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  • 1
    Keywords: CELLS ; EXPRESSION ; imaging ; GENE ; GENE-EXPRESSION ; PROTEINS ; ACTIVATION ; DNA ; recombination ; INDUCTION ; RECOGNITION ; gene expression ; VARIABILITY ; VIRUS-INFECTION ; SINGLE-CELL ; RIG-I ; BACTERIAL ARTIFICIAL CHROMOSOMES ; BETA GENE-EXPRESSION ; interferon regulatory factor-7 ; live-cell microscopy ; multi-scale modelling
    Abstract: The cellular recognition of viruses evokes the secretion of type-I interferons (IFNs) that induce an antiviral protective state. By live-cell imaging, we show that key steps of virus-induced signal transduction, IFN-beta expression, and induction of IFN-stimulated genes (ISGs) are stochastic events in individual cells. The heterogeneity in IFN production is of cellular-and not viral-origin, and temporal unpredictability of IFN-beta expression is largely due to cell-intrinsic noise generated both upstream and downstream of the activation of nuclear factor-kappa B and IFN regulatory factor transcription factors. Subsequent ISG induction occurs as a stochastic all-or-nothing switch, where the responding cells are protected against virus replication. Mathematical modelling and experimental validation show that reliable antiviral protection in the face of multi-layered cellular stochasticity is achieved by paracrine response amplification. Achieving coherent responses through intercellular communication is likely to be a more widely used strategy by mammalian cells to cope with pervasive stochasticity in signalling and gene expression.
    Type of Publication: Journal article published
    PubMed ID: 22617958
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  • 2
    Keywords: CANCER ; BIODISTRIBUTION ; POSITRON-EMISSION-TOMOGRAPHY ; DECAY ; IMMUNO-PET ; EMITTERS
    Abstract: Fast progressing immuno-PET gives reasons to develop new potential medium-long and long-lived radioisotopes. One of the promising candidates is Nb-90. It has a half-life of 14.6 h, which allows visualizing and quantifying processes with medium and slow kinetics, such as tumor accumulation of antibodies and antibodies fragments or polymers and other nanoparticles. Nb-90 exhibits a high positron branching of 53% and an optimal energy of beta(+) emission of E-mean = 0.35 MeV only. Consequently, efficient radionuclide production routes and Nb-V labeling techniques are required. Nb-90 was produced by the Zr-90(p,n)Nb-90 nuclear reaction on natural zirconium targets. No-carrier-added (n.c.a.) Nb-90 was separated from the zirconium target via a multi-step separation procedure including extraction steps and ion-exchange chromatography. Protein labeling was exemplified using the bifunctional chelator desferrioxamine attached to the monoclonal antibody rituximab. Desferrioxamine was coupled to rituximab via two different routes, by the use of N-succinyl-desferrioxamine (N-suc-Df) and by means of the bifunctional derivative p-isothiocyanatobenzyl-desferrioxarnine B (Df-Bz-NCS), respectively. Following antibody modification, labeling with Nb-90 was performed in HEPES buffer at pH 7 at room temperature. In vitro stability of the radiolabeled conjugates was tested in saline buffer at room temperature and in fetal calf serum (FCS) at 37 degrees C. The selected production route led to a high yield of 145 +/- 10 MBq/mu Ah of Nb-90 with high radioisotopic purity of 〉97%. This yield may allow for large scale production of about 10 GBq Nb-90. The separation procedure resulted in 76-81% yield. The Zr/Nb-90 decontamination factor reaches 10(7). Subsequent radiolabeling of the two different conjugates with Nb-90 gave high yields; after one hour incubation at room temperature, more than 90% of Nb-90-Df-mAb was formed in both cases. At room temperature in aqueous solution, both Nb-90-Df-mAb constructs were more than 99% stable over a period of 18d. The developed production and separation strategy provided Nb-90 with purity appropriate for radiolabeling applications. Labeling and stability studies proved the applicability of Nb-90 as a potential positron emitter for immuno-PET.
    Type of Publication: Journal article published
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  • 3
    Keywords: EXPRESSION ; CELL ; Germany ; GENERATION ; SYSTEM ; SYSTEMS ; SITE ; SITES ; GENE ; RNA ; transcription ; LINES ; TRANSDUCTION ; INFECTION ; CELL-LINES ; SEQUENCE ; SEQUENCES ; FREQUENCY ; PERFORMANCE ; VECTORS ; DESIGN ; VECTOR ; PROMOTER ; genetics ; CELL-LINE ; LINE ; GENE-THERAPY ; retroviral vector ; RETROVIRAL VECTORS ; MURINE LEUKEMIA-VIRUS ; cell lines ; INTEGRATION ; DETERMINANTS ; TECHNOLOGY ; LOCI ; VECTOR INTEGRATION ; Genetic ; RECOMBINANT RETROVIRUSES ; TITER
    Abstract: The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line
    Type of Publication: Journal article published
    PubMed ID: 20222806
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  • 4
    Keywords: CANCER ; THERAPY ; MONOCLONAL-ANTIBODIES ; PHARMACOKINETICS ; TUMOR ANGIOGENESIS ; DRUG DEVELOPMENT ; THERAPEUTICS
    Abstract: Fast progressing immuno- PET asks to explore new radionuclides. One of the promising candidates is Nb-90. It has a half-life of 14.6 h that allows visualizing and quantifying biological processes with medium and slow kinetics, such as tumor accumulation of antibodies and antibodies fragments or drug delivery systems and nanoparticles. Nb-90 exhibits a positron branching of 53% and an average kinetic energy of emitted positrons of.. mean = 0.35MeV. Currently, radionuclide production routes and Nb V labeling techniques are explored to turn this radionuclide into a useful imaging probe. However, efficient separation of Nb-90 from irradiated targets remains in challenge. Ion exchange based separation of Nb-90 from zirconium targets was investigated in systems AG 1 x 8 - HCl/H2O2 and UTEVA-HCl. Nb-95 (t(1/2) = 35.0 d), Zr-95 (t(1/2) = 64.0 d) and (92m) Nb (t(1/2) = 10.15 d) were chosen for studies on distribution coefficients. Separation after AG 1 x 8 anion exchange yields 99% of Nb-90/95. Subsequent use of a solid-phase extraction step on UTEVA resin further decontaminates Nb-90/95 from traces of zirconium with yields 95% of Nb-90/95. A semi-automated separation takes one hour to obtain an overall recovery of Nb-90/95 of 90%. The amount of Zr was reduced by factor of 10(8). The selected separation provides rapid preparation (〈 1h) of high purity Nb-90 appropriate for the synthesis of Nb-90-radiopharmaceuticals, relevant for purposes of immuno-PET. Applying the radioniobium obtained, Nb-90/95-labeling of a monoclonal antibody (rituximab) modified with desferrioxamine achieved labeling yields of〉 90% after 1 h incubation at room temperature.
    Type of Publication: Journal article published
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  • 5
    Abstract: The application of radionuclide-labeled biomolecules such as monoclonal antibodies or antibody fragments for imaging purposes is called immunoscintigraphy. More specifically, when the nuclides used are positron emitters, such as zirconium-89, the technique is referred to as immuno-PET. Currently, there is an urgent need for radionuclides with a half-life which correlates well with the biological kinetics of the biomolecules under question and which can be attached to the proteins by robust labeling chemistry. (90)Nb is a promising candidate for in vivo immuno-PET, due its half-life of 14.6h and low beta(+) energy of Emean=0.35MeV per decay. (95)Nb on the other hand, is a convenient alternative for longer-term ex vivo biodistribution studies, due to its longer half-life of (t(1/2)=35days) and its convenient, lower-cost production (reactor-based production). In this proof-of-principle work, the monoclonal antibody bevacizumab (Avastin((R))) was labeled with (95/90)Nb and in vitro and in vivo stability was evaluated in normal Swiss mice and in tumor-bearing SCID mice. Initial ex vivo experiments with (95)Nb-bevacizumab showed adequate tumor uptake, however at the same time high uptake in the liver, spleen and kidneys was observed. In order to investigate whether this behavior is due to instability of ()Nb-bevacizumab or to the creation of other ()Nb species in vivo, we performed biodistribution studies of (95)Nb-oxalate, (95)Nb-chloride and (95)Nb-Df. These potential metabolite species did not show any specific uptake, apart from bone accumulation for (95)Nb-oxalate and (95)Nb-chloride, which, interestingly, may serve as an "indicator" for the release of (90)Nb from labeled biomolecules. Concerning the initial uptake of (95)Nb-bevacizumab in non-tumor tissue, biodistribution of a higher specific activity radiolabeled antibody sample did show only negligible uptake in the liver, spleen, kidneys or bones. In-vivo imaging of a tumor-bearing SCID mouse after injection with (90)Nb-bevacizumab was acquired on an experimental small-animal PET camera, and indeed showed localization of the radiotracer in the tumor area. It is the first time that such results are described in the literature, and indicates promise of application of (90)Nb-labeled antibodies for the purposes of immuno-PET.
    Type of Publication: Journal article published
    PubMed ID: 27150030
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  • 6
    Keywords: RECEPTOR ; EXPRESSION ; Germany ; transcription ; RESPONSES ; INFECTION ; INDUCTION ; ANTIGEN ; CONTRAST ; fibroblasts ; virus ; MOUSE ; BETA ; TRANSFORMATION ; INTERFERON ; ADULT ; RE ; immortalization ; secretion ; MICE LACKING ; INFLAMMATORY RESPONSES ; INTERFERON REGULATORY FACTOR-3 ; ALPHA PRODUCTION ; ANTIVIRAL DEFENSE ; I IFN GENES ; interferon regulatory factor ; mouse fibroblast ; POSITIVE FEEDBACK ; type I interferon ; virus induction
    Abstract: Type I interferons (IFNs) have been shown to be involved in many immune defence and inflammatory responses. We here show that IFN-beta plays an absolute essential role in the efficient induction of all type I IFNs after infection of primary embryonic as well as primary adult fibroblasts with Sendai virus. In contrast, after immortalization of such fibroblasts with SV40 large T antigen, IFN-alpha 4 can be induced independently of IFN-beta. However, efficient secretion of type I IFNs even in immortalized fibroblasts is only found when the complete signalling loop is induced by IFN-beta. (c) 2005 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16091286
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  • 7
    Keywords: PEPTIDE ; RECEPTOR ; CANCER ; IN-VITRO ; tumor ; carcinoma ; Germany ; IN-VIVO ; LUNG ; VITRO ; DENSITY ; imaging ; lung cancer ; LUNG-CANCER ; TISSUE ; TUMORS ; MICE ; TIME ; PATIENT ; RAT ; CONTRAST ; BINDING ; BREAST ; BREAST-CANCER ; ACID ; ASSAY ; NUDE-MICE ; LINE ; LOCALIZATION ; BIODISTRIBUTION ; STRATEGIES ; PET ; nuclear medicine ; TRACER ; targeting ; MOLECULAR-BASIS ; RE ; SOMATOSTATIN ANALOG ; PROSTATE-CANCER CELLS ; ASSAYS ; INTERNALIZATION ; RADIOLIGAND ; Ga-68 ; bombesin analog ; EXPRESSING TUMORS ; gastrin-releasing peptide receptor imaging ; PEPTIDE RECEPTORS ; RADIOCHEMICAL INVESTIGATIONS ; VITRO/IN VIVO
    Abstract: Bombesin (BN), a 14-amino-acid peptide, shows high affinity for the human gastrin-releasing peptide receptor (GRP-r), which is overexpressed on several types of cancer, including prostate, breast, gastrointestinal, and small cell lung cancer. Thus, radiolabeled BN or BN analogs may prove to be specific tracers for diagnostic and therapeutic targeting of GRP-r-positive tumors in nuclear medicine. This study evaluated a novel BN analog labeled with the positron emitter Ga-68 for receptor imaging with PET. Methods: DOTA-PEG(2)-[D-Tyr(6), beta-Ala(11),Thi(13), Nle(14)] BN(6-14) amide (BZH3) (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid; PEG is ethyleneglycol (2-aminoethyl)carboxymethyl ether) was synthetized using the Fmoc strategy and radiolabeled with either Ga-67 or Lu-177 for in vitro and biodistribution experiments. Ga-68 for PET was obtained from a Ge-68/Ga-68 generator. In vitro binding, internalization, and efflux were determined using the pancreatic tumor cell line AR42J. Biodistribution of the peptide as a function of time and dose was studied in AR42J tumor-bearing mice. Results: In vitro assays demonstrated a high affinity of Ga-67-BZH3 (dissociation constant = 0.46 nmol/L), a rapid internalization (70% of total cell-associated activity was endocytosed after a 15-min incubation), and an intracellular retention half-life (t(1/2)) of the 67Ga activity of 16.5 +/- 2.4 h. Biodistribution indicated a dose-dependent uptake in the tumor and a prolonged tumor residence time (t(1/2) similar to 16 h). Clearance from GRP-r-negative tissues was fast, resulting in high tumor-to-tissue ratios as early as 1 h after injection. Replacing Ga-67 by Lu-177, a therapeutic radionuclide, for peptide labeling resulted in a slightly reduced (similar to 20%) tumor uptake and tumor residence time of Lu-177-BZH3. In contrast, Lu-177 decline in the pancreas was significantly accelerated by a factor of similar to 3 compared with that of Ga-67. PET of mice with Ga-68-BZH3 clearly delineated tumors in the mediastinal area. Conclusion: The promising in vivo data of Ga-68-BZH3 indicate its potential for an improved localization of GRP-r-positive tumors and also suggest its application in patients. PET may also be favorably used for GRP-r density determination, a prerequisite for therapeutic applications
    Type of Publication: Journal article published
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  • 8
    Keywords: EXPRESSION ; GENE ; MICE ; IMMUNE-RESPONSES ; INTERFERON-GAMMA ; TUMOR-METASTASIS ; ANTIVIRAL DEFENSE ; NECTIN-2 CD112 ; DNAM-1 ; PVR CD155 ; REGULATORY FACTOR-I ; TRANSCRIPTION FACTOR IRF-1
    Abstract: IFN-gamma promotes tumoral immune surveillance, but its involvement in controlling metastases is less clear. Using a mouse model of pulmonary metastases, we show that local IFN-gamma treatment inhibits formation of metastases through its regulation of IRF-1 in tumor cells. IRF-1 is an IFN-gamma-induced transcription factor pivotal in the regulation of infection and inflammation. IRF-1 blockade abolished the inhibitory effect of IFN-gamma on tumor metastases, whereas ectopic expression of IRF-1 phenocopied the inhibitory effects of IFN-gamma. IRF-1 did not affect the survival of tumor cells in the circulation or their infiltration into lungs, but it was essential to support the pulmonary attraction and activation of natural killer (NK) cells. Depleting NK cells from mice abolished the protective effect of IFN-gamma or IRF-1 on metastases. In addition, cytotoxicity assays revealed that tumor cells expressing IRF-1 were targeted more effectively by NK cells than IRF-1 nonexpressing tumor cells. Moreover, NK cells isolated from lungs inoculated with IRF-1-expressing tumor cells exhibit a greater cytotoxic activity. Mechanistic investigations revealed that IRF-1-induced NK cell cytotoxicity was independent of perforin and granzyme B but dependent on the NK cell activating receptor DNAM-1. Taken together, our findings establish IRF-1 as an essential mediator of the cross-talk between tumor cells and NK cells that mediate immune surveillance in the metastatic niche.
    Type of Publication: Journal article published
    PubMed ID: 21900395
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