Refine
Has Fulltext
- yes (21)
Is part of the Bibliography
- yes (21)
Year of publication
- 2013 (21) (remove)
Document Type
- Journal article (16)
- Doctoral Thesis (5)
Language
- English (21) (remove)
Keywords
- Arteriosklerose (2)
- Dendritische Zelle (2)
- Maus (2)
- atherosclerosis (2)
- expression (2)
- macrophages (2)
- membrane potential (2)
- mice (2)
- neuronal dendrites (2)
- 2-photon microscopy (1)
- Active zone (1)
- Aktive Zone (1)
- Aorta (1)
- Atherosclerosis (1)
- CCR2 (1)
- CD84 (1)
- Cluster (1)
- Conditioning (1)
- Cranial window (1)
- DNA origami (1)
- Dendritic cells (1)
- Drosophila (1)
- E1 (1)
- Enoyl-Reduktase (1)
- Enoyl-acyl-carrier-protein-Reductase (1)
- Entzündung (1)
- Fettsäurebiosynthese (1)
- Fettsäurestoffwechsel (1)
- Fluorescence (1)
- Fragiles X Syndrom (1)
- GPVI (1)
- Genexpression (1)
- HIF1alpha (1)
- Hypoxia (1)
- Hypoxie (1)
- In vivo imaging (1)
- Inhibition (1)
- Kristallstruktur (1)
- Learning and memory (1)
- Lernen und Gedächtnis (1)
- Lipide (1)
- Lung cancer (1)
- Lungenkrebs (1)
- Malignant effusion (1)
- Metabotropic glutamate receptor (1)
- Metalloproteinase (1)
- Metalloproteinasen (1)
- Molekulare Marker (1)
- Mouse model (1)
- Mushroom body (1)
- Neurobiologie (1)
- Neurogenetik (1)
- Neurons (1)
- Oncolytic virotherapy (1)
- Pilzkörper (1)
- SDS polyacrylamide gel electrophoresis (1)
- Staphylococcus aureus (1)
- Synapse (1)
- T-cells (1)
- TGN1412 (1)
- Taufliege (1)
- Thrombozyt (1)
- VEGF (1)
- Vascular endothelial Growth Factor (1)
- Wirkstoff (1)
- Zelle (1)
- Zentralnervensystem (1)
- accumulation (1)
- actin (1)
- anions (1)
- antagonists (1)
- anthrax (1)
- antibiotics (1)
- antioxidant function (1)
- astrocytes (1)
- atheriosclerotic lesions (1)
- atomic force microscopy (AFM) (1)
- atopic dermatitis (1)
- bacterial meningitis (1)
- bioconjugation (1)
- biosensors (1)
- blue native polyacrylamide gel electrophoresis (1)
- bone-marrow (1)
- borrelia burgdorferi (1)
- brain damage (1)
- carcinoma (1)
- cell membranes (1)
- cell wall (1)
- cell walls (1)
- cell-cycle arrest (1)
- cerebellum (1)
- chemokine (1)
- chloroquine (1)
- cholesterol-dependent cytolysin (1)
- complex (1)
- conjugation (1)
- conservation (1)
- conserving surgery (1)
- corynebacteria (1)
- corynebacterium diphtheriae (1)
- cytokine release (1)
- damage responses (1)
- dendritic cells (1)
- dentate gyrus (1)
- detergents (1)
- developmental cycle (1)
- embryos (1)
- enoyl reductase (1)
- escherichia coli (1)
- fatty acid biosynthesis (1)
- fluorescence imaging (1)
- fluorescence microscopy (1)
- fractalkine (1)
- glutamate (1)
- gram negative bacteria (1)
- granule cells (1)
- hippocampus (1)
- histology (1)
- human interleukin-4 (1)
- hydrodynamics (1)
- hypercholeterolemia (1)
- hyperlipedemic mice (1)
- in vivo imaging (1)
- inflammation (1)
- inflammatory sites (1)
- intoxication (1)
- ligases (1)
- lipid bilayer (1)
- magnetic resonance imaging (1)
- marcophages (1)
- mastectomy (1)
- matrix protein porin (1)
- membrane (1)
- meningitis (1)
- metastases (1)
- miR-17~92 (1)
- miRNS (1)
- mobilization (1)
- molecular mass (1)
- monoclonal antibodies (1)
- monocyte (1)
- murine (1)
- mutant p53 (1)
- mycobacterium smegmatis (1)
- nanoelectronics (1)
- nanolithography (1)
- nanomedicine (1)
- nanorobot (1)
- nanotechnology (1)
- neddylation (1)
- neocortex (1)
- neuronal differentiation (1)
- neurons (1)
- oncolytic viruses (1)
- outer membrane proteins (1)
- oxazolone colitis (1)
- p53-dependent apoptosis (1)
- p53-inducible regulator (1)
- pathway (1)
- permeability (1)
- platelets (1)
- pneumolysin (1)
- pore-forming toxin (1)
- protein NEDD8 (1)
- protochlamydia amoebophila (1)
- purification (1)
- radiation-therapy (1)
- radii (1)
- recruitment (1)
- responses (1)
- restoration (1)
- rheumatoid arthritis (1)
- senescence (1)
- signal peptides (1)
- single channel analysis (1)
- single channel recording (1)
- single molecule (1)
- sodium-iodide symporter (1)
- stage-i (1)
- subsets (1)
- synapses (1)
- system (1)
- thyroid-cancer (1)
- toxins (1)
- tumorigenesis (1)
Institute
- Rudolf-Virchow-Zentrum (21) (remove)
Background: Animal models of human inflammatory diseases have limited predictive quality for human clinical trials for various reasons including species specific activation mechanisms and the immunological background of the animals which markedly differs from the genetically heterogeneous and often aged patient population.
Objective: Development of an animal model allowing for testing therapeutics targeting pathways involved in the development of Atopic Dermatitis (AD) with better translatability to the patient.
Methods: NOD-scid IL2R \(\gamma^{null}\) mice engrafted with human peripheral blood mononuclear cells (hPBMC) derived from patients suffering from AD and healthy volunteers were treated with IL-4 and the antagonistic IL-4 variant R121/Y124D (Pitrakinra). Levels of human (h) IgE, amount of B-, T- and plasma-cells and ratio of CD4 : CD8 positive cells served as read out for induction and inhibition of cell proliferation and hIgE secretion. Results were compared to in vitro analysis.
Results: hIgE secretion was induced by IL-4 and inhibited by the IL-4 antagonist Pitrakinra in vivo when formulated with methylcellulose. B-cells proliferated in response to IL-4 in vivo; the effect was abrogated by Pitrakinra. IL-4 shifted CD4 : CD8 ratios in vitro and in vivo when hPBMC derived from healthy volunteers were used. Pitrakinra reversed the effect. Human PBMC derived from patients with AD remained inert and engrafted mice reflected the individual responses observed in vitro.
Conclusion: NOD-scid IL2R \(\gamma^{null}\) mice engrafted with human PBMC reflect the immunological history of the donors and provide a complementary tool to in vitro studies. Thus, studies in this model might provide data with better translatability from bench to bedside.
Introduction: Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model.
Methods: GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 mu Ci of I-124-iodide.
Results: Viral infectivity, measured by green fluorescent protein (GFP) expression, was time-and concentrationdependent. All cell lines showed less than 10% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( < 10,000-fold increase from the initial viral dose) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm(3) versus 168 mm(3) in untreated controls (P < 0.05).
Conclusions: This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors.
Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53(E177R) mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.
We used a novel approach of cytostatically induced leucocyte depletion and subsequent reconstitution with leucocytes deprived of classical \((inflammatory/Gr1^{hi})\) or non-classical \((resident/Gr1^{lo})\) monocytes to dissect their differential role in atheroprogression under high-fat diet (HFD). Apolipoprotein E-deficient \((Apoe^{-/-})\) mice lacking classical but not non-classical monocytes displayed reduced lesion size and macrophage and apoptotic cell content. Conversely, HFD induced a selective expansion of classical monocytes in blood and bone marrow. Increased CXCL1 levels accompanied by higher expression of its receptor CXCR2 on classical monocytes and inhibition of monocytosis by CXCL1-neutralization indicated a preferential role for the CXCL1/CXCR2 axis in mobilizing classical monocytes during hypercholesterolemia. Studies correlating circulating and lesional classical monocytes in gene-deficient \(Apoe^{-/-}\) mice, adoptive transfer of gene-deficient cells and pharmacological modulation during intravital microscopy of the carotid artery revealed a crucial function of CCR1 and CCR5 but not CCR2 or \(CX_3CR1\) in classical monocyte recruitment to atherosclerotic vessels. Collectively, these data establish the impact of classical monocytes on atheroprogression, identify a sequential role of CXCL1 in their mobilization and CCR1/CCR5 in their recruitment.
The eukaryotic actin cytoskeleton is an evolutionarily well-established pathogen target, as a large number of bacterial factors disturb its dynamics to alter the function of the host cells. These pathogenic factors modulate or mimic actin effector proteins or they modify actin directly, leading to an imbalance of the precisely regulated actin turnover. Here, we show that the pore-forming, cholesterol-dependent cytolysin pneumolysin (PLY), a major neurotoxin of Streptococcus pneumoniae, has the capacity to bind actin directly and to enhance actin polymerisation in vitro. In cells, the toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by Förster resonance energy transfer. PLY was capable of exerting its effect on actin through the lipid bilayer of giant unilamellar vesicles, but only when its pore competence was preserved. The dissociation constant of G-actin binding to PLY in a biochemical environment was 170–190 nM, which is indicative of a high-affinity interaction, comparable to the affinity of other intracellular actin-binding factors. Our results demonstrate the first example of a direct interaction of a pore-forming toxin with cytoskeletal components, suggesting that the cross talk between pore-forming cytolysins and cells is more complex than previously thought.
Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display
(2013)
The C-terminal peptides of ubiquitin (UB) and UB-like proteins (UBLs) play a key role in their recognition by the specific activating enzymes (E1s) to launch their transfer through the respective enzymatic cascades thus modifying cellular proteins. UB and Nedd8, a UBL regulating the activity of cullin-RING UB ligases, only differ by one residue at their C-termini; yet each has its specific E1 for the activation reaction. It has been reported recently that UAE can cross react with Nedd8 to enable its passage through the UB transfer cascade for protein neddylation. To elucidate differences in UB recognition by UAE and NAE, we carried out phage selection of a UB library with randomized C-terminal sequences based on the catalytic formation of UB similar to NAE thioester conjugates. Our results confirmed the previous finding that residue 72 of UB plays a "gate-keeping" role in E1 selectivity. We also found that diverse sequences flanking residue 72 at the UB C-terminus can be accommodated by NAE for activation. Furthermore heptameric peptides derived from the C-terminal sequences of UB variants selected for NAE activation can function as mimics of Nedd8 to form thioester conjugates with NAE and the downstream E2 enzyme Ubc12 in the Nedd8 transfer cascade. Once the peptides are charged onto the cascade enzymes, the full-length Nedd8 protein is effectively blocked from passing through the cascade for the critical modification of cullin. We have thus identified a new class of inhibitors of protein neddylation based on the profiles of the UB C-terminal sequences recognized by NAE.
Nanotechnological applications increasingly exploit the selectivity and processivity of biological molecules. Integration of biomolecules such as proteins or DNA into nano-systems typically requires their conjugation to surfaces, for example of carbon-nanotubes or fluorescent quantum dots. The bioconjugated nanostructures exploit the unique strengths of both their biological and nanoparticle components and are used in diverse, future oriented research areas ranging from nanoelectronics to biosensing and nanomedicine. Atomic force microscopy imaging provides valuable, direct insight for the evaluation of different conjugation approaches at the level of the individual molecules. Recent technical advances have enabled high speed imaging by AFM supporting time resolutions sufficient to follow conformational changes of intricately assembled nanostructures in solution. In addition, integration of AFM with different spectroscopic and imaging approaches provides an enhanced level of information on the investigated sample. Furthermore, the AFM itself can serve as an active tool for the assembly of nanostructures based on bioconjugation. AFM is hence a major workhorse in nanotechnology; it is a powerful tool for the structural investigation of bioconjugation and bioconjugation-induced effects as well as the simultaneous active assembly and analysis of bioconjugation-based nanostructures.
Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum
(2013)
This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures.
Background
Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy.
Methodology/Principal Findings
The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors.
Conclusions/Significance
These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.
Abstract
Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage.
Author Summary
Bacterial meningitis is one of the most devastating brain diseases. Among the bacteria that cause meningitis, Streptococcus pneumoniae is the most common. Meningitis predominantly affects children, especially in the Third World, and most of them do not survive. Those that do survive often suffer permanent brain damage and hearing problems. The exact morphological substrates of brain damage in Streptococcus pneumoniae meningitis remain largely unknown. In our experiments, we found that the brain cortex of patients with meningitis demonstrated a loss of synapses (the contact points among neurons, responsible for the processes of learning and memory), and we identified the major pneumococcal neurotoxin pneumolysin as a sufficient cause of this loss. The effect was not direct but was mediated by the brain neurotransmitter glutamate, which was released upon toxin binding by one of the non-neuronal cell types of the brain – the astrocytes. Pneumolysin initiated calcium influx in astrocytes and subsequent glutamate release. Glutamate damaged the synapses via NMDA-receptors – a mechanism similar to the damage occurring in brain ischemia. Thus, we show that synaptic loss is present in pneumococcal meningitis, and we identify the toxic bacterial protein pneumolysin as the major factor in this process. These findings alter our understanding of bacterial meningitis and establish new therapeutic strategies for this fatal disease.