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- ItemBio-responsive polymer hydrogels homeostatically regulate blood coagulation(London : Nature Publishing Group, 2013) Maitz, Manfred F.; Freudenberg, U.; Tsurkan, M.V.; Fischer, M.; Beyrich, T.; Werner, C.Bio-responsive polymer architectures can empower medical therapies by engaging molecular feedback-response mechanisms resembling the homeostatic adaptation of living tissues to varying environmental constraints. Here we show that a blood coagulation-responsive hydrogel system can deliver heparin in amounts triggered by the environmental levels of thrombin, the key enzyme of the coagulation cascade, which - in turn - becomes inactivated due to released heparin. The bio-responsive hydrogel quantitatively quenches blood coagulation over several hours in the presence of pro-coagulant stimuli and during repeated incubation with fresh, non-anticoagulated blood. These features enable the introduced material to provide sustainable, autoregulated anticoagulation, addressing a key challenge of many medical therapies. Beyond that, the explored concept may facilitate the development of materials that allow the effective and controlled application of drugs and biomolecules.
- ItemAnti-Prion Drug mPPIg5 Inhibits PrPC Conversion to PrPSc(San Francisco, CA : Public Library of Science, 2013) McCarthy, J.M.; Franke, M.; Resenberger, U.K.; Waldron, S.; Simpson, J.C.; Tatzelt, J.; Appelhans, D.; Rogers, M.S.Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrPSc, an abnormal isoform of the cellular protein PrPC, is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrPSc in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrPC to PrPSc conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future.
- 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.
- ItemWhole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography(San Francisco, CA : Public Library of Science, 2013) Baudoin, J.-P.; Jerome, W.G.; Kübel, C.; de Jonge, N.Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters.
- ItemAntimicrobial Efficacy of Two Surface Barrier Discharges with Air Plasma against In Vitro Biofilms(San Francisco, CA : Public Library of Science, 2013) Matthes, R.; Bender, C.; Schlüter, R.; Koban, I.; Bussiahn, R.; Reuter, S.; Lademann, J.; Weltmann, K.-D.; Kramer, A.The treatment of infected wounds is one possible therapeutic aspect of plasma medicine. Chronic wounds are often associated with microbial biofilms which limit the efficacy of antiseptics. The present study investigates two different surface barrier discharges with air plasma to compare their efficacy against microbial biofilms with chlorhexidine digluconate solution (CHX) as representative of an important antibiofilm antiseptic. Pseudomonas aeruginosa SG81 and Staphylococcus epidermidis RP62A were cultivated on polycarbonate discs. The biofilms were treated for 30, 60, 150, 300 or 600 s with plasma or for 600 s with 0.1% CHX, respectively. After treatment, biofilms were dispensed by ultrasound and the antimicrobial effects were determined as difference in the number of the colony forming units by microbial culture. A high antimicrobial efficacy on biofilms of both plasma sources in comparison to CHX treatment was shown. The efficacy differs between the used strains and plasma sources. For illustration, the biofilms were examined under a scanning electron microscope before and after treatment. Additionally, cytotoxicity was determined by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with L929 mouse fibroblast cell line. The cell toxicity of the used plasma limits its applicability on human tissue to maximally 150 s. The emitted UV irradiance was measured to estimate whether UV could limit the application on human tissue at the given parameters. It was found that the UV emission is negligibly low. In conclusion, the results support the assumption that air plasma could be an option for therapy of chronic wounds.
- ItemCardio-respiratory coordination increases during sleep apnea(San Francisco, CA : Public Library of Science (PLoS), 2014) Riedl, M.; Müller, A.; Kraemer, J.F.; Penzel, T.; Kurths, J.; Wessel, N.Cardiovascular diseases are the main source of morbidity and mortality in the United States with costs of more than $170 billion. Repetitive respiratory disorders during sleep are assumed to be a major cause of these diseases. Therefore, the understanding of the cardio-respiratory regulation during these events is of high public interest. One of the governing mechanisms is the mutual influence of the cardiac and respiratory oscillations on their respective onsets, the cardiorespiratory coordination (CRC). We analyze this mechanism based on nocturnal measurements of 27 males suffering from obstructive sleep apnea syndrome. Here we find, by using an advanced analysis technique, the coordigram, not only that the occurrence of CRC is significantly more frequent during respiratory sleep disturbances than in normal respiration (p-value<10-51) but also more frequent after these events (p-value<10-15). Especially, the latter finding contradicts the common assumption that spontaneous CRC can only be observed in epochs of relaxed conditions, while our newly discovered epochs of CRC after disturbances are characterized by high autonomic stress. Our findings on the connection between CRC and the appearance of sleep-disordered events require a substantial extension of the current understanding of obstructive sleep apneas and hypopneas.
- ItemAlterations in Event Related Potentials (ERP) associated with tinnitus distress and attention(Dordrecht [u.a.] : Springer Science + Business Media B.V, 2008) Delb, W.; Strauss, D.J.; Low, Y.F.; Seidler, H.; Rheinschmitt, A.; Wobrock, T.; D'Amelio, R.Tinnitus related distress corresponds to different degrees of attention paid to the tinnitus. Shifting attention to a signal other than the tinnitus is therefore particularly difficult for patients with high tinnitus related distress. As attention effects on Event Related Potentials (ERP) have been shown this should be reflected in ERP measurements (N100, phase locking). In order to prove this hypothesis single sweep ERP recordings were obtained in 41 tinnitus patients as well as 10 control subjects during a period of time when attention was shifted to a tone (attended) and during a second phase (unattended) when they did not focus attention to the tone. Whereas tinnitus patients with low distress showed a significant reduction in both N100 amplitude and phase locking when comparing the attended and unattended measurement condition a group of patients with high tinnitus related distress did not show such ERP alterations. Using single sweep ERP measurements the results of our study show, that attention in high tinnitus related distress patients is captured by their tinnitus significantly more than in low distress patients. Furthermore our results provide the basis for future neurofeedback based tinnitus therapies aiming at maximizing the ability to shift attention away from the tinnitus. © 2008 The Author(s).
- 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.
- 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.
- ItemInsulin adsorption to catheter materials used for intensive insulin therapy in critically ill patients: Polyethylene versus polyurethane - possible cause of variation in glucose control?(Sharjah : Bentham Science Publishers B.V., 2014) Ley, S.C.; Ammann, J.; Herder, C.; Dickhaus, T.; Hartmann, M.; Kindgen-Milles, D.Introduction: Restoring and maintaining normoglycemia by intensified insulin therapy in critically ill patients is a matter of ongoing debate since the risk of hypoglycemia may outweigh positive effects on morbidity and mortality. In this context, adsorption of insulin to different catheter materials may contribute to instability of glucose control. We studied the adsorption of insulin to different tubing materials in vitro and the effects on glycemic control in vivo. Materials and Methods: In vitro experiments: A syringe pump was filled with 50 IU insulin diluted to 50 ml saline. A flow of 2 ml/h was perfused through polyethylene (PET) or polyurethane (PUR) tubing. Insulin concentrations were measured at the end of the tube for 24 hours using Bradford's protein assay. In vivo study: In a randomized double-blinded cross-over design, 10 intensive care patients received insulin via PET and PUR tubes for 24 hours each, targeting blood glucose levels of 80-150 mg/dl. We measured blood glucose levels, the insulin dose required to maintain target levels, and serum insulin and C-peptide levels. Results: In vitro experiments: After the start of the insulin infusion, only 20% (median, IQR 20-27) (PET) and 22% (IQR 16-27) (PUR) of the prepared insulin concentration were measured at the end of the 2 meter tubing. Using PET, after one hour infusion the concentration increased to 34% (IQR 29-36) and did not increase significantly during the next 24 hours (39% (IQR 39-40)). Using PUR, higher concentrations were detected than for PET at every measurement from 1 hour (82% (IQR 70-86)) to 24 hours (79% (IQR 64-87)). In vivo study: Glycemic control was effective and not different between groups. Significantly higher volumes of insulin solution had to be infused with PET compared to PUR (median PET 70.0 (IQR 56-82) ml vs. PUR 42 (IQR 31-63) ml; p=0.0015). Serum insulin concentrations did not decrease significantly one hour after changing to PET or PUR tubing. Conclusion: Polyurethane tubing systems allow application of insulin with significantly lower adsorption rates than polyethylene tubing systems. As a consequence, less insulin solution has to be infused to patients for effective blood glucose control. Tubing material of the insulin infusion may be crucial for safe and effective glycemic control in critically ill patients.