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- 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.
- ItemVinculin binding angle in podosomes revealed by high resolution microscopy(San Francisco, CA : Public Library of Science, 2014) Walde, M.; Monypenny, J.; Heintzmann, R.; Jones, G.E.; Cox, S.Podosomes are highly dynamic actin-rich adhesive structures formed predominantly by cells of the monocytic lineage, which degrade the extracellular matrix. They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin. We have characterised the structure of podosomes in macrophages, particularly the structure of the ring, using three super-resolution fluorescence microscopy techniques: stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy. Rather than being round, as previously assumed, we found the vinculin ring to be created from relatively straight strands of vinculin, resulting in a distinctly polygonal shape. The strands bind preferentially at angles between 116° and 135°. Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth.
- ItemComplement activation by carbon nanotubes and its influence on the phagocytosis and cytokine response by macrophages(Amsterdam [u.a.] : Elsevier, 2014) Pondman, K.M.; Sobik, M.; Nayak, A.; Tsolaki, A.G.; Jäkel, A.; Flahaut, E.; Hampel, S.; ten Haken, B.; Sim, R.B.; Kishore, U.Carbon nanotubes (CNTs) have promised a range of applications in biomedicine. Although influenced by the dispersants used, CNTs are recognized by the innate immune system, predominantly by the classical pathway of the complement system. Here, we confirm that complement activation by the CNT used continues up to C3 and C5, indicating that the entire complement system is activated including the formation of membrane-attack complexes. Using recombinant forms of the globular regions of human C1q (gC1q) as inhibitors of CNT-mediated classical pathway activation, we show that C1q, the first recognition subcomponent of the classical pathway, binds CNTs via the gC1q domain. Complement opsonisation of CNTs significantly enhances their uptake by U937 cells, with concomitant downregulation of pro-inflammatory cytokines and up-regulation of anti-inflammatory cytokines in both U937 cells and human monocytes. We propose that CNT-mediated complement activation may cause recruitment of cellular infiltration, followed by phagocytosis without inducing a pro-inflammatory immune response. From the Clinical Editor: This study highlights the importance of the complement system in response to carbon nanontube administration, suggesting that the ensuing complement activation may cause recruitment of cellular infiltration, followed by phagocytosis without inducing a pro-inflammatory immune response.
- ItemGrowth induction and low-oxygen apoptosis inhibition of human CD34 + progenitors in collagen gels(New York, NY : Hindawi, 2013) Avitabile, D.; Salchert, K.; Werner, C.; Capogrossi, M.C.; Pesce, M.Various reports have indicated low survival of injected progenitors into unfavorable environments such as the ischemic myocardium or lower limb tissues. This represents a major bottleneck in stem-cell-based cardiovascular regenerative medicine. Strategies to enhance survival of these cells in recipient tissues have been therefore sought to improve stem cell survival and ensure long-term engraftment. In the present contribution, we show that embedding human cord blood-derived CD34+ cells into a collagen I-based hydrogel containing cytokines is a suitable strategy to promote stem cell proliferation and protect these cells from anoxia-induced apoptosis.
- 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.
- 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.
- ItemMicromotor-mediated sperm constrictions for improved swimming performance(Berlin ; Heidelberg : Springer, 2021) Striggow, Friedrich; Nadporozhskaia, Lidiia; Friedrich, Benjamin M.; Schmidt, Oliver G.; Medina-Sánchez, MarianaSperm-driven micromotors, consisting of a single sperm cell captured in a microcap, utilize the strong propulsion generated by the flagellar beat of motile spermatozoa for locomotion. It enables the movement of such micromotors in biological media, while being steered remotely by means of an external magnetic field. The substantial decrease in swimming speed, caused by the additional hydrodynamic load of the microcap, limits the applicability of sperm-based micromotors. Therefore, to improve the performance of such micromotors, we first investigate the effects of additional cargo on the flagellar beat of spermatozoa. We designed two different kinds of microcaps, which each result in different load responses of the flagellar beat. As an additional design feature, we constrain rotational degrees of freedom of the cell’s motion by modifying the inner cavity of the cap. Particularly, cell rolling is substantially reduced by tightly locking the sperm head inside the microcap. Likewise, cell yawing is decreased by aligning the micromotors under an external static magnetic field. The observed differences in swimming speed of different micromotors are not so much a direct consequence of hydrodynamic effects, but rather stem from changes in flagellar bending waves, hence are an indirect effect. Our work serves as proof-of-principle that the optimal design of microcaps is key for the development of efficient sperm-driven micromotors.
- 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.
- ItemGeometry-Driven Cell Organization Determines Tissue Growths in Scaffold Pores: Consequences for Fibronectin Organization(San Francisco, CA : Public Library of Science, 2013) Joly, P.; Duda, G.N.; Schöne, M.; Welzel, P.B.; Freudenberg, U.; Werner, C.; Petersen, A.To heal tissue defects, cells have to bridge gaps and generate new extracellular matrix (ECM). Macroporous scaffolds are frequently used to support the process of defect filling and thus foster tissue regeneration. Such biomaterials contain micro-voids (pores) that the cells fill with their own ECM over time. There is only limited knowledge on how pore geometry influences cell organization and matrix production, even though it is highly relevant for scaffold design. This study hypothesized that 1) a simple geometric description predicts cellular organization during pore filling at the cell level and that 2) pore closure results in a reorganization of ECM. Scaffolds with a broad distribution of pore sizes (macroporous starPEG-heparin cryogel) were used as a model system and seeded with primary fibroblasts. The strategies of cells to fill pores could be explained by a simple geometrical model considering cells as tensioned chords. The model matched qualitatively as well as quantitatively by means of cell number vs. open cross-sectional area for all pore sizes. The correlation between ECM location and cell position was higher when the pores were not filled with tissue (Pearson's coefficient ρ = 0.45±0.01) and reduced once the pores were closed (ρ = 0.26±0.04) indicating a reorganization of the cell/ECM network. Scaffold pore size directed the time required for pore closure and furthermore impacted the organization of the fibronectin matrix. Understanding how cells fill micro-voids will help to design biomaterial scaffolds that support the endogenous healing process and thus allow a fast filling of tissue defects.