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- ItemGuidance of mesenchymal stem cells on fibronectin structured hydrogel films(San Francisco, California, US : PLOS, 2014) Kasten, Annika; Naser, Tamara; Brüllhoff, Kristina; Fiedler, Jörg; Müller, Petra; Möller, Martin; Rychly, Joachim; Groll, Jürgen; Brenner, Rolf E.; Engler, Adam J.Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 µm and 80 µm and spacings between 5 µm and 20 µm that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.
- ItemLiver Dysfunction and Phosphatidylinositol-3-Kinase Signalling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis(San Francisco, CA : Public Library of Science, 2012) Recknagel, P.; Gonnert, F.A.; Westermann, M.; Lambeck, S.; Lupp, A.; Rudiger, A.; Dyson, A.; Carré, J.E.; Kortgen, A.; Krafft, C.; Popp, J.; Sponholz, C.; Fuhrmann, V.; Hilger, I.; Claus, R.A.; Riedemann, N.C.; Wetzker, R.; Singer, M.; Trauner, M.; Bauer, M.Background: Hepatic dysfunction and jaundice are traditionally viewed as late features of sepsis and portend poor outcomes. We hypothesized that changes in liver function occur early in the onset of sepsis, yet pass undetected by standard laboratory tests. Methods and Findings: In a long-term rat model of faecal peritonitis, biotransformation and hepatobiliary transport were impaired, depending on subsequent disease severity, as early as 6 h after peritoneal contamination. Phosphatidylinositol-3-kinase (PI3K) signalling was simultaneously induced at this time point. At 15 h there was hepatocellular accumulation of bilirubin, bile acids, and xenobiotics, with disturbed bile acid conjugation and drug metabolism. Cholestasis was preceded by disruption of the bile acid and organic anion transport machinery at the canalicular pole. Inhibitors of PI3K partially prevented cytokine-induced loss of villi in cultured HepG2 cells. Notably, mice lacking the PI3Kγ gene were protected against cholestasis and impaired bile acid conjugation. This was partially confirmed by an increase in plasma bile acids (e.g., chenodeoxycholic acid [CDCA] and taurodeoxycholic acid [TDCA]) observed in 48 patients on the day severe sepsis was diagnosed; unlike bilirubin (area under the receiver-operating curve: 0.59), these bile acids predicted 28-d mortality with high sensitivity and specificity (area under the receiver-operating curve: CDCA: 0.77; TDCA: 0.72; CDCA+TDCA: 0.87). Conclusions: Liver dysfunction is an early and commonplace event in the rat model of sepsis studied here; PI3K signalling seems to play a crucial role. All aspects of hepatic biotransformation are affected, with severity relating to subsequent prognosis. Detected changes significantly precede conventional markers and are reflected by early alterations in plasma bile acids. These observations carry important implications for the diagnosis of liver dysfunction and pharmacotherapy in the critically ill. Further clinical work is necessary to extend these concepts into clinical practice. Please see later in the article for the Editors' Summary.
- ItemIn vitro model of metastasis to bone marrow mediates prostate cancer castration resistant growth through paracrine and extracellular matrix factors(San Francisco, CA : Public Library of Science, 2012) Lescarbeau, R.M.; Seib, F.P.; Prewitz, M.; Werner, C.; Kaplan, D.L.The spread of prostate cancer cells to the bone marrow microenvironment and castration resistant growth are key steps in disease progression and significant sources of morbidity. However, the biological significance of mesenchymal stem cells (MSCs) and bone marrow derived extracellular matrix (BM-ECM) in this process is not fully understood. We therefore established an in vitro engineered bone marrow tissue model that incorporates hMSCs and BM-ECM to facilitate mechanistic studies of prostate cancer cell survival in androgen-depleted media in response to paracrine factors and BM-ECM. hMSC-derived paracrine factors increased LNCaP cell survival, which was in part attributed to IGFR and IL6 signaling. In addition, BM-ECM increased LNCaP and MDA-PCa-2b cell survival in androgen-depleted conditions, and induced chemoresistance and morphological changes in LNCaPs. To determine the effect of BM-ECM on cell signaling, the phosphorylation status of 46 kinases was examined. Increases in the phosphorylation of MAPK pathway-related proteins as well as sustained Akt phosphorylation were observed in BM-ECM cultures when compared to cultures grown on plasma-treated polystyrene. Blocking MEK1/2 or the PI3K pathway led to a significant reduction in LNCaP survival when cultured on BM-ECM in androgen-depleted conditions. The clinical relevance of these observations was determined by analyzing Erk phosphorylation in human bone metastatic prostate cancer versus non-metastatic prostate cancer, and increased phosphorylation was seen in the metastatic samples. Here we describe an engineered bone marrow model that mimics many features observed in patients and provides a platform for mechanistic in vitro studies.
- ItemProteinase-activated receptor-2 agonist activates anti-influenza mechanisms and modulates IFNγ induced antiviral pathways in human neutrophils(London : Hindawi, 2013) Feld, Micha; Shpacovitch, Victoria; Ehrhardt, Christina; Fastrich, Michaela; Goerge, Tobias; Ludwig, Stephan; Steinhoff, MartinProteinase-activated receptor-2 (PAR2) is expressed by human leukocytes and participates in the development of inflammatory diseases. Recent studies demonstrated an ability of PAR2 agonist to enhance IFNγ-induced antiviral responses of human leukocytes. However, the precise cellular antiviral defense mechanisms triggered in leukocytes after stimulation with IFNγ and/or PAR2 agonist remain elusive. Therefore, we aimed to identify neutrophil defense mechanisms involved in antiviral resistance. Here we demonstrated that PAR2 agonist enhanced IFNγ-related reduction of influenza A virus (IAV) replication in human neutrophils. PAR2-mediated decrease in IAV replication was associated with reduced NS-1 transcription. Moreover, PAR2-dependent neutrophil activation resulted in enhanced myeloperoxidase degranulation and extracellular myeloperoxidase disrupted IAV. The production of ROS was elevated in response to PAR2 activation. Interestingly, IFNγ did not influence both effects: PAR2 agonist-triggered myeloperoxidase (MPO) release and reactive oxygen species (ROS) production, which are known to limit IAV infections. In contrast, orthomyxovirus resistance gene A (MxA) protein expression was synergistically elevated through PAR2 agonist and IFNγ in neutrophils. Altogether, these findings emphasize two PAR2-controlled antiviral mechanisms that are independent of or modulated by IFNγ.
- ItemNonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength(San Francisco, California, US : PLOS, 2016) Lu-Walther, Hui-Wen; Hou, Wenya; Kielhorn, Martin; Arai, Yoshiyuki; Nagai, Takeharu; Kessels, Michael M.; Qualmann, Britta; Heintzmann, RainerStructured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.
- ItemDifferential influence of components resulting from atmospheric-pressure plasma on integrin expression of human HaCaT keratinocytes(New York, NY : Hindawi, 2013) Haertel, B.; Straßenburg, S.; Oehmigen, K.; Wende, K.; Von Woedtke, T.; Lindequist, U.Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells.
- ItemEffects of new beta-type Ti-40Nb implant materials, brain-derived neurotrophic factor, acetylcholine and nicotine on human mesenchymal stem cells of osteoporotic and non osteoporotic donors(San Francisco, CA : Public Library of Science (PLoS), 2018) Kauschke, V.; Gebert, A.; Calin, M.; Eckert, J.; Scheich, S.; Heiss, C.; Lips, K.S.Introduction Treatment of osteoporotic fractures is still challenging and an urgent need exists for new materials, better adapted to osteoporotic bone by adjusted Young’s modulus, appropriate surface modification and pharmaceuticals. Materials and methods Titanium-40-niobium alloys, mechanically ground or additionally etched and titanium-6-alu-minium-4-vanadium were analyzed in combination with brain-derived neurotrophic factor, acetylcholine and nicotine to determine their effects on human mesenchymal stem cells in vitro over 21 days using lactate dehydrogenase and alkaline phosphatase assays, live cell imaging and immunofluorescence microscopy. Results Cell number of human mesenchymal stem cells of osteoporotic donors was increased after 14 d in presence of ground titanium-40-niobium or titanium-6-aluminium-4-vanadium, together with brain-derived neurotrophic factor. Cell number of human mesenchymal stem cells of non osteoporotic donors increased after 21 d in presence of titanium-6-aluminium-4-vanadium without pharmaceuticals. No significant increase was measured for ground or etched titanium-40-niobium after 21 d. Osteoblast differentiation of osteoporotic donors was significantly higher than in non osteoporotic donors after 21 d in presence of etched, ground titanium-40-niobium or titanium-6-aluminium-4-vanadium accompanied by all pharmaceuticals tested. In presence of all alloys tested brain-derived neurotrophic factor, acetylcholine and nicotine increased differentiation of cells of osteoporotic donors and accelerated it in non osteoporotic donors. Conclusion We conclude that ground titanium-40-niobium and brain-derived neurotrophic factor might be most suitable for subsequent in vivo testing.
- ItemTissue Tolerable Plasma (TTP) induces apoptosis in pancreatic cancer cells in vitro and in vivo(London : BioMed Central, 2012) Partecke, L.I.; Evert, K.; Haugk, J.; Doering, F.; Normann, L.; Diedrich, S.; Weiss, F.-U.; Evert, M.; Huebner, N.O.; Guenther, C.; Heidecke, C.D.; Kramer, A.; Bussiahn, R.; Weltmann, K.-D.; Pati, O.; Bender, C.; von Bernstorff, W.Background: The rate of microscopic incomplete resections of gastrointestinal cancers including pancreatic cancer has not changed considerably over the past years. Future intra-operative applications of tissue tolerable plasmas (TTP) could help to address this problem. Plasma is generated by feeding energy, like electrical discharges, to gases. The development of non-thermal atmospheric plasmas displaying spectra of temperature within or just above physiological ranges allows biological or medical applications of plasmas.Methods: We have investigated the effects of tissue tolerable plasmas (TTP) on the human pancreatic cancer cell line Colo-357 and PaTu8988T and the murine cell line 6606PDA in vitro (Annexin-V-FITC/DAPI-Assay and propidium iodide DNA staining assay) as well as in the in vivo tumour chorio-allantoic membrane (TUM-CAM) assay using Colo-357.Results: TTP of 20 seconds (s) induced a mild elevation of an experimental surface temperature of 23.7 degree Celsius up to 26.63+/-0.40 degree Celsius. In vitro TTP significantly (p=0.0003) decreased cell viability showing the strongest effects after 20s TTP. Also, TTP effects increased over time levelling off after 72 hours (30.1+/-4.4% of dead cells (untreated control) versus 78.0+/-9.6% (20s TTP)). However, analyzing these cells for apoptosis 10s TTP revealed the largest proportion of apoptotic cells (34.8+/-7.2%, p=0.0009 versus 12.3+/-6.6%, 20s TTP) suggesting non-apoptotic cell death in the majority of cells after 20s TTP. Using solid Colo-357 tumours in the TUM-CAM model TUNEL-staining showed TTP-induced apoptosis up to a depth of tissue penetration (DETiP) of 48.8+/-12.3μm (20s TTP, p<0.0001). This was mirrored by a significant (p<0.0001) reduction of Ki-67+ proliferating cells (80.9+/-13.2% versus 37.7+/-14.6%, p<0.0001) in the top cell layers as well as typical changes on HE specimens. The bottom cell layers were not affected by TTP.Conclusions: Our data suggest possible future intra-operative applications of TTP to reduce microscopic residual disease in pancreatic cancer resections. Further promising applications include other malignancies (central liver/lung tumours) as well as synergistic effects combining TTP with chemotherapies. Yet, adaptations of plasma sources as well as of the composition of effective components of TTP are required to optimize their synergistic apoptotic actions.
- ItemRecombinant phage coated 1D Al2O3 nanostructures for controlling the adhesion and proliferation of endothelial cells(New York [u.a.] : Hindawi, 2015) Lee, Juseok; Jeon, Hojeong; Haidar, Ayman; Abdul-Khaliq, Hashim; Veith, Michael; Aktas, Cenk; Kim, YoungjunA novel synthesis of a nanostructured cell adhesive surface is investigated for future stent developments. One-dimensional (1D) Al2O3 nanostructures were prepared by chemical vapor deposition of a single source precursor. Afterwards, recombinant filamentous bacteriophages which display a short binding motif with a cell adhesive peptide (RGD) on p3 and p8 proteins were immobilized on these 1D Al2O3 nanostructures by a simple dip-coating process to study the cellular response of human endothelial EA hy.926. While the cell density decreased on as-deposited 1D Al2O3 nanostructures, we observed enhanced cell proliferation and cell-cell interaction on recombinant phage overcoated 1D Al2O3 nanostructures. The recombinant phage overcoating also supports an isotropic cell spreading rather than elongated cell morphology as we observed on as-deposited Al2O3 1D nanostructures.
- ItemGraphene oxide functional nanohybrids with magnetic nanoparticles for improved vectorization of doxorubicin to neuroblastoma cells(Basel : MDPI AG, 2019) Lerra, L.; Farfalla, A.; Sanz, B.; Cirillo, G.; Vittorio, O.; Voli, F.; Grand, M.L.; Curcio, M.; Nicoletta, F.P.; Dubrovska, A.; Hampel, S.; Iemma, F.; Goya, G.F.With the aim to obtain a site-specific doxorubicin (DOX) delivery in neuroblastoma SH-SY5Y cells, we designed an hybrid nanocarrier combining graphene oxide (GO) and magnetic iron oxide nanoparticles (MNPs), acting as core elements, and a curcumin–human serum albumin conjugate as functional coating. The nanohybrid, synthesized by redox reaction between the MNPs@GO system and albumin bioconjugate, consisted of MNPs@GO nanosheets homogeneously coated by the bioconjugate as verified by SEM investigations. Drug release experiments showed a pH-responsive behavior with higher release amounts in acidic (45% at pH 5.0) vs. neutral (28% at pH 7.4) environments. Cell internalization studies proved the presence of nanohybrid inside SH-SY5Y cytoplasm. The improved efficacy obtained in viability assays is given by the synergy of functional coating and MNPs constituting the nanohybrids: while curcumin moieties were able to keep low DOX cytotoxicity levels (at concentrations of 0.44–0.88 µM), the presence of MNPs allowed remote actuation on the nanohybrid by a magnetic field, increasing the dose delivered at the target site.