• Are resorbable implants about to become a reality?

      Peuster, Matthias; Beerbaum, Phillip; Bach, Friedrich-Wilhelm; Hauser, Hansjoerg; Clinic for Congenital Heart Defects and Cardiovascular Implant Research Unit, Heart and Diabetes Center, Ruhr-University Bochum, Bad Oeynhausen, Germany. mpeuster@hdz-nrw.de (2006-04)
    • Bimodal and hysteretic expression in mammalian cells from a synthetic gene circuit.

      May, Tobias; Eccleston, Lee; Herrmann, Sabrina; Hauser, Hansjörg; Goncalves, Jorge; Wirth, Dagmar; Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2008)
      In order to establish cells and organisms with predictable properties, synthetic biology makes use of controllable, synthetic genetic devices. These devices are used to replace or to interfere with natural pathways. Alternatively, they may be interlinked with endogenous pathways to create artificial networks of higher complexity. While these approaches have been already successful in prokaryotes and lower eukaryotes, the implementation of such synthetic cassettes in mammalian systems and even animals is still a major obstacle. This is mainly due to the lack of methods that reliably and efficiently transduce synthetic modules without compromising their regulation properties. To pave the way for implementation of synthetic regulation modules in mammalian systems we utilized lentiviral transduction of synthetic modules. A synthetic positive feedback loop, based on the Tetracycline regulation system was implemented in a lentiviral vector system and stably integrated in mammalian cells. This gene regulation circuit yields a bimodal expression response. Based on experimental data a mathematical model based on stochasticity was developed which matched and described the experimental findings. Modelling predicted a hysteretic expression response which was verified experimentally. Thereby supporting the idea that the system is driven by stochasticity. The results presented here highlight that the combination of three independent tools/methodologies facilitate the reliable installation of synthetic gene circuits with predictable expression characteristics in mammalian cells and organisms.
    • Caveolin-1 influences human influenza A virus (H1N1) multiplication in cell culture.

      Sun, Lijing; Hemgård, Gun-Viol; Susanto, Sony A; Wirth, Manfred; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2010)
      The threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication.
    • Characterisation of bovine leukocyte Ig-like receptors.

      Hogan, Louise; Bhuju, Sabin; Jones, Des C; Laing, Ken; Trowsdale, John; Butcher, Philip; Singh, Mahavir; Vordermeier, Martin; Allen, Rachel L; Centre for Infection, Division of Clinical Sciences, St George's, University of London, Cranmer Terrace, London, United Kingdom. p0904768@sgul.ac.uk (2012)
      Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.
    • Comparison of in vitro and in vivo protein release from hydrogel systems.

      Wöhl-Bruhn, Stefanie; Badar, Muhammad; Bertz, Andreas; Tiersch, Brigitte; Koetz, Joachim; Menzel, Henning; Mueller, Peter P; Bunjes, Heike; Technische Universität Braunschweig, Institute of Pharmaceutical Technology, Mendelssohnstraße 1, 38106 Braunschweig, Germany. (2012-08-20)
      Hydrogel systems based on hydroxyethyl starch-polyethylene glycol methacrylate (HES-P(EG)(6)MA) or hydroxyethyl starch methacrylate (HES-MA) were used to assess the protein release behavior. Here, we analyzed the in vitro release of FITC-anti-human antibodies incorporated in either HES-P(EG)(6)MA or HES-MA hydrogel delivery systems in PBS or human serum. In addition, hydrogel disks and microparticles prepared from the two polymers were subcutaneously implanted in BALB/c mice. The in vivo release of FITC-IgG was non-invasively monitored by an in vivo imaging system (IVIS 200) over a time period of up to 3 months. The imaging system allowed to asses individual animals over time, therefore only a small number of animals was required to obtain high quality data. The reduction in fluorescence intensity at the site of administration was compared to in vitro release profiles. These investigations demonstrated a sustained release from HES-MA hydrogel disks compared to rapidly degrading HES-P(EG)(6)MA disks and microparticles. The sustained release from HES-MA disks could be further optimized by using increased polymer concentrations. Human serum as in vitro release medium reflected better the in vivo release from HES-P(EG)(6)MA systems than PBS, suggesting that the presence of organic substances like proteins or lipids may play a significant role for the release kinetics.
    • An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo.

      Jenke, Bok Hee C; Fetzer, Christian P; Stehle, Isa M; Jönsson, Franziska; Fackelmayer, Frank O; Conradt, Harald; Bode, Jürgen; Lipps, Hans J; Institute of Cell Biology, Stockumer Strasse 10, University of Witten/Herdecke, D-58448 Witten. (2002-04)
      pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.
    • Essential role of CCL2 in clustering of splenic ERTR-9+ macrophages during infection of BALB/c mice by Listeria monocytogenes.

      Jablonska, Jadwiga; Dittmar, Kurt E; Kleinke, Tanja; Buer, Jan; Weiss, Siegfried; Molecular Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. jja@gbf.de (2007-01)
      Early interactions between pathogens and host cells are often decisive for the subsequent course of infection. Here we investigated early events during infection by Listeria monocytogenes, a ubiquitously occurring facultative intracellular microorganism that exhibits severe pathogenicity, mainly in immunocompromised individuals. We show that the inflammatory chemokine CCL2 is highly up-regulated early after Listeria infection in spleens of BALB/c mice. ERTR-9+ macrophages of the marginal zone were identified as the only infected cells and exclusive producers of CCL2 at the early time point. Consequently, clusters of different cell types were formed around infected ERTR-9+ cells. Metallophilic MOMA-1+ marginal zone macrophages were, however, excluded from the clusters and migrated into the B-cell follicles. Depletion of CCL2 during infection resulted in a different composition of cell clusters in the spleen and increased the mortality rate of treated mice. Interestingly, ERTR-9+ macrophages no longer were part of clusters in such mice but remained at their original location in the marginal zone.
    • High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

      Hemmen, Katherina; Reinl, Tobias; Buttler, Kerstin; Behler, Friederike; Dieken, Hauke; Jänsch, Lothar; Wilting, Jörg; Weich, Herbert A; Department of Gene Regulation, HZI, Build. D, Inhoffenstr. 7, 38124, Braunschweig, Germany. katharina.hemmen@ewetel.net (2011-05)
      Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.
    • Human bone marrow mesenchymal stem cells: a systematic reappraisal via the genostem experience.

      Charbord, Pierre; Livne, Erella; Gross, Gerhard; Häupl, Thomas; Neves, Nuno M; Marie, Pierre; Bianco, Paolo; Jorgensen, Christian (2011-03)
      Genostem (acronym for "Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side") has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.
    • Identification of IRF-8 and IRF-1 target genes in activated macrophages.

      Dror, Natalie; Alter-Koltunoff, Michal; Azriel, Aviva; Amariglio, Ninette; Jacob-Hirsch, Jasmine; Zeligson, Sharon; Morgenstern, Avigail; Tamura, Tomohiko; Hauser, Hansjörg; Rechavi, Gideon; et al. (2007-01)
      Interferon regulatory factor 1 (IRF-1) and IRF-8, also known as interferon consensus sequence binding protein (ICSBP), are important regulators of macrophage differentiation and function. These factors exert their activities through the formation of heterocomplexes. As such, they are coactivators of various interferon-inducible genes in macrophages. To gain better insights into the involvement of these two transcription factors in the onset of the innate immune response and to identify their regulatory network in activated macrophages, DNA microarray was employed. Changes in the expression profile were analyzed in peritoneal macrophages from wild type mice and compared to IRF-1 and IRF-8 null mice, before and following 4 h exposure to IFN-gamma and LPS. The expression pattern of 265 genes was significantly changed (up/down) in peritoneal macrophages extracted from wild type mice following treatment with IFN-gamma and LPS, while no changes in the expression levels of these genes were observed in samples of the same cell-type from both IRF-1 and IRF-8 null mice. Among these putative target genes, numerous genes are involved in macrophage activity during inflammation. The expression profile of 10 of them was further examined by quantitative RT-PCR. In addition, the promoter regions of three of the identified genes were analyzed by reporter gene assay for the ability to respond to IRF-1 and IRF-8. Together, our results suggest that both IRF-1 and IRF-8 are involved in the transcriptional regulation of these genes. We therefore suggest a broader role for IRF-1 and IRF-8 in macrophages differentiation and maturation, being important inflammatory mediators.
    • Innovative strategies for treatment of soft tissue injuries in human and animal athletes.

      Hoffmann, Andrea; Gross, Gerhard; Department of Molecular Biotechnology, Helmholtz Centre for Infection Research, Braunschweig, Germany. Andrea.Hoffmann@helmholtz-hzi.de (2009)
      Our aim is to review the recent progress in the management of musculoskeletal disorders. We will cover novel therapeutic approaches based on growth factors, gene therapy and cells, including stem cells, which may be combined with each other as appropriate. We focus mainly on the treatment of soft tissue injuries - muscle, cartilage, and tendon/ligament for both human and animal athletes. The need for innovative strategies results from the fact that despite all efforts, the current strategies for cartilage and tendon/ligament still result in the formation of functionally and biomechanically inferior tissues after injury (a phenomenon called 'repair' as opposed to proper 'regeneration'), whereas the outcome for muscle is more favorable. Innovative approaches are urgently needed not only to enhance the outcome of conservative or surgical procedures but also to speed up the healing process from the very long disabling periods, which is of special relevance for athletes.
    • Integrated strategy for the production of therapeutic retroviral vectors.

      Carrondo, Manuel; Panet, Amos; Wirth, Dagmar; Coroadinha, Ana Sofia; Cruz, Pedro; Falk, Haya; Schucht, Roland; Dupont, Francis; Geny-Fiamma, Cécile; Merten, Otto-Wilhelm; et al. (2011-03)
      The broad application of retroviral vectors for gene delivery is still hampered by the difficulty to reproducibly establish high vector producer cell lines generating sufficient amounts of highly concentrated virus vector preparations of high quality. To enhance the process for producing clinically relevant retroviral vector preparations for therapeutic applications, we have integrated novel and state-of-the-art technologies in a process that allows rapid access to high-efficiency vector-producing cells and consistent production, purification, and storage of retroviral vectors. The process has been designed for various types of retroviral vectors for clinical application and to support a high-throughput process. New modular helper cell lines that permit rapid insertion of DNA encoding the therapeutic vector of interest at predetermined, optimal chromosomal loci were developed to facilitate stable and high vector production levels. Packaging cell lines, cultivation methods, and improved medium composition were coupled with vector purification and storage process strategies that yield maximal vector infectivity and stability. To facilitate GMP-grade vector production, standard of operation protocols were established. These processes were validated by production of retroviral vector lots that drive the expression of type VII collagen (Col7) for the treatment of a skin genetic disease, dystrophic epidermolysis bullosa. The potential efficacy of the Col7-expressing vectors was finally proven with newly developed systems, in particular in target primary keratinocyte cultures and three-dimensional skin tissues in organ culture.
    • Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid--an experimental study in rats.

      Schliephake, Henning; Weich, Herbert A; Dullin, Christian; Gruber, Rudolf; Frahse, Sarah; Department of Oral and Maxillofacial Surgery, George-Augusta-University, Robert-Koch-Strasse 40, 37075 Göttingen, Germany. schliephake.henning@med.uni-goettingen.de (2008-01)
      The aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-DL-lactic acid discs were produced and loaded with 48 and 96 microg rhBMP-2 and inserted into non-healing defects of the mandible of 45 Wistar rats. Fifteen rats received implants with 96 microg rhBMP-2 (Group 2), 48 microg rhBMP-2 (Group 1) and blank implants without BMP (Group 0) each on one side of the mandible. Unfilled defects of the same size on the contralateral sides of the mandibles served as empty controls. After 6, 13 and 26 weeks, implants of each group were retrieved from five animals each and submitted to flat panel detector computed tomography. Bone formation and thickness of augmentation was assessed by computer-assisted histomorphometry. In Group 2 significantly more bone was produced than in Group 1. Implants of Group 1 induced significantly more bone than the blank controls only after 6 weeks, whereas the difference was not significant after 13 and 26 weeks. Differences between Group 2 and Group 1 were clearly significant after 26 weeks. The thickness of bone tissue was maintained in Group 2 whereas it decreased in Group 1 and was negligible in Group 0. It is concluded that the PLA implants with 96 microg rhBMP-2 were able to bridge a non-healing defect in the rat mandible and maintained the thickness of an augmented volume. However, continuous supply of osteogenic signals appears to be required to compensate for adverse effects during polymer degradation.
    • Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels.

      Schniedermann, Judith; Rennecke, Moritz; Buttler, Kerstin; Richter, Georg; Städtler, Anna-Maria; Norgall, Susanne; Badar, Muhammad; Barleon, Bernhard; May, Tobias; Wilting, Jörg; et al. (2010)
      Postnatal endothelial progenitor cells (EPCs) have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated.
    • NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element.

      Reboll, Marc René; Oumard, André; Gazdag, Aniko Carla; Renger, Isabelle; Ritter, Birgit; Schwarzer, Michael; Hauser, Hansjoerg; Wood, Monika; Yamada, Michiyuki; Resch, Klaus; et al. (2007-08)
      The mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.
    • Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration.

      Schucht, Roland; Lydford, Simon; Andzinski, Lisa; Zauers, Jeannette; Cooper, James; Hauser, Hansjörg; Wirth, Dagmar; May, Tobias; Department of Gene Regulation and Differentiation, HZI-Helmholtz Centre for Infection Research, Braunschweig, Germany. roland.schucht@inscreenex.com (2011-03)
      The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.
    • Screening of photochemically grafted polymer films for compatibility with osteogenic precursor cells.

      Adden, Nina; Hoffmann, Andrea; Gross, Gerhard; Windhagen, Henning; Thorey, Fritz; Menzel, Henning; Institut für Technische Chemie, Abt. TC Makromolekularer Stoffe, Technische Universität Braunschweig, Hans-Sommer-Strasse 10, D-38106 Braunschweig, Germany. (2007)
      Surfaces of biomaterials often do not have the ideal properties for direct application in vivo. Although titanium and its alloys show a good biocompatibility, in some applications there is still need to improve the osteoblast adhesion to titanium implants. A polymeric surface coating is an ideal solution because the polymer can be adjusted to the needs of the application and can be bound to the surface by the photochemical grafting method. Therefore, 22 different polymers were tested for their compatibility using a murine mesenchymal progenitor cell line and three polymers were identified for which more elaborate investigations are reasonable. It was investigated whether or not the results of the cell culture test can be correlated with, e.g., the wetting properties. Indeed it was found that a contact angle above approx. 45 degrees was necessary for good cell adhesion and proliferation. However, otherwise no clear correlation between the contact angle hysteresis or the functionalities of the polymers and the cell growth was observed.