TY - JOUR A1 - Bröker, Katharine A1 - Sinelnikov, Evgeny A1 - Gustavus, Dirk A1 - Schumacher, Udo A1 - Pörtner, Ralf A1 - Hoffmeister, Hans A1 - Lüth, Stefan A1 - Dammermann, Werner T1 - Mass Production of Highly Active NK Cells for Cancer Immunotherapy in a GMP Conform Perfusion Bioreactor JF - Frontiers in Bioengineering and Biotechnology N2 - NK cells have emerged as promising candidates for cancer immunotherapy, especially due to their ability to fight circulating tumor cells thereby preventing metastases formation. Hence several studies have been performed to generate and expand highly cytotoxic NK cells ex vivo, e.g., by using specific cytokines to upregulate both their proliferation and surface expression of distinct activating receptors. Apart from an enhanced activity, application of NK cells as immunotherapeutic agent further requires sufficient cell numbers and a high purity. All these parameters depend on a variety of different factors including the starting material, additives like cytokines as well as the culture system. Here we analyzed PBMC-derived NK cells of five anonymized healthy donors expanded under specific conditions in an innovative perfusion bioreactor system with respect to their phenotype, IFN gamma production, and cytotoxicity in vitro. Important features of the meander type bioreactors used here are a directed laminar flow of medium and control of relevant process parameters. Cells are cultivated under "steady state" conditions in perfusion mode. Our data demonstrate that expansion of CD3(+) T cell depleted PBMCs in our standardized system generates massive amounts of highly pure (>85%) and potent anticancer active NK cells. These cells express a variety of important receptors driving NK cell recruitment, adhesion as well as activation. More specifically, they express the chemokine receptors CXCR3, CXCR4, and CCR7, the adhesion molecules L-selectin, LFA-1, and VLA-4, the activating receptors NKp30, NKp44, NKp46, NKG2D, DNAM1, and CD16 as well as the death ligands TRAIL and Fas-L. Moreover, the generated NK cells show a strong IFN gamma expression upon cultivation with K562 tumor cells and demonstrate a high cytotoxicity toward leukemic as well as solid tumor cell lines in vitro. Altogether, these characteristics promise a high clinical potency of thus produced NK cells awaiting further evaluation. KW - natural killer cells (NK cells) KW - cytotoxicity KW - tumor immunity KW - immunotherapy KW - perfusion bioreactor KW - GMP KW - mass production process Y1 - 2019 U6 - https://doi.org/10.3389/fbioe.2019.00194 SN - 2296-4185 VL - 7 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Adem, Fozia A. A1 - Kuete, Victor A1 - Mbaveng, Armelle T. A1 - Heydenreich, Matthias A1 - Koch, Andreas A1 - Ndakala, Albert A1 - Irungu, Beatrice A1 - Yenesew, Abiy A1 - Efferth, Thomas T1 - Cytotoxic flavonoids from two Lonchocarpus species JF - Natural Product Research N2 - A new isoflavone, 4′-prenyloxyvigvexin A (1) and a new pterocarpan, (6aR,11aR)-3,8-dimethoxybitucarpin B (2) were isolated from the leaves of Lonchocarpus bussei and the stem bark of Lonchocarpus eriocalyx, respectively. The extract of L. bussei also gave four known isoflavones, maximaisoflavone H, 7,2′-dimethoxy-3′,4′-methylenedioxyisoflavone, 6,7,3′-trimethoxy-4′,5′-methylenedioxyisoflavone, durmillone; a chalcone, 4-hydroxylonchocarpin; a geranylated phenylpropanol, colenemol; and two known pterocarpans, (6aR,11aR)-maackiain and (6aR,11aR)-edunol. (6aR,11aR)-Edunol was also isolated from the stem bark of L. eriocalyx. The structures of the isolated compounds were elucidated by spectroscopy. The cytotoxicity of the compounds was tested by resazurin assay using drug-sensitive and multidrug-resistant cancer cell lines. Significant antiproliferative effects with IC50 values below 10 μM were observed for the isoflavones 6,7,3′-trimethoxy-4′,5′-methylenedioxyisoflavone and durmillone against leukemia CCRF-CEM cells; for the chalcone, 4-hydroxylonchocarpin and durmillone against its resistant counterpart CEM/ADR5000 cells; as well as for durmillone against the resistant breast adenocarcinoma MDA-MB231/BCRP cells and resistant gliobastoma U87MG.ΔEGFR cells. KW - Lonchocarpus bussei KW - Lonchocarpus eriocalyx KW - Leguminosae KW - isoflavone KW - pterocarpan KW - cytotoxicity Y1 - 2019 U6 - https://doi.org/10.1080/14786419.2018.1462179 SN - 1478-6419 SN - 1478-6427 VL - 33 IS - 18 SP - 2609 EP - 2617 PB - Routledge, Taylor & Francis Group CY - Abingdon ER - TY - JOUR A1 - Awan, Asad Bashir A1 - Schiebel, Juliane A1 - Boehm, Alexander A1 - Nitschke, Joerg A1 - Sarwar, Yasra A1 - Schierack, Peter A1 - Ali, Aamir T1 - Association of biofilm formation and cytotoxic potential with multidrug resistance in clinical isolates of pseudomonas aeruginosa JF - EXCLI Journal N2 - Multidrug resistant (MDR) Pseudomonas aeruginosa having strong biofilm potential and virulence factors are a serious threat for hospitalized patients having compromised immunity In this study, 34 P. aeruginosa isolates of human origin (17 MDR and 17 non-MDR clinical isolates) were checked for biofilm formation potential in enriched and minimal media. The biofilms were detected using crystal violet method and a modified software package of the automated VideoScan screening method. Cytotoxic potential of the isolates was also investigated on HepG2, LoVo and T24 cell lines using automated VideoScan technology. Pulse field gel electrophoresis revealed 10 PFGE types in MDR and 8 in non-MDR isolates. Although all isolates showed biofilm formation potential, strong biofilm formation was found more in enriched media than in minimal media. Eight MDR isolates showed strong biofilm potential in both enriched and minimal media by both detection methods. Strong direct correlation between crystal violet and VideoScan methods was observed in identifying strong biofilm forming isolates. High cytotoxic effect was observed by 4 isolates in all cell lines used while 6 other isolates showed high cytotoxic effect on T24 cell line only. Strong association of multidrug resistance was found with biofilm formation as strong biofilms were observed significantly higher in MDR isolates (p-value < 0.05) than non-MDR isolates. No significant association of cytotoxic potential with multidrug resistance or biofilm formation was found (p-value > 0.05). The MDR isolates showing significant cytotoxic effects and strong biofilm formation impose a serious threat for hospitalized patients with weak immune system. KW - Pseudomonas aeruginosa KW - multidrug resistance KW - biofilm KW - cytotoxicity KW - VideoScan technology Y1 - 2019 U6 - https://doi.org/10.17179/excli2018-1948 SN - 1611-2156 VL - 18 SP - 79 EP - 90 PB - Leibniz Research Centre for Working Environment and Human Factors CY - Dortmund ER -