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Abou-Elkacem L.,RWTH Aachen | Gremse F.,RWTH Aachen | Barth S.,Institute for Applied Medical Engineering | Barth S.,Fraunhofer Institute for Molecular Biology and Applied Ecology | And 4 more authors.
Anticancer Research | Year: 2011

Aim: To compare magnetic resonance imaging (MRI), micro-computed tomography (μCT), optical reflectance imaging (ORI) and caliper measurements for subcutaneous tumor detection and size assessment. Materials and Methods: HCT 116-green- (GFP)-red-fluorescent protein (RFP) tumor volumes were measured in vivo by calipers and by ORI, MRI and μCT over 15 days and validated ex vivo. The method correlating best with the ex vivo tumor volumes was used as reference for longitudinal in vivo correlations. Results: MRI and ORI detected tumors at day 1 post-injection, μCT after 3 days. The in vivo MRI data correlated best with the ex vivo tumor volumes (r 2=0.96), followed by μCT (r 2=0.93). Thus, MRI was chosen as the reference. μCT-(r 2=0.90), in vivo caliper data (r 2=0.80) and fluorescence intensities (GFP:r 2=0.71; RFP:r 2=0.75) highly correlated with MRI-data, whereas fluorescent areas (GFP:r 2=0.26; RFP:r 2=030) poorly correlated. Conclusion: MRI sensitively detects tumors and precisely determines their size; μCT is an accurate alternative for larger tumors; ORI is as sensitive as MRI, but overestimates small tumor sizes; and fluorescence intensity correlates better with tumor volume than fluorescence area.


Losasso V.,German Research School for Simulation science | Schiffer S.,Institute for Applied Medical Engineering | Barth S.,Institute for Applied Medical Engineering | Barth S.,Fraunhofer Institute for Molecular Biology and Applied Ecology | And 2 more authors.
Proteins: Structure, Function and Bioinformatics | Year: 2012

Human granzyme B (hGB) is a serine protease involved in immune-mediated apoptosis. Its cytotoxicity makes it potentially applicable in cancer therapy. However, the effectiveness of hGB can be hampered by the cytosolic expression of a natural protein inhibitor, human Serpin B9 (hSB9). Here, we used computational approaches to identify hGB mutations that can affect its binding to hSB9 without significantly decreasing its catalytic efficiency. Alanine-scanning calculations allowed us to identify residues of hGB important for the interaction with hSB9. Some variants were selected, and molecular dynamic simulations on the mutated hGB in complex with hSB9 in aqueous solution were carried out to investigate the effect of these variants on the stability of the complex. The R28K, R201A, and R201K mutants significantly destabilized the interaction of the protein with hSB9. Consistently, all of these variants also retained their activity in the presence of the Serpin B9 inhibitor in subsequent in vitro assays of wild-type and mutated hGB. In particular, the activity of R201K hGB with and without Serpin B9 is very similar to that of the wild-type protein. Hence, R201K hGB emerges as a promising species for antitumoral therapy applications. © 2012 Wiley Periodicals, Inc.


Recker T.,RWTH Aachen | Haamann D.,RWTH Aachen | Schmitt A.,RWTH Aachen | Kuster A.,RWTH Aachen | And 4 more authors.
Bioconjugate Chemistry | Year: 2011

Cytokines are important mediators coordinating inflammation and wound healing in response to tissue damage and infection. Therefore, immobilization of cytokines on the surface of biomaterials is a promising approach to improve biocompatibility. Soluble cytokines signal through receptors on the cell surface leading to cell differentiation, proliferation, or other effector functions. Random immobilization of cytokines on surfaces will result in a large fraction of inactive protein due to impaired cytokine-receptor interaction. We developed a strategy that combined (i) directed covalent coupling of cytokines, (ii) quantification of coupling efficiency through fluorescence detection, and (iii) a reliable protease cleavage assay to control orientation of coupling. For this purpose, fusion proteins of the SNAP-tag followed by an enterokinase recognition site, yellow fluorescent protein (YFP), and the cytokine of interest being either interleukin-6 (IL-6) or oncostatin M (OSM) were generated. The SNAP-tag is a derivative of O 6-alkylguanine-DNA alkyltransferase that couples itself covalently to benzylguanine. Bioactivities of the SNAP-YFP-cytokines were shown to be comparable with the nontagged cytokines. Efficient coupling of SNAP-YFP-cytokines to benzylguanine-modified beads was demonstrated by flow cytometry. The fact that enterokinase treatment released most of the fluorescence from the beads is indicative for directed coupling and only marginal adsorptive binding. Cellular responses to SNAP-YFP-cytokine beads were analyzed in cellular lysates and by confocal microscopy indicating that the directionally immobilized cytokines are fully signaling competent with respect to the activation of ERK and STAT3. The strategy presented here is generally applicable for the directed covalent immobilization of fluorescently labeled proteins including the convenient and reliable control of coupling efficiency and orientation. © 2011 American Chemical Society.


Schirrmann T.,TU Braunschweig | Steinwand M.,TU Braunschweig | Wezler X.,TU Braunschweig | Ten Haaf A.,Justus Liebig University | And 3 more authors.
BioDrugs | Year: 2014

Hodgkin's lymphoma (HL) and ALK+ anaplastic large-cell lymphoma (ALCL) have become highly curable due to the success of modern regimens of chemotherapy and radiotherapy. However, up to one-third of the patients experience relapse or do not respond to first-line therapy, and half of them relapse again after secondary therapy with limited options for further treatment. In the last 15 years, monoclonal antibodies (mAbs) directed to surface receptors became a new and valuable therapeutic option in many hematologic malignancies. Due to its restricted expression on normal activated lymphocytes and its high expression on malignant cells, CD30 represents an attractive target molecule for HL and ALCL therapy. However, unconjugated CD30 mAbs have demonstrated a lack of objective clinical responses in patients with recurrent HL. CD30 exhibits complex signaling pathways, and binding of its natural ligand or anti-CD30 mAbs can induce apoptosis but may also promote proliferation and activation depending on the cellular context. Moreover, CD30 rapidly internalizes after crosslinking, which counteracts efficient recruitment of immunologic effectors but also provides the opportunity to transfer cytotoxic payloads coupled to CD30-specific mAbs into the tumor cells. Several tumor targeting approaches have been studied, including radio-immunoconjugates, immunotoxins, immunoRNases, immunokinases, and antibody drug conjugates (ADCs). In 2011, the ADC brentuximab-vedotin, consisting of the CD30-specific chimeric mAb cAC10 and the potent tubulin toxin monomethyl auristatin E, gained regulatory approval as a well tolerated and highly active drug in patients with refractory and relapsed HL and ALCL. SGN-35 is on the way to being incorporated in the standard management of CD30+ lymphoma with significant therapeutic impact. This review gives a critical overview about anti-CD30 therapies with unconjugated, engineered, and conjugated mAbs and the therapeutic challenges of treatment of CD30+ lymphoma. © 2013 Springer International Publishing.


Otto J.,RWTH Aachen | Binnebsel M.,RWTH Aachen | Pietsch S.,RWTH Aachen | Anurov M.,Moscow State University | And 7 more authors.
Journal of Investigative Surgery | Year: 2010

Background: Currently, absorbable meshes are used as temporary closure in case of laparostoma. Unfortunately the multifilament polyglycolic acid (PG) meshes with small pores reveal little elasticity acting rather as a fluid barrier than permitting drainage of intra-abdominal fluids. Therefore, a new mesh was constructed of absorbable polydioxanon monofilaments (PDS) with increased porosity and longer degradation time. Material and Methods: For evaluation of the tissue response the new PDS mesh was implanted as abdominal wall replacement in each five rats for 7, 21, or 90 days, respectively, and compared to a PG mesh. Histological analysis included HE staining with measurement of the size of the granuloma and immunoshistochemistry for TUNEL, Ki67, TNF-R2, MMP-2, YB1, FVIII, gas6, AXL. Parameters for neovascularization and nerve ingrowth were analyzed. Results: The inflammatory and fibrotic tissue reaction is attenuated with PDS in comparison to PG, e.g., the size of the granuloma was smaller with less cell turnover, and less remodeling as represented by, e.g., reduction of apoptosis, expression of MMP-2, or TNF-R2. The number of ingrowing nerves and vessels explored via AXL, gas6, and factor VIII was increased in the PDS mesh. Conclusion: The results from the present investigation showed that a mesh can be constructed of monofilament PDS that induce significant less inflammatory and fibrotic reaction, however permits fluid drainage and preserves elasticity. © 2010 Informa Healthcare USA, Inc.


Amaral F.,Helmholtz Institute | Egger C.,Helmholtz Institute | Steinseifer U.,Helmholtz Institute | Schmitz-Rode T.,Institute for Applied Medical Engineering
Artificial Organs | Year: 2013

Assuming that blood has a constant viscosity is a common practice when designing rotary blood pumps (RBPs), where shear stresses are generally higher than in the human body. This eases the design and allows numerical simulations and bench top experiments to be performed with Newtonian fluids. However, specific flow conditions may cause a change in cell distribution leading to an apparent lower blood viscosity. It has been observed that decreasing the vessel diameters and increasing flow velocities contribute to this effect. Because a hydrodynamic bearing operates under flow conditions following this pattern, it is important to verify whether this effect also takes place when this type of bearing is applied to a RBP. Because the operation of a hydrodynamic bearing depends directly on the fluid viscosity, a local change in cell distribution in the bearing gap can be reflected in changes in the bearing performance. In this work, a spiral groove hydrodynamic bearing was tested with porcine blood in a specially built test rig. The generated suspension force, cross flow, and bearing torque were recorded and compared with the reference response when using a solution of water and glycerol. Experiments with porcine blood yielded lower suspension forces, lower flows, and lower bearing torques than when using the glycerol solution. An explanation could be a lower apparent viscosity due to inhomogeneity of blood cell concentrations. Therefore, it is crucial to consider the effective blood viscosity when designing hydrodynamic bearings for RBPs and performing experiments. © 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation.

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