibilität von Titan und der nachgewiesenen bakteriostatischen Wirkung von Silber ist dies ein hervorragender Werkstoff , welcher schädliche Bakterien um das Implantat herum eliminiert und trotzdem ein ungehindertes Einwachsen des Implantats in den Knochen erlaubt. KW - Silber-Titan-Legierung KW - Osteoblasten KW - Zytotoxizitätsprüfung KW - bronchiale Epithelzellen HBE 16 KW - PVD-Verfahren KW - silver-titanium-alloy KW - osteoblasts KW - cytotoxicity KW - human respiratory epithelial cells Y1 - 2004 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-11021 ER - TY - JOUR A1 - Gordon, Sarah A1 - Daneshian, Mardas A1 - Bouwstra, Joke A1 - Caloni, Francesca A1 - Constant, Samuel A1 - Davies, Donna E. A1 - Dandekar, Gudrun A1 - Guzman, Carlos A. A1 - Fabian, Eric A1 - Haltner, Eleonore A1 - Hartung, Thomas A1 - Hasiwa, Nina A1 - Hayden, Patrick A1 - Kandarova, Helena A1 - Khare, Sangeeta A1 - Krug, Harald F. A1 - Kneuer, Carsten A1 - Leist, Marcel A1 - Lian, Guoping A1 - Marx, Uwe A1 - Metzger, Marco A1 - Ott, Katharina A1 - Prieto, Pilar A1 - Roberts, Michael S. A1 - Roggen, Erwin L. A1 - Tralau, Tewes A1 - van den Braak, Claudia A1 - Walles, Heike A1 - Lehr, Claus-Michael T1 - Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology JF - ALTEX: Alternatives to Animal Experimentation N2 - Models of the outer epithelia of the human body namely the skin, the intestine and the lung have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report. KW - on-a-chip KW - asthmatic bronchial epithelium KW - vesicle-based barrier KW - pulmonary drug-delivery KW - epithelial cell culture KW - cytotoxicity KW - transport studies KW - permeability KW - in vitro models KW - air-liquid interface KW - respiratory syncytial virus KW - reconstructed human epidermis KW - artificial membrane-permeability KW - embryonic stem cells Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144275 VL - 32 IS - 4 ER - TY - JOUR A1 - Scherzad, Agmal A1 - Meyer, Till A1 - Ickrath, Pascal A1 - Gehrke, Thomas Eckhart A1 - Bregenzer, Maximillian A1 - Hagen, Rudolf A1 - Dembski, Sofia A1 - Hackenberg, Stephan T1 - Cultivation of hMSCs in human plasma prevents the cytotoxic and genotoxic potential of ZnO-NP in vitro JF - Applied Sciences N2 - Zinc oxide nanoparticles (ZnO-NPs) are commonly used for industrial applications. Consequently, there is increasing exposure of humans to them. The in vitro analysis of cytotoxicity and genotoxicity is commonly performed under standard cell culture conditions. Thus, the question arises of how the results of genotoxicity and cytotoxicity experiments would alter if human plasma was used instead of cell culture medium containing of fetal calf serum (FCS). Human mesenchymal stem cells (hMSCs) were cultured in human plasma and exposed to ZnO-NPs. A cultivation in expansion medium made of DMEM consisting 10% FCS (DMEM-EM) served as control. Genotoxic and cytotoxic effects were evaluated with the comet and MTT assay, respectively. hMSC differentiation capacity and ZnO-NP disposition were evaluated by histology and transmission electron microscopy (TEM). The protein concentration and the amount of soluble Zn2+ were measured. The cultivation of hMSCs in plasma leads to an attenuation of genotoxic and cytotoxic effects of ZnO-NPs compared to control. The differentiation capacity of hMSCs was not altered. The TEM showed ZnO-NP persistence in cytoplasm in both groups. The concentrations of protein and Zn2+ were higher in plasma than in DMEM-EM. In conclusion, the cultivation of hMSCs in plasma compared to DMEM-EM leads to an attenuation of cytotoxicity and genotoxicity in vitro. KW - ZnO-NP KW - mesenchymal stem cells KW - genotoxicity KW - cytotoxicity KW - human plasma Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193063 SN - 2076-3417 VL - 9 IS - 23 ER - TY - JOUR A1 - Ickrath, Pascal A1 - Wagner, Martin A1 - Scherzad, Agmal A1 - Gehrke, Thomas A1 - Burghartz, Marc A1 - Hagen, Rudolf A1 - Radeloff, Katrin A1 - Kleinsasser, Norbert A1 - Hackenberg, Stephan T1 - Time-Dependent Toxic and Genotoxic Effects of Zinc Oxide Nanoparticles after Long-Term and Repetitive Exposure to Human Mesenchymal Stem Cells JF - International Journal of Environmental Research and Public Health N2 - Zinc oxide nanoparticles (ZnO-NP) are widely spread in consumer products. Data about the toxicological characteristics of ZnO-NP is still under controversial discussion. The human skin is the most important organ concerning ZnO-NP exposure. Intact skin was demonstrated to be a sufficient barrier against NPs; however, defect skin may allow NP contact to proliferating cells. Within these cells, stem cells are the most important toxicological target for NPs. The aim of this study was to evaluate the genotoxic and cytotoxic effects of ZnO-NP at low-dose concentrations after long-term and repetitive exposure to human mesenchymal stem cells (hMSC). Cytotoxic effects of ZnO-NP were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Furthermore, genotoxicity was evaluated by the comet assay. For long-term observation over 6 weeks, transmission electron microscopy (TEM) was applied. The results of the study indicated cytotoxic effects of ZnO-NP beginning at high concentrations of 50 μg/mL and genotoxic effects in hMSC exposed to 1 and 10 μg/mL ZnO-NP. Repetitive exposure enhanced cyto- but not genotoxicity. Intracellular NP accumulation was observed up to 6 weeks. The results suggest cytotoxic and genotoxic potential of ZnO-NP. Even low doses of ZnO-NP may induce toxic effects as a result of repetitive exposure and long-term cellular accumulation. This data should be considered before using ZnO-NP on damaged skin. KW - zinc oxide KW - ZnO KW - nanoparticles KW - cytotoxicity KW - toxicity KW - genotoxicity Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-169932 VL - 14 IS - 12 ER - TY - JOUR A1 - Abd El-Aziz, Asmaa M. A1 - El-Maghraby, Azza A1 - Ewald, Andrea A1 - Kandil, Sherif H. T1 - In-vitro cytotoxicity study: cell viability and cell morphology of carbon nanofibrous scaffold/hydroxyapatite nanocomposites JF - Molecules N2 - Electrospun carbon nanofibers (CNFs), which were modified with hydroxyapatite, were fabricated to be used as a substrate for bone cell proliferation. The CNFs were derived from electrospun polyacrylonitrile (PAN) nanofibers after two steps of heat treatment: stabilization and carbonization. Carbon nanofibrous (CNF)/hydroxyapatite (HA) nanocomposites were prepared by two different methods; one of them being modification during electrospinning (CNF-8HA) and the second method being hydrothermal modification after carbonization (CNF-8HA; hydrothermally) to be used as a platform for bone tissue engineering. The biological investigations were performed using in-vitro cell counting, WST cell viability and cell morphology after three and seven days. L929 mouse fibroblasts were found to be more viable on the hydrothermally-modified CNF scaffolds than on the unmodified CNF scaffolds. The biological characterizations of the synthesized CNF/HA nanofibrous composites indicated higher capability of bone regeneration. KW - HA modifiedCNF membranes KW - cytotoxicity KW - WST test KW - cell counting KW - cell viability KW - cell morphology Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234037 SN - 1420-3049 VL - 26 IS - 6 ER - TY - JOUR A1 - Wilhelms, Benedikt A1 - Broscheit, Jens A1 - Shityakov, Sergey T1 - Chemical analysis and molecular modelling of cyclodextrin-formulated propofol and its sodium salt to improve drug solubility, stability and pharmacokinetics (cytogenotoxicity) JF - Pharmaceuticals N2 - Propofol is a widely used general anesthetic in clinical practice, but its use is limited by its water-insoluble nature and associated pharmacokinetic and pharmacodynamic limitations. Therefore, researchers have been searching for alternative formulations to lipid emulsion to address the remaining side effects. In this study, novel formulations for propofol and its sodium salt Na-propofolat were designed and tested using the amphiphilic cyclodextrin (CD) derivative hydroxypropyl-β-cyclodextrin (HPβCD). The study found that spectroscopic and calorimetric measurements suggested complex formation between propofol/Na-propofolate and HPβCD, which was confirmed by the absence of an evaporation peak and different glass transition temperatures. Moreover, the formulated compounds showed no cytotoxicity and genotoxicity compared to the reference. The molecular modeling simulations based on molecular docking predicted a higher affinity for propofol/HPβCD than for Na-propofolate/HPβCD, as the former complex was more stable. This finding was further confirmed by high-performance liquid chromatography. In conclusion, the CD-based formulations of propofol and its sodium salt may be a promising option and a plausible alternative to conventional lipid emulsions. KW - propofol KW - anaesthesiology KW - HPβCD KW - \(^1\)H-NMR spectroscopy KW - calorimetry KW - molecular modelling KW - cytotoxicity KW - genotoxicity Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313705 SN - 1424-8247 VL - 16 IS - 5 ER -