82 results on '"Hohenadl C"'
Search Results
2. UV-B Irradiated Cell Lines Execute Programmed Cell Death in Various Forms
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Hagenhofer, M., Germaier, H., Hohenadl, C., Rohwer, P., Kalden, J. R., and Herrmann, M.
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- 1998
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3. Nucleo-cytoplasmic export of Mouse mammary tumour virus RNAs: YSF-66
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Mullner, M., Salmons, B., Gunzburg, W., Hohenadl, C., and Indik, S.
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- 2009
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4. Human herpesvirus-8 (HHV-8) gene expression in Kaposi's sarcoma (KS) primary lesions: an in situ hybridization study
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Stürzl, M, Wunderlich, A, Ascherl, G, Hohenadl, C, Monini, P, Zietz, C, Browning, PJ, Neipel, F, Biberfeld, P, and Ensoli, B
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- 1999
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5. Infection With Human Immunodeficiency Virus-1 Increases Expression of Vascular Endothelial Cell Growth Factor in T Cells: Implications for Acquired Immunodeficiency Syndrome-Associated Vasculopathy
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Ascherl, G., Hohenadl, C., Schatz, O., Shumay, E., Bogner, J., Eckhart, L., Tschachler, E., Monini, P., Ensoli, B., and Stürzl, M.
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- 1999
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6. Immune escape in colorectal carcinoma: role of the IFN-gamma pathway
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Stürzl, M, Hohenadl, C, Lipnik, K, Ocker, M, Naschberger, E, Schellerer, V, Croner, R, and Britzen-Laurent, N
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ddc: 610 ,610 Medical sciences ,Medicine - Abstract
Introduction: The presence of a Th1 adaptive immune response has been associated with improved clinical outcome and survival of patients with colorectal carcinoma (CRC). The Th1 adaptive immune response is mediated by IFN-γ. Using guanylate-binding protein 1 (GBP-1) as a well established marker[for full text, please go to the a.m. URL], 128. Kongress der Deutschen Gesellschaft für Chirurgie
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- 2011
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7. Clinical impact of secreted hGBP-1
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Gonin-Laurent, N, Lipnik, K, Naschberger, E, Meyer, N, Reipschläger, S, Hohenadl, C, Hohenberger, W, and Stürzl, M
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ddc: 610 - Published
- 2008
8. Secretion and Uptake of Recombinant GBP-1: A New Approach for an Anti-Angiogenic Tumor Therapy
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Metzner, M, Hupe, M, Naschberger, E, Thurau, M, Hohenadl, C, Lipnik, K, Mörtinger, T, Hohenberger, W, and Stürzl, M
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ddc: 610 - Published
- 2006
9. Foot-and-mouth disease virus leader proteinase Purification of the Lb form and determination of its cleavage site on eIF-4γ
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Kirchweger, R., Ziegler, E., Lamphear, B. J., Waters, D., Liebig -, H. D., Sommergruber, W., Francisco Sobrino, Hohenadl, C., Blaas, D., Rhoads, R. E., and Skern, T.
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viruses - Abstract
Many picornaviruses cause a dramatic decrease in the translation of cellular mRNAs in the infected cell, without affecting the translation of their own RNA. Specific proteolysis of protein synthesis initiation factor eIF-4γ occurs during infection with rhinoviruses, enteroviruses, and aphthoviruses, apparently leading to an inability of the ribosomes to bind capped mRNAs. Cleavage of eIF-4γ in human rhinoviruses and enteroviruses is carried out by the viral 2A proteinase; in aphthoviruses (i.e.;foot-and- mouth disease viruses), the leader proteinase is responsible for this reaction. We describe here the purification to homogeneity of the Lb form of the leader proteinase expressed in Escherichia coli. The primary cleavage products of eIF-4γ obtained in vitro with purified leader or 2A proteinase are electrophoretically indistinguishable from those found during infection in vivo. However, additional proteolysis products of eIF-4γ are observed with the leader proteinase and the human rhinovirus type 2 2A proteinase in vitro. The cleavage site of the leader proteinase in eIF-4γ from rabbit reticulocyte was determined by sequencing the purified C-terminal cleavage product by automated Edman degradation. The cleavage site is between Gly-479 and Arg-480 and thus differs from that of rhinovirus and enterovirus 2A proteinases, which cleave between Arg-486 and Gly-487.
- Published
- 1994
10. Primary Effusion Lymphoma Cells Undergoing Human Herpesvirus Type 8 Productive Infection Produce C-Type Retroviral Particles.
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Tinari, A., Superti, F., Ammendolia, M.G., Chiozzini, C., Hohenadl, C., Leone, P., Nappi, F., Nicoletti, M., Borsetti, A., Marchetti, M., Ensoli, B., and Monini, P.
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- 2008
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11. Expression of K13/v-FLIP gene of human herpesvirus 8 and apoptosis in Kaposi's sarcoma spindle cells.
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Stürzl, M, Hohenadl, C, Zietz, C, Castanos-Velez, E, Wunderlich, A, Ascherl, G, Biberfeld, P, Monini, P, Browning, P J, and Ensoli, B
- Abstract
Background: Human herpesvirus 8 (HHV8) infection is associated with all forms of Kaposi's sarcoma (KS). The HHV8 genome locus ORFK13-72-73 (ORF = open reading frame) encodes proteins that may be important in HHV8-mediated pathogenesis, i.e., the latency-associated nuclear antigen (encoded by ORF73), viral-cyc-D (v-cyc-D), a viral homologue of cellular cyclin D (encoded by ORF72), and viral-FLIP (v-FLIP), a homologue of the cellular FLICE (Fas-associated death domain-like interleukin 1 beta-converting enzyme) inhibitory protein (encoded by ORFK13; is an inhibitor of apoptosis [programmed cell death]). Through differential splicing events, this locus expresses individual RNA transcripts that encode all three proteins (tricistronic transcripts) or just two of them (v-FLIP and v-cyc-D; bicistronic transcripts). We examined expression of these transcripts in KS tissues.Methods: We collected tissues from patients with KS of different stages. By use of an optimized in situ hybridization procedure, we examined different ORFK13-72-73 locus transcripts in HHV8-infected cells in skin lesions and in one adjacent lymph node. Apoptosis in KS lesions was determined by use of an in situ assay.Results and Conclusions: Our results indicate the following: 1) Transcripts from the ORFK13-72-73 locus appear to be spliced differentially in latently infected KS cells in skin lesions and in HHV8-infected cells in lymph nodes; specifically, ORFK13-ORF72 bicistronic transcripts were expressed abundantly in KS cells, whereas ORFK13-ORF72-ORF73 tricistronic transcripts were detected only in lymph node cells. 2) Sequences encoding the antiapoptotic protein v-FLIP are expressed at very low levels in early KS lesions, but expression increases dramatically in late-stage lesions. 3) The increase in expression of v-FLIP-encoding transcripts is associated with a reduction in apoptosis in KS lesions.Implications: These data suggest that functional v-FLIP is produced in vivo and that antiapoptotic mechanisms may be involved in the rapid growth of KS lesions, where only a few cells undergoing mitosis are generally observed. [ABSTRACT FROM AUTHOR]- Published
- 1999
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12. Clinical: Other.
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Engelson, E.S., Kotler, D.P., Tan, Y.X., Agin, D., Wang, J., Pierson, R.N., Diaz, P.T., King, M.A., Pacht, E.R., Wewers, M.D., Gadek, J.E., Neal, D., Ascherl, G., Hohenadl, C., Schatz, O., Shumay, E., Bogner, J., and Eckhart, L.
- Abstract
Reports global developments related to AIDS as of August 1999. Association of changes in body composition with a syndrome of profound body habitus and metabolic alterations; Pathophysiology of pulmonary diffusion impairment in HIV infection; Risk factors for bleeding and mortality in HIV-infected patients with acute lower GI hemorrhage.
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- 1999
13. Molecular studies on enteroviral heart disease: patterns of acute and persistent infections.
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Kandolf, R., Klingel, K., Mertsching, H., Canu, A., Hohenadl, C., Zell, R., Reimann, B. Y., Heim, A., McManus, B. M., Foulis, A. K., Schultheiss, H.-P., Erdmann, E., and Riecker, G.
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- 1991
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14. Strand-specific detection of enteroviral RNA in myocardial tissue by in situ hybridization
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Hohenadl, C., Klingel, K., Mertsching, J., Hofschneider, P.H., and Kandolf, R.
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- 1991
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15. UVB irradiation of human keratinocytes causes enhanced expression of endogenous retroviral sequences.
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Hohenadl, C., Germaier, H., Erfie, V., Hehlmann, R., and Leib-Mösch, C.
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- 1996
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16. Transcription of endogenous retroviral sequences in human keratinocytes is activated by UVB-irradiation
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Germaier, H., Hohenadl, C., Erfle, V., Leib-Mösch, C., Walchner, M., and Kind, P.
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- 1998
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17. Long-Term Biocompatibility of a Highly Viscously Thiol-Modified Cross-Linked Hyaluronate as a Novel Vitreous Body Substitute.
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Hurst J, Rickmann A, Heider N, Hohenadl C, Reither C, Schatz A, Schnichels S, Januschowski K, and Spitzer MS
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Purpose: In surgical ophthalmology, the treatment of complicated retinal and vitreous diseases is one of the central challenges. For this purpose, the vitreous body is removed as part of the standard therapy and replaced by a temporary tamponade to stabilize the position of the retina. Since the tamponading properties of previous materials such as silicone oils, gases, or semi-fluorinated alkanes are a combination of their surface tension and their buoyancy vector, they cannot completely fill the vitreous cavity. The aim of this work was to test in vivo a novel vitreous body substitute (ViBos strong) based on cross-linked hyaluronic acid for its compatibility. Methods: A pars plana vitrectomy with posterior vitreous detachment was performed in the right eye of 18 pigmented rabbits, with subsequent injection of ViBos strong. Follow-up examination included slit-lamp examination, funduscopy, intraocular pressure measurements (IOP), optical coherence tomography (OCT), and electroretinogram (ERG) measurements. The rabbits were sacrificed at three different time points (1, 3, and 6 months; each 6 animals) and examined macroscopically and prepared for histological examination (HE staining) and immunohistochemistry (Brn3a and glial fibrillary acidic protein (GFAP)). Results: ViBos strong demonstrated good intraoperative handling and remained stable for at least 1 month and degraded slowly over 6 months. IOP was within clinical acceptable values at all follow-up examinations. Retinal function was well preserved after instillation of the hydrogel and comparable to the untreated eye after 6 months in OCT, ERG, and histological examinations. An increase in the GFAP expression was found in the surgery eyes, with a peak in the 3-month group. The Brn3a expression was not significantly affected by vitrectomy with ViBos strong. Conclusion: Highly viscously thiol-modified cross-linked hyaluronate showed a good biocompatibility in rabbit eyes over 6 months after vitrectomy, making it a promising potential as a vitreous substitute., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hurst, Rickmann, Heider, Hohenadl, Reither, Schatz, Schnichels, Januschowski and Spitzer.)
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- 2022
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18. Deep learning differentiates between healthy and diabetic mouse ears from optical coherence tomography angiography images.
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Pfister M, Stegmann H, Schützenberger K, Schäfer BJ, Hohenadl C, Schmetterer L, Gröschl M, and Werkmeister RM
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- Algorithms, Animals, Case-Control Studies, Diagnosis, Differential, Female, Image Processing, Computer-Assisted, Machine Learning, Mice, ROC Curve, Reproducibility of Results, Angiography, Deep Learning, Diabetes Mellitus, Experimental diagnostic imaging, Diabetes Mellitus, Experimental pathology, Ear blood supply, Ear diagnostic imaging, Tomography, Optical Coherence methods
- Abstract
We trained a deep learning algorithm to use skin optical coherence tomography (OCT) angiograms to differentiate between healthy and type 2 diabetic mice. OCT angiograms were acquired with a custom-built OCT system based on an akinetic swept laser at 1322 nm with a lateral resolution of ∼13 μm and using split-spectrum amplitude decorrelation. Our data set consisted of 24 stitched angiograms of the full ear, with a size of approximately 8.2 × 8.2 mm, evenly distributed between healthy and diabetic mice. The deep learning classification algorithm uses the ResNet v2 convolutional neural network architecture and was trained on small patches extracted from the full ear angiograms. For individual patches, we obtained a cross-validated accuracy of 0.925 and an area under the receiver operating characteristic curve (ROC AUC) of 0.974. Averaging over multiple patches extracted from each ear resulted in the correct classification of all 24 ears., (© 2021 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals LLC on behalf of New York Academy of Sciences.)
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- 2021
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19. Transferability study of the BINACLE (binding and cleavage) assay for in vitro determination of botulinum neurotoxin activity.
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Behrensdorf-Nicol HA, Bonifas U, Klimek J, Hanschmann KM, Dorner BG, Hohenadl C, Töllner L, Kegel B, and Krämer B
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- Animals, Biological Assay trends, Humans, Mice, Protein Binding, Proteolysis, Reproducibility of Results, Biological Assay methods, Botulinum Toxins analysis, Botulinum Toxins metabolism, Clinical Laboratory Techniques methods
- Abstract
The muscle-relaxing effects of the botulinum neurotoxin (BoNT) serotypes A and B are widely used in clinical and aesthetic medicine. The standard method for measuring the biological activity of pharmaceutical BoNT products is a mouse bioassay. In line with the European Directive 2010/63/EU, a replacement by an animal-free method would be desirable. Whereas the existing approved in vitro methods for BoNT activity measurements are product-specific and not freely available for all users, the "binding and cleavage" (BINACLE) assay could become a widely applicable alternative. This method quantifies active BoNT molecules based on their specific receptor-binding and proteolytic properties and can be applied to all BoNT products on the European market. Here we describe the results of a transferability study, in which identical BoNT samples were tested in the BINACLE assay in four laboratories. All participants successfully performed the method and observed clear dose-response relationships. Assay variability was within an acceptable range. These data indicate that the BoNT BINACLE assay is robust and can be straightforwardly transferred between laboratories. They thus provide an appropriate basis for future studies to further substantiate the suitability of the BINACLE assay for the potency determination of BoNT products., (Copyright © 2020 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)
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- 2020
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20. Cutaneous optical coherence tomography for longitudinal volumetric assessment of intradermal volumes in a mouse model.
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Schuetzenberger K, Pfister M, Messner A, Garhöfer G, Hohenadl C, Pfeiffenberger U, Schmetterer L, and Werkmeister RM
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- Animals, Biopsy, Cone-Beam Computed Tomography, Female, Hydrogels, Imaging, Three-Dimensional, Immunohistochemistry, Mice, Skin pathology, Skin diagnostic imaging, Tomography, Optical Coherence
- Abstract
Clinical evaluation of skin lesions requires precise and reproducible technologies for their qualitative and quantitative assessment. In this study, we investigate the applicability of a custom-built dermatologic OCT system for longitudinal assessment of intradermal volumes in a mouse model. The OCT, based on an akinetic swept laser working at 1310 nm was employed for visualization and quantification of intradermal deposits of three different hyaluronic acid-based hydrogel formulations - one commercial and two test substances. Hydrogels were applied in 22 BALB/c mice, and measurements were performed over a six-month time period. All hydrogels increased in volume within the first weeks and degraded steadily thereafter. The half-lifes of the test hydrogels (27.2 ± 13.6 weeks for Hydrogel 1, 31.5 ± 17.2 weeks for Hydrogel 2) were higher in comparison to the commercially available HA hydrogel (21.4 ± 12.0 weeks), although differences were not significant. The sphericity parameter was used for evaluation of the deposit geometry. While on the injection day the sphericities were similar (~0.75 ± 0.04), at later time points significant differences between the different test substances were found (T24: PRV 0.59 ± 0.09, Hydrogel 1 0.70 ± 0.11, Hydrogel 2 0.78 ± 0.07; p ≤ 0.012 for all pairs). This study shows the applicability of OCT imaging for quantitative assessment of the volumetric behavior of intradermal deposits in vivo.
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- 2020
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21. Comparison of optical coherence tomography and high frequency ultrasound imaging in mice for the assessment of skin morphology and intradermal volumes.
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Schuetzenberger K, Pfister M, Messner A, Froehlich V, Garhoefer G, Hohenadl C, Schmetterer L, and Werkmeister RM
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- Animals, Mice, Mice, Inbred BALB C, Models, Animal, Reproducibility of Results, Skin diagnostic imaging, Ultrasonography instrumentation, Skin anatomy & histology, Tomography, Optical Coherence methods, Ultrasonography methods
- Abstract
Optical coherence tomography (OCT) and high-frequency ultrasound (HFUS), two established imaging modalities in the field of dermatology, were evaluated and compared regarding their applicability for visualization of skin tissue morphology and quantification of murine intradermal structures. The accuracy and reproducibility of both methods were assessed ex vivo and in vivo using a standardized model for intradermal volumes based on injected soft tissue fillers. OCT revealed greater detail in skin morphology, allowing for detection of single layers due to the superior resolution. Volumetric data measured by OCT (7.9 ± 0.3 μl) and HFUS (7.7 ± 0.5 μl) were in good agreement and revealed a high accuracy when compared to the injected volume of 7.98 ± 0.8 µl. In vivo, OCT provided a higher precision (relative SD: 26% OCT vs. 42% HFUS) for the quantification of intradermal structures, whereas HFUS offered increased penetration depth enabling the visualization of deeper structures. A combination of both imaging technologies might be valuable for tumor assessments or other dermal pathologies in clinical settings.
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- 2019
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22. Ex vivo biophysical characterization of a hydrogel-based artificial vitreous substitute.
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Januschowski K, Schnichels S, Hurst J, Hohenadl C, Reither C, Rickmann A, Pohl L, Bartz-Schmidt KU, and Spitzer MS
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- Animals, Biophysical Phenomena, Cattle, Cross-Linking Reagents, Electroretinography, Hyaluronic Acid chemistry, Hydrogels chemistry, In Vitro Techniques, Intraocular Pressure, Materials Testing, Refractometry, Retina physiology, Rheology, Sus scrofa, Vitrectomy, Vitreous Body physiology, Vitreous Body surgery, Biocompatible Materials chemistry, Vitreous Body chemistry
- Abstract
Purpose: To characterize the biophysical properties of an artificial vitreous body substitute (VBS), which consists of a biocompatible, cross-linked, hyaluronic acid (HA)-based hydrogel, by analysing the VBS's influence on intraocular pressure (IOP) and retinal integrity in distinct ex vivo eye models in order to evaluate the its potential for in vivo biocompatibility testing., Methods: Pig eyes were obtained immediately postmortem, and VBS was injected after core-vitrectomy. IOP was followed for 24 h (n = 5). VBS influence on retinal integrity was investigated using isolated bovine retinas superfused with an oxygen saturated nutrient solution. An electroretinogram (ERG) was recorded on explanted bovine retinae using silver/silver chloride electrodes; after application of VBS for 2 min, a washout period of 70 min was employed. The percentage of a-and b-wave reduction at the end of the washout phase was compared to baseline values (n = 5). Data were calculated throughout as the mean and the standard deviation. qRT-PCR (Bax/Bcl-2-ratio, GFAP- and PGP9.5-levels) or western blot analysis was used to test for toxicity of Princess Volume after 24 h (and β-3 tubulin with GAPDH as a control gene). Significance was estimated by Student´s t-test; p ≤0.05 was considered to be statistically significant., Results: The IOP increased non-significantly by 10% after 24 h. Short-term biocompatibility testing using isolated superfused bovine retinas showed neither significant reductions of the b-wave nor the a-wave amplitudes (b-wave reduction 14.2%, p>0.05; a-wave reduction 23.9%, p>0.05). qRT-PCR and western blot analysis did not reveal significant toxicity after 24 h., Conclusions: The manufactured HA-based hydrogel showed highly favourable biophysical characteristics in the explored ex vivo models, justifying in vivo studies enabling the assessment of biocompatibility., Competing Interests: Parts of the experiments were financed by Croma Pharma GmbH, Austria. CH and CR are employed by Croma Pharma GmbH. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.
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- 2019
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23. Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal-Limbal and Human Conjunctival Epithelial Cells.
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Schuerer N, Stein E, Inic-Kanada A, Pucher M, Hohenadl C, Bintner N, Ghasemian E, Montanaro J, and Barisani-Asenbauer T
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- Buffers, Cell Line, Cell Survival drug effects, Humans, Ophthalmic Solutions chemistry, Pharmaceutical Preparations chemistry, Time Factors, Conjunctiva cytology, Epithelial Cells drug effects, Limbus Corneae cytology, Ophthalmic Solutions toxicity
- Abstract
Purpose: To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal-limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model., Methods: HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT-based assay. The morphology of cells was also investigated., Results: HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers., Conclusions: We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface.
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- 2017
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24. Efficacy of two different thiol-modified crosslinked hyaluronate formulations as vitreous replacement compared to silicone oil in a model of retinal detachment.
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Schnichels S, Schneider N, Hohenadl C, Hurst J, Schatz A, Januschowski K, and Spitzer MS
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- Animals, Biocompatible Materials adverse effects, Biocompatible Materials chemistry, Cross-Linking Reagents chemistry, Hyaluronic Acid adverse effects, Hyaluronic Acid chemistry, Hydrogels adverse effects, Hydrogels chemistry, Rabbits, Retina drug effects, Retina pathology, Silicone Oils adverse effects, Silicone Oils chemistry, Sulfhydryl Compounds chemistry, Biocompatible Materials therapeutic use, Hyaluronic Acid therapeutic use, Hydrogels therapeutic use, Retinal Detachment surgery, Silicone Oils therapeutic use, Vitreous Body surgery
- Abstract
The efficacy of two novel artificial vitreous body substitutes (VBS) consisting of highly biocompatible thiolated cross-linked hyaluronic acid (HA)-based hydrogels in comparison to silicone oil in a model of retinal detachment was investigated. Pars plana vitrectomy (23G) was performed in the right eye of 24 pigmented rabbits. Retinal detachment of two quadrants was induced by creating a small retinotomy near the vascular arcade and injecting balanced salt solution (BSS) subretinally. The retina was reattached by injecting air, which was followed by increasing the infusion pressure, and the retinal tear was treated by endolaser photocoagulation. At the end of the procedure, the eye was filled either with 5000-cs silicone oil (after fluid air exchange) or the respective hydrogel (with two different viscosities). Follow-up examination included slit lamp examination, funduscopy, intraocular pressure measurements (IOP), optical coherence tomography (OCT) and electroretinogram (ERG) measurements. After a maximum follow-up of four weeks both eyes were removed, examined macroscopically, photographed, and prepared for histology. Of the eight rabbits that received silicone oil, seven (87.5%) developed a recurrent retinal detachment with pronounced proliferative vitreoretinopathy within the first two weeks after surgery. In contrast, in the hydrogel treated eyes, the retina stayed attached in the majority of the cases (73.3%). IOP and retinal morphology were normal as long as the retina remained re-attached. In conclusions, this model of retinal detachment, both thiolated crosslinked hyaluronate hydrogels showed superior efficacy when compared to silicone oil. These hydrogels have a promising potential as novel vitreous body substitutes.
- Published
- 2017
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25. Effect of Topically Administered Chitosan- N -acetylcysteine on Corneal Wound Healing in a Rabbit Model.
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Fischak C, Klaus R, Werkmeister RM, Hohenadl C, Prinz M, Schmetterer L, and Garhöfer G
- Abstract
Purpose: The present study was performed to investigate the effect of topically administered chitosan- N -acetylcysteine (C-NAC) on corneal wound healing in a rabbit model., Methods: A total of 20 New Zealand White rabbits were included in the randomized, masked, placebo-controlled experiment. A monocular epithelial debridement was induced by manual scraping under general anesthesia. Animals were randomized to receive either C-NAC two times daily or placebo. Monitoring of corneal wound healing was performed with ultra-high-resolution optical coherence tomography (OCT) and epithelial fluorescein staining. Measurements were done immediately after and up to 72 hours after wound induction., Results: No difference in wound size was found immediately after surgical debridement between the C-NAC group and the placebo group. Wound healing was significantly faster in the C-NAC group compared to the placebo group ( p < 0.01 for both methods). A good correlation was found between the OCT technique and the epithelial fluorescein staining in terms of wound size ( r = 0.94)., Conclusions: Administration of C-NAC containing eye drops twice daily leads to a faster corneal wound healing in a rabbit model of corneal debridement as compared to placebo. Ultra-high-resolution OCT is considered a noninvasive, dye-free alternative to conventional fluorescein staining in assessing corneal wound healing also in humans.
- Published
- 2017
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26. Immunogenicity and protective efficacy of a Vero cell culture-derived whole-virus H7N9 vaccine in mice and guinea pigs.
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Wodal W, Schwendinger MG, Savidis-Dacho H, Crowe BA, Hohenadl C, Fritz R, Brühl P, Portsmouth D, Karner-Pichl A, Balta D, Grillberger L, Kistner O, Barrett PN, and Howard MK
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- Animals, Antibodies, Viral immunology, Antibody Formation, Chlorocebus aethiops, Female, Guinea Pigs, Hemagglutination Inhibition Tests, Hemagglutinin Glycoproteins, Influenza Virus immunology, Immunoglobulin G analysis, Influenza A Virus, H1N1 Subtype immunology, Influenza A Virus, H1N1 Subtype physiology, Influenza A Virus, H7N9 Subtype physiology, Interferon-gamma analysis, Interleukin-4 analysis, Mice, Mice, Inbred DBA, Neuraminidase antagonists & inhibitors, Neuraminidase metabolism, Orthomyxoviridae Infections immunology, Orthomyxoviridae Infections mortality, Th1 Cells immunology, Th1 Cells metabolism, Th2 Cells immunology, Th2 Cells metabolism, Vero Cells, Influenza A Virus, H7N9 Subtype immunology, Influenza Vaccines immunology, Orthomyxoviridae Infections prevention & control
- Abstract
Background: A novel avian H7N9 virus with a high case fatality rate in humans emerged in China in 2013. We evaluated the immunogenicity and protective efficacy of a candidate Vero cell culture-derived whole-virus H7N9 vaccine in small animal models., Methods: Antibody responses induced in immunized DBA/2J mice and guinea pigs were evaluated by hemagglutination inhibition (HI), microneutralization (MN), and neuraminidase inhibition (NAi) assays. T-helper cell responses and IgG subclass responses in mice were analyzed by ELISPOT and ELISA, respectively. Vaccine efficacy against lethal challenge with wild-type H7N9 virus was evaluated in immunized mice. H7N9-specific antibody responses induced in mice and guinea pigs were compared to those induced by a licensed whole-virus pandemic H1N1 (H1N1pdm09) vaccine., Results: The whole-virus H7N9 vaccine induced dose-dependent H7N9-specific HI, MN and NAi antibodies in mice and guinea pigs. Evaluation of T-helper cell responses and IgG subclasses indicated the induction of a balanced Th1/Th2 response. Immunized mice were protected against lethal H7N9 challenge in a dose-dependent manner. H7N9 and H1N1pdm09 vaccines were similarly immunogenic., Conclusions: The induction of H7N9-specific antibody and T cell responses and protection against lethal challenge suggest that the Vero cell culture-derived whole-virus vaccine would provide an effective intervention against the H7N9 virus.
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- 2015
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27. Neuraminidase-Inhibiting Antibody Response to H5N1 Virus Vaccination in Chronically Ill and Immunocompromised Patients.
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Fritz R, Hetzelt N, Ilk R, Hohenadl C, van der Velden MV, Aichinger G, Portsmouth D, Kistner O, Howard MK, Barrett PN, and Kreil TR
- Abstract
Neuraminidase-inhibiting (NAi) antibodies have been reported to be an independent correlate of protection from influenza disease, but the NAi antibody response to influenza vaccination has never been assessed in chronically ill or immunocompromised participants. Using an enzyme-linked lectin assay, we demonstrated that 2 immunizations with a Vero cell culture-derived, whole-virus H5N1 A/Vietnam vaccine induces NAi antibodies in 94.3% of chronically ill and 83.8% of immunocompromised participants. A booster with a heterologous A/Indonesia H5N1 vaccine induced comparable NAi antibody titers in both groups and resulted in 100% seropositivity. These data support prepandemic H5N1 vaccination strategies for these highly vulnerable risk groups.
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- 2014
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28. Hyperimmune intravenous immunoglobulin containing high titers of pandemic H1N1 hemagglutinin and neuraminidase antibodies provides dose-dependent protection against lethal virus challenge in SCID mice.
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Hohenadl C, Wodal W, Kerschbaum A, Fritz R, Howard MK, Farcet MR, Portsmouth D, McVey JK, Baker DA, Ehrlich HJ, Barrett PN, and Kreil TR
- Subjects
- Animals, Disease Models, Animal, Dose-Response Relationship, Drug, Immunocompromised Host, Mice, Mice, SCID, Neuraminidase immunology, Survival Analysis, Treatment Outcome, Viral Proteins immunology, Antibodies, Viral administration & dosage, Hemagglutinin Glycoproteins, Influenza Virus immunology, Immunoglobulins, Intravenous administration & dosage, Influenza A Virus, H1N1 Subtype immunology, Neuraminidase antagonists & inhibitors, Orthomyxoviridae Infections prevention & control, Viral Proteins antagonists & inhibitors
- Abstract
Background: Convalescent plasma and fractionated immunoglobulins have been suggested as prophylactic or therapeutic interventions during an influenza pandemic., Findings: Intravenous immunoglobulin (IVIG) preparations manufactured from human plasma collected before the 2009 H1N1 influenza pandemic, and post-pandemic hyperimmune (H)-IVIG preparations were characterized with respect to hemagglutination inhibition (HI), microneutralization (MN) and neuraminidase-inhibiting (NAi) antibody titers against pandemic H1N1 (pH1N1) and seasonal H1N1 (sH1N1) viruses. The protective efficacy of the IVIG and H-IVIG preparations was evaluated in a SCID mouse challenge model.Substantial levels of HI, MN and NAi antibodies against pH1N1 (GMTs 1:45, 1:204 and 1: 727, respectively) and sH1N1 (GMTs 1:688, 1:4,946 and 1:312, respectively) were present in pre-pandemic IVIG preparations. In post-pandemic H-IVIG preparations, HI, MN and NAi antibody GMTs against pH1N1 were 1:1,280, 1:11,404 and 1:2,488 (28-, 56- and 3.4-fold enriched), respectively, compared to pre-pandemic IVIG preparations (p < 0.001). Post-pandemic H-IVIG (HI titer 1:1,280) provided complete protection from lethality of SCID mice against pH1N1 challenge (100% of mice survived for 29 days post-challenge). Pre-pandemic IVIG (HI titer 1:70) did not provide significant protection against pH1N1 challenge (50% of mice survived 29 days post-challenge compared to 40% survival in the buffer control group). There was a highly significant correlation between circulating in vivo HI and MN antibody titers and survival (p < 0001)., Conclusion: The substantial enrichment of HA- and NA-specific antibodies in H-IVIG and the efficacious protection of SCID mice against challenge with pH1N1 suggests H-IVIG as a promising intervention against pandemic influenza for immunocompromised patients and other risk groups.
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- 2014
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29. GBP-1 acts as a tumor suppressor in colorectal cancer cells.
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Britzen-Laurent N, Lipnik K, Ocker M, Naschberger E, Schellerer VS, Croner RS, Vieth M, Waldner M, Steinberg P, Hohenadl C, and Stürzl M
- Subjects
- Animals, Cell Line, Tumor, Colorectal Neoplasms pathology, GTP-Binding Proteins genetics, Humans, Mice, RNA Interference, Colorectal Neoplasms physiopathology, GTP-Binding Proteins physiology, Genes, Tumor Suppressor
- Abstract
The human guanylate-binding protein 1 (GBP-1) is among the proteins the most highly induced by interferon-γ (IFN-γ) in every cell type investigated as yet. In vivo, GBP-1 expression is associated with the presence of inflammation and has been observed in autoimmune diseases, inflammatory bowel diseases (IBD) and cancer. In colorectal carcinoma (CRC), the expression of GBP-1 in the desmoplastic stroma has been previously reported to correlate with the presence of an IFN-γ-dominated T helper type 1 (Th1) micromilieu and with an increased cancer-related 5-year survival. In the present study, the analysis of GBP-1 expression in a series of 185 CRCs by immunohistochemistry confirmed that GBP-1 is expressed in stroma cells of CRCs and revealed a significantly less frequent expression in tumor cells, which was contradictory with the broad inducibility of GBP-1. Furthermore, three of six CRC cell lines treated with IFN-γ were unable to express GBP-1 indicating that colorectal tumor cells tend to downregulate GBP-1. On the contrary, non-transformed colon epithelial cells strongly expressed GBP-1 in vitro in presence of IFN-γ and in vivo in inflammatory bowel diseases. Reconstitution of GBP-1 expression in a negative CRC cell line inhibited cell proliferation, migration and invasion. Using RNA interference, we showed that GBP-1 mediates the antitumorigenic effects of IFN-γ in CRC cells. In addition, GBP-1 was able to inhibit tumor growth in vivo. Altogether, these results suggested that GBP-1 acts directly as a tumor suppressor in CRC and the loss of GBP-1 expression might indicate tumor evasion from the IFN-γ-dominated Th1 immune response.
- Published
- 2013
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30. A cell culture-derived whole-virus H9N2 vaccine induces high titer antibodies against hemagglutinin and neuraminidase and protects mice from severe lung pathology and weight loss after challenge with a highly virulent H9N2 isolate.
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Wodal W, Falkner FG, Kerschbaum A, Gaiswinkler C, Fritz R, Kiermayr S, Portsmouth D, Savidis-Dacho H, Coulibaly S, Piskernik C, Hohenadl C, Howard MK, Kistner O, Barrett PN, and Kreil TR
- Subjects
- Animals, Antibodies, Neutralizing blood, Antibodies, Viral blood, Dose-Response Relationship, Immunologic, Female, Guinea Pigs, Hemagglutination Inhibition Tests, Lung pathology, Lung virology, Mice, Mice, Inbred BALB C, Neutralization Tests, Orthomyxoviridae Infections immunology, Weight Loss, Hemagglutinin Glycoproteins, Influenza Virus immunology, Influenza A Virus, H9N2 Subtype immunology, Influenza Vaccines immunology, Neuraminidase immunology, Orthomyxoviridae Infections prevention & control
- Abstract
Background: Influenza viruses of subtype A/H9N2 are enzootic in poultry across Asia and the Middle East and are considered to have pandemic potential. The development of new vaccine manufacturing technologies is a cornerstone of influenza pandemic preparedness., Methods: A non-adjuvanted whole-virus H9N2 vaccine was developed using Vero cell culture manufacturing technology. The induction of hemagglutination inhibition (HI) and virus-neutralizing antibodies was assessed in CD1 mice and guinea pigs. A highly sensitive enzyme-linked lectin assay was used to investigate the induction of antibodies capable of inhibiting the enzymatic activity of the H9N2 neuraminidase. Protective efficacy against virus replication in the lung after challenge with the homologous virus was evaluated in BALB/c mice by a TCID(50) assay, and prevention of virus replication in the lung and associated pathology were evaluated by histology and immunohistochemistry. To investigate the ability of the vaccine to prevent severe disease, BALB/c mice were challenged with a highly virulent mouse-adapted H9N2 isolate which was generated by multiple lung-to-lung passage of wild-type virus., Results: The vaccine elicited high titers of functional H9N2-specific HA antibodies in both mice and guinea pigs, as determined by HI and virus neutralization assays. High titer H9N2-specific neuraminidase inhibiting (NAi) antibodies were also induced in both species. Vaccinated mice were protected from lung virus replication in a dose-dependent manner after challenge with the homologous H9N2 virus. Immunohistochemical analyses confirmed the lack of virus replication in the lung and an associated substantial reduction in lung pathology. Dose-dependent protection from severe weight loss was also provided after challenge with the highly virulent mouse-adapted H9N2 virus., Conclusions: The induction of high titers of H9N2-specific HI, virus-neutralizing and NAi antibodies and dose-dependent protection from virus replication and severe disease in animal models suggest that the Vero cell culture-derived whole-virus vaccine will provide an effective intervention in the event of a H9N2 pandemic situation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
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31. Magnetic field-controlled gene expression in encapsulated cells.
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Ortner V, Kaspar C, Halter C, Töllner L, Mykhaylyk O, Walzer J, Günzburg WH, Dangerfield JA, Hohenadl C, and Czerny T
- Subjects
- Genes, Reporter genetics, Green Fluorescent Proteins genetics, HEK293 Cells, Heat-Shock Proteins genetics, Humans, Hyperthermia, Induced, Luciferases genetics, Polymers chemistry, Promoter Regions, Genetic genetics, RNA, Messenger metabolism, Ferric Compounds administration & dosage, Gene Expression Regulation, Magnetic Fields, Metal Nanoparticles administration & dosage
- Abstract
Cell and gene therapies have an enormous range of potential applications, but as for most other therapies, dosing is a critical issue, which makes regulated gene expression a prerequisite for advanced strategies. Several inducible expression systems have been established, which mainly rely on small molecules as inducers, such as hormones or antibiotics. The application of these inducers is difficult to control and the effects on gene regulation are slow. Here we describe a novel system for induction of gene expression in encapsulated cells. This involves the modification of cells to express potential therapeutic genes under the control of a heat inducible promoter and the co-encapsulation of these cells with magnetic nanoparticles. These nanoparticles produce heat when subjected to an alternating magnetic field; the elevated temperatures in the capsules then induce gene expression. In the present study we define the parameters of such systems and provide proof-of-principle using reporter gene constructs. The fine-tuned heating of nanoparticles in the magnetic field allows regulation of gene expression from the outside over a broad range and within short time. Such a system has great potential for advancement of cell and gene therapy approaches., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2012
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32. The 5' leader sequence of mouse mammary tumor virus enhances expression of the envelope and reporter genes.
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Hohenadl C, Gunzburg WH, Salmons B, and Indik S
- Subjects
- Active Transport, Cell Nucleus, Animals, Cell Line, Genes, Reporter, Humans, Mammary Tumor Virus, Mouse genetics, Models, Molecular, Nucleic Acid Conformation, RNA Stability, RNA, Messenger metabolism, RNA, Viral metabolism, Viral Envelope Proteins genetics, 5' Untranslated Regions, Mammary Tumor Virus, Mouse physiology, Viral Envelope Proteins biosynthesis, Virus Replication
- Abstract
Mouse mammary tumor virus (MMTV) is a complex betaretrovirus, which utilizes a Rev-like auxiliary protein Rem to export the unspliced viral RNA from the nucleus. MMTV env mRNA appears to be exported via a distinct, Rem-independent, mechanism. Here, we analysed the effect of an extensively folded region coinciding with the 5' leader sequence on env gene expression. We found that the presence of the 5' leader stimulates expression of the envelope protein. Enhanced Env production was accompanied by increased cytoplasmic levels of env mRNA. The 5' leader promotes nucleocytoplasmic translocation and increases stability of env mRNA. The region responsible for this effect was mapped to the distal part of the 5' leader. Furthermore, the 5' leader inserted in the sense orientation into a heterologous luciferase expression construct increased luciferase activity.
- Published
- 2012
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33. A vero cell-derived whole-virus H5N1 vaccine effectively induces neuraminidase-inhibiting antibodies.
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Fritz R, Sabarth N, Kiermayr S, Hohenadl C, Howard MK, Ilk R, Kistner O, Ehrlich HJ, Barrett PN, and Kreil TR
- Subjects
- Adolescent, Adult, Animals, Chlorocebus aethiops, Humans, Influenza Vaccines administration & dosage, Influenza, Human blood, Middle Aged, Vero Cells, Young Adult, Antibodies, Viral blood, Influenza A Virus, H5N1 Subtype immunology, Influenza Vaccines immunology, Influenza, Human immunology, Neuraminidase antagonists & inhibitors
- Abstract
A Vero cell-derived whole-virus H5N1 influenza vaccine has been shown to induce neutralizing antibodies directed against the hemagglutinin (HA) protein of diverse H5N1 strains in animal studies and clinical trials. However, neuraminidase-inhibiting (NAi) antibodies can reduce viral spread and may be of particular importance in the event of an H5N1 pandemic, where immunity due to HA antibodies is likely absent in the general population. Here we demonstrate the effective induction of NAi antibody titers after H5N1 vaccination in humans. In contrast to the immune response directed toward HA, a single vaccine dose induced a strong NAi response that was not significantly boosted by a second dose, most probably due to priming by previous vaccination or infection with seasonal influenza viruses. After 2 immunizations, seroconversion rates based on antibody titers against HA and NA were similar, indicating the induction of equally strong immune responses against both proteins by this H5N1 vaccine.
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- 2012
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34. Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors.
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Anliker B, Abel T, Kneissl S, Hlavaty J, Caputi A, Brynza J, Schneider IC, Münch RC, Petznek H, Kontermann RE, Koehl U, Johnston IC, Keinänen K, Müller UC, Hohenadl C, Monyer H, Cichutek K, and Buchholz CJ
- Subjects
- AC133 Antigen, Animals, Antigens, CD genetics, Antigens, CD20 genetics, Cells, Cultured, Glycoproteins genetics, Hippocampus metabolism, Humans, Mice, Mice, Inbred C57BL, Peptides genetics, Receptors, AMPA genetics, Endothelial Cells metabolism, Gene Transfer Techniques, Genetic Vectors, Hematopoietic Stem Cells metabolism, Lentivirus genetics, Neurons metabolism
- Abstract
We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105(+) endothelial cells, human CD133(+) hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133(+) cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.
- Published
- 2010
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35. Interferon gamma-induced human guanylate binding protein 1 inhibits mammary tumor growth in mice.
- Author
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Lipnik K, Naschberger E, Gonin-Laurent N, Kodajova P, Petznek H, Rungaldier S, Astigiano S, Ferrini S, Stürzl M, and Hohenadl C
- Subjects
- Adenocarcinoma blood supply, Adenocarcinoma genetics, Adenocarcinoma metabolism, Adenocarcinoma therapy, Animals, Blotting, Western, Cell Growth Processes physiology, Cell Line, Tumor, Doxycycline pharmacology, Female, GTP-Binding Proteins biosynthesis, GTP-Binding Proteins genetics, Hemoglobins metabolism, Histocytochemistry, Humans, Lymphocytes, Tumor-Infiltrating cytology, Mammary Neoplasms, Experimental blood supply, Mammary Neoplasms, Experimental genetics, Mammary Neoplasms, Experimental metabolism, Mice, Mice, Inbred BALB C, Neovascularization, Pathologic metabolism, Transduction, Genetic, Transfection, Vascular Endothelial Growth Factor A metabolism, Xenograft Model Antitumor Assays, GTP-Binding Proteins metabolism, Interferon-gamma metabolism, Mammary Neoplasms, Experimental therapy
- Abstract
Interferon gamma (IFN-gamma) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-gamma are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-gamma-mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1-expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1-expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-gamma-mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-gamma-induced antitumoral defense system.
- Published
- 2010
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36. Molecularly characterised xenograft tumour mouse models: valuable tools for evaluation of new therapeutic strategies for secondary liver cancers.
- Author
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Mischek D, Steinborn R, Petznek H, Bichler C, Zatloukal K, Stürzl M, Günzburg WH, and Hohenadl C
- Subjects
- Adenocarcinoma genetics, Adenocarcinoma pathology, Aged, Animals, Female, Humans, Immunohistochemistry, Liver Neoplasms pathology, Male, Mice, Mice, SCID, Middle Aged, Neoplasm Transplantation, Reverse Transcriptase Polymerase Chain Reaction, Statistics, Nonparametric, Adenocarcinoma secondary, Gene Expression Regulation, Neoplastic, Liver Neoplasms genetics, Liver Neoplasms secondary, Xenograft Model Antitumor Assays
- Abstract
To develop and evaluate new therapeutic strategies for the treatment of human cancers, well-characterised preclinical model systems are a prerequisite. To this aim, we have established xenotransplantation mouse models and corresponding cell cultures from surgically obtained secondary human liver tumours. Established xenograft tumours were patho- and immunohistologically characterised, and expression levels of cancer-relevant genes were quantified in paired original and xenograft tumours and the derivative cell cultures applying RT-PCR-based array technology. Most of the characteristic morphological and immunohistochemical features of the original tumours were shown to be maintained. No differences were found concerning expression of genes involved in cell cycle regulation and oncogenesis. Interestingly, cytokine and matrix metalloproteinase encoding genes appeared to be expressed differentially. Thus, the established models are closely reflecting pathohistological and molecular characteristics of the selected human tumours and may therefore provide useful tools for preclinical analyses of new antitumour strategies in vivo.
- Published
- 2009
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37. Guanylate binding protein-1 inhibits spreading and migration of endothelial cells through induction of integrin alpha4 expression.
- Author
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Weinländer K, Naschberger E, Lehmann MH, Tripal P, Paster W, Stockinger H, Hohenadl C, and Stürzl M
- Subjects
- Endothelial Cells drug effects, Fibronectins metabolism, Gene Expression, Humans, Integrin alpha4 metabolism, Interleukin-1beta pharmacology, Tumor Necrosis Factor-alpha pharmacology, Up-Regulation, Endothelial Cells physiology, GTP-Binding Proteins physiology, Integrin alpha4 biosynthesis
- Abstract
Human guanylate binding protein-1 (GBP-1) is a large GTPase that is induced by inflammatory cytokines and acts antiangiogenically through the inhibition of endothelial cell proliferation and migration. In this study, we detected that GBP-1-expressing cells show a significantly reduced spreading and migration on fibronectin matrices. Investigating possible mechanisms of these effects, we found that integrin alpha(4) (ITGA4) was consistently up-regulated at both the RNA and protein level in GBP-1-expressing cell cultures. Inhibition of cell spreading and migration by GBP-1 was dependent on the binding of ITGA4 to fibronectin. The inflammatory cytokines IL-1beta and TNF-alpha induced ITGA4 expression in HUVECs and inhibited spreading and migration. Knockdown of GBP-1 by shRNA abrogated inflammatory cytokine induced ITGA4 expression and restored spreading and migration capabilities of the cells. These results show that inhibition of endothelial cell spreading and migration by inflammatory cytokines is mediated by GBP-1 through induction of ITGA4 expression. Endothelial cell migration is a key process during angiogenesis. Therefore, ITGA4 may be a novel molecular target to modulate angiogenesis in human disease.
- Published
- 2008
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38. Angiostatic immune reaction in colorectal carcinoma: Impact on survival and perspectives for antiangiogenic therapy.
- Author
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Naschberger E, Croner RS, Merkel S, Dimmler A, Tripal P, Amann KU, Kremmer E, Brueckl WM, Papadopoulos T, Hohenadl C, Hohenberger W, and Stürzl M
- Subjects
- Adult, Aged, Aged, 80 and over, Biomarkers, Tumor analysis, Chemokine CXCL10 analysis, Colorectal Neoplasms blood supply, Colorectal Neoplasms drug therapy, Colorectal Neoplasms mortality, Female, Humans, Immunohistochemistry, Interferon-gamma physiology, Male, Middle Aged, Neoplasm Staging, Retrospective Studies, Angiogenesis Inhibitors therapeutic use, Colorectal Neoplasms immunology, GTP-Binding Proteins analysis, Th1 Cells immunology
- Abstract
Angiogenesis and inflammation are the 2 major stroma reactions in colorectal carcinoma (CRC). Guanylate binding protein-1 (GBP-1) is a key mediator of angiostatic effects of inflammation. Therefore, we hypothesized that GBP-1 may be a biomarker of intrinsic angiostasis associated with an improved outcome in CRC patients. GBP-1 was strongly expressed in endothelial cells and immune cells in the desmoplastic stroma of 32% of CRC as determined by immunohistochemical investigation of 388 sporadic CRC. Cancer-related 5-year survival was highly significant (p < 0.001) increased (16.2%) in patients with GBP-1-positive CRC. Multivariate analysis showed that GBP-1 is an independent prognostic factor indicating a reduction of the relative risk of cancer-related death by the half (p = 0.032). A comparative transcriptome analysis (22,215 probe sets) of GBP-1-positive (n = 12) and -negative (n = 12) tumors showed that particularly IFN-gamma-induced genes including the major antiangiogenic chemokines CXCL9, CXCL10 and CXCL11 were coexpressed with GBP-1. Altogether our findings indicated that GBP-1 may be a novel biomarker and an active component of a Th-1-like angiostatic immune reaction in CRC. This reaction may affect patient's response to antiangiogenic therapy and the identification of such tumors may provide a novel criterion for patient selection. Moreover, the induction of a Th-1-like angiostatic immune reaction may be a promising approach for the clinical treatment of CRC., ((c) 2008 Wiley-Liss, Inc.)
- Published
- 2008
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39. Reconstituting retroviral (ReCon) vectors facilitating delivery of cytotoxic genes in cancer gene therapy approaches.
- Author
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Brandtner EM, Kodajova P, Hlavaty J, Jandl G, Tabotta W, Salmons B, Günzburg WH, and Hohenadl C
- Subjects
- Animals, Base Sequence, Cell Death, Genetic Engineering, HeLa Cells, Humans, Mice, Molecular Sequence Data, NIH 3T3 Cells, Neoplasms pathology, Organ Specificity, Polyadenylation, Promoter Regions, Genetic genetics, Sequence Deletion, Transduction, Genetic, Cytotoxins genetics, Genetic Therapy methods, Genetic Vectors genetics, Neoplasms genetics, Neoplasms therapy, Retroviridae genetics
- Abstract
Background: We have previously described the generation of reconstituting retroviral (ReCon) vectors designed for cancer gene therapy using cytotoxic gene products. The unique vector structure with a promoter physically separated from the transgene allows generation of stable virus producer cells irrespective of the toxic gene. The mechanism of synthesis of DNA from retroviral RNA dictates that infection leads to the reconstitution of functional expression cassettes in the target cell., Methods: To improve vector titres, a cytomegalovirus enhancer was inserted upstream of the 5'-long-terminal repeat (LTR); the Woodchuck hepatitis virus post-transcriptional regulatory element and an elongated attachment site upstream of the 3'-LTR were included. In addition, a bacterial origin of replication was deleted and a functional internal polyadenylation signal mutated. Transcriptional targeting was attempted by introducing mammary tissue-specific promoters such as the U3 region of mouse mammary tumour virus or the promoter of the whey acidic protein encoding gene. All modifications were analysed in detail with respect to virus production and infectivity. Finally, the vector was armed with the lambda-holin encoding gene and transduced cells were analysed for cytotoxic effects., Results: Distinct modifications of the vector resulted in a titre improvement of more than 560-fold. Compatibility of the optimized vector with targeted cellular promoters was demonstrated. When equipped with the cytotoxic gene, stable producer cells could be successfully established and high titre virus infection resulted in rigorous target cell killing., Conclusions: The ReCon vector in its optimized form is an attractive tool for cancer gene therapy approaches.
- Published
- 2008
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40. Quantification and characterization of autotransduction in retroviral vector producer cells.
- Author
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Brandtner EM, Kodajova P, Knapp E, Ertl R, Tabotta W, Salmons B, Günzburg WH, and Hohenadl C
- Subjects
- Cell Line, Gene Products, env genetics, Gene Products, env metabolism, Humans, Transfection, Genetic Therapy methods, Genetic Vectors, Retroviridae genetics, Transduction, Genetic
- Abstract
Gene therapy has evolved into a tempting strategy for the management of cancer and other life-threatening diseases. Various approaches employ retroviral vectors to deliver the therapeutic gene. The profound knowledge about retrovirus biology allows the generation of increasingly advanced vector systems as well as an accurate assessment and management of potential safety risks. This study focuses on the genetic stability of retrovirus producer cells as a basic safety requirement and its compromise by autotransduction. It has been shown previously that protection of retroviral packaging systems by superinfection interference is not guaranteed. The current study provides insight into the extent of autotransduction and the time point at which it occurs, and examines strategies to antagonize it. Therefore, a reconstituting vector system was used that obviates transgene expression in virus producer cells by physically separating transgene and promoter. Just on infection two functional expression cassettes are reconstituted, causing highly efficient transgene expression in transduced cells. Equipped with an enhanced green fluorescent protein-encoding gene, this vector allowed accurate quantification of autotransduced cells, which were then isolated by fluorescence-activated cell sorting and further characterized. Sequencing of recloned integrated vector copies demonstrated that high transgene expression levels were strictly associated with the presence of reverse-transcribed vector copies. Envelope protein expression levels, however, were found to be equal in autotransduced and noninfected virus producer cells. Finally, the occurrence of autotransduction could be assigned to an early time point after transfection and was successfully blocked by azidothymidine treatment, yielding a stable and homogeneous population of noninfected retrovirus producer cells.
- Published
- 2008
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41. Mouse mammary tumor virus promoter-containing retroviral promoter conversion vectors for gene-directed enzyme prodrug therapy are functional in vitro and in vivo.
- Author
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Klein R, Ruttkowski B, Schwab S, Peterbauer T, Salmons B, Günzburg WH, and Hohenadl C
- Subjects
- Animals, Genetic Vectors genetics, Mice, Treatment Outcome, Gene Targeting methods, Genetic Therapy methods, Mammary Neoplasms, Experimental drug therapy, Mammary Neoplasms, Experimental genetics, Prodrugs therapeutic use, Promoter Regions, Genetic genetics, Retroviridae genetics
- Abstract
Gene directed-enzyme prodrug therapy (GDEPT) is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1) metabolizes the prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon) vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV) promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC(50) values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.
- Published
- 2008
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42. Bacteriophage-encoded toxins: the lambda-holin protein causes caspase-independent non-apoptotic cell death of eukaryotic cells.
- Author
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Agu CA, Klein R, Lengler J, Schilcher F, Gregor W, Peterbauer T, Bläsi U, Salmons B, Günzburg WH, and Hohenadl C
- Subjects
- Apoptosis, Bacteriophage lambda metabolism, Cell Line, Tumor pathology, Endoplasmic Reticulum metabolism, HeLa Cells pathology, Humans, Membrane Potentials drug effects, Mitochondria drug effects, Mitochondria metabolism, Viral Proteins metabolism, Viral Proteins pharmacology, Bacteriophage lambda pathogenicity, Caspases metabolism, Eukaryotic Cells pathology, Necrosis, Viral Proteins toxicity
- Abstract
The bacteriophage-encoded holin proteins are known to promote bacterial cell lysis by forming lesions within the cytoplasmic membrane. Recently, we have shown that the bacteriophage lambda-holin protein exerts cytotoxic activity also in eukaryotic cells accounting for a reduced tumour growth in vivo. In order to elucidate the mechanisms of lambda-holin-induced mammalian cell death, detailed biochemical and morphological analyses were performed. Colocalization analyses by subcellular fractionation and organelle-specific fluorescence immunocytochemistry indicated the presence of the lambda-holin protein in the endoplasmic reticulum and in mitochondria. Functional studies using the mitochondria-specific fluorochrome JC-1 demonstrated a loss of mitochondrial transmembrane potential in response to lambda-holin expression. Morphologically, these cells exhibited unfragmented nuclei but severe cytoplasmic vacuolization representing signs of oncosis/necrosis rather than apoptosis. Consistently, Western blot analyses indicated neither an activation of effector caspases 3 and 7 nor cleavage of the respective substrate poly(ADP-ribose) polymerase (PARP) in an apoptosis-specific manner. These findings suggest that the lambda-holin protein mediates a caspase-independent non-apoptotic mode of cell death.
- Published
- 2007
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43. Unique features of different members of the human guanylate-binding protein family.
- Author
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Tripal P, Bauer M, Naschberger E, Mörtinger T, Hohenadl C, Cornali E, Thurau M, and Stürzl M
- Subjects
- Cells, Cultured, Endothelial Cells metabolism, Endothelium, Vascular cytology, Endothelium, Vascular metabolism, GTP-Binding Proteins genetics, Gene Expression physiology, Humans, Multigene Family genetics, GTP-Binding Proteins chemistry, GTP-Binding Proteins physiology, Multigene Family physiology
- Abstract
Guanylate-binding proteins (GBPs) are the most abundant cellular proteins expressed in response to interferon-gamma (IFN-gamma), with seven highly homologous members in humans, termed HuGBP-1 to HuGBP-7. To date, differential features that may indicate differential functions of these proteins have not been described. Here, we investigated the expression and subcellular localization of the different HuGBPs in endothelial cells (EC). IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) induced the expression of HuGBP-1, HuGBP-2, and HuGBP-3 at similar high levels. In contrast, expression of HuGBP-4 and HuGBP-5 was robustly induced only by IFN-gamma and not by TNF-alpha and IL-1beta. Expression of HuGBP-6 and HuGBP-7 was not detected in EC under the various conditions examined. Investigating subcellular localization of the EC-expressed HuGBPs, HuGBP-1, HuGBP-3, and HuGBP-5 were exclusively detected in the cytoplasm, whereas HuGBP-2 and HuGBP-4 displayed a nucleocytoplasmic distribution. Treatment of the cells with IFN-gamma and aluminum fluoride caused rapid enrichment of HuGBP-1 and HuGBP-2 in the Golgi apparatus, as demonstrated by time-lapse microscopy and fluorescence analyses of GFP-tagged HuGBPs. HuGBP-3 and HuGBP-4 were never detected in the Golgi apparatus, whereas HuGBP-5 was constitutively enriched in this cytosolic compartment, irrespective of stimulation. These results assign a characteristic pattern of expression and subcellular localization to each of the HuGBPs, indicating for the first time that these proteins may have different cellular functions.
- Published
- 2007
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44. Hypoxia- and radiation-inducible, breast cell-specific targeting of retroviral vectors.
- Author
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Lipnik K, Greco O, Scott S, Knapp E, Mayrhofer E, Rosenfellner D, Günzburg WH, Salmons B, and Hohenadl C
- Subjects
- Animals, Cell Line, Disease Models, Animal, Gene Expression Regulation, Genes, Reporter, Green Fluorescent Proteins biosynthesis, Green Fluorescent Proteins genetics, Humans, Immunohistochemistry, Mice, Mice, SCID, Microscopy, Fluorescence, Neoplasm Transplantation, Sensitivity and Specificity, Transplantation, Heterologous, X-Rays, Breast Neoplasms therapy, Cell Hypoxia, Genetic Therapy methods, Genetic Vectors, Milk Proteins genetics, Promoter Regions, Genetic drug effects, Promoter Regions, Genetic radiation effects, Retroviridae
- Abstract
To facilitate a more efficient radiation and chemotherapy of mammary tumours, synthetic enhancer elements responsive to hypoxia and ionizing radiation were coupled to the mammary-specific minimal promoter of the murine whey acidic protein (WAP) encoding gene. The modified WAP promoter was introduced into a retroviral promoter conversion (ProCon) vector. Expression of a transduced reporter gene in response to hypoxia and radiation was analysed in stably infected mammary cancer cell lines and an up to 9-fold increase in gene expression demonstrated in comparison to the respective basic vector. Expression analyses in vitro, moreover, demonstrated a widely preserved mammary cell-specific promoter activity. For in vivo analyses, xenograft tumours consisting of infected human mammary adenocarcinoma cells were established in SCID/beige mice. Immunohistochemical analyses demonstrated a hypoxia-specific, markedly increased WAP promoter-driven expression in these tumours. Thus, this retroviral vector will facilitate a targeted gene therapeutic approach exploiting the unique environmental condition in solid tumours.
- Published
- 2006
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- View/download PDF
45. WPRE-mediated enhancement of gene expression is promoter and cell line specific.
- Author
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Klein R, Ruttkowski B, Knapp E, Salmons B, Günzburg WH, and Hohenadl C
- Subjects
- Animals, Cell Line, Cells, Cultured, Dogs, Genetic Vectors genetics, Green Fluorescent Proteins metabolism, Humans, Leukemia Virus, Murine genetics, Mice, Organ Specificity, RNA, Messenger genetics, RNA, Messenger metabolism, Virus Assembly, Gene Expression, Hepatitis B Virus, Woodchuck genetics, Promoter Regions, Genetic genetics
- Abstract
The success of gene therapy approaches relies on sufficiently high levels of expression of the therapeutic gene. However, if tissue specific or tumour specific gene expression is desired, a lower level of transgene expression usually has to be accepted due to the weakness of the majority of available tissue or tumour specific promoters. This obstacle can in part be overcome by the insertion of viral cis-acting elements that enhance gene expression in various expression vector contexts regardless of the respective promoter. We designed a series of murine leukaemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors that contain the woodchuck hepatitis post-transcriptional regulatory element (WPRE) and evaluated its use by measuring enhanced green fluorescent protein (EGFP) levels and viral titres. In viral vector packaging cells, when the EGFP encoding gene was transcribed from the MLV promoter, incorporation of the WPRE resulted in a marked improvement of the vectors in terms of EGFP expression and virus titres. However, in infected cells after promoter conversion had taken place, the effect of the WPRE became promoter and cell line dependent. When the EGFP gene was transcribed from the heterologous mouse mammary tumour virus (MMTV) promoter the same beneficial role of the WPRE on transgene expression was observed in all eight cell lines tested. In contrast, when EGFP gene expression was driven by the murine whey acidic protein (WAP) promoter, the positive effect of the WPRE could only be observed in two cell lines whereas expression was actually reduced in the six other cell lines tested. This decrease of EGFP expression was not only demonstrated at the protein level but also manifested on the RNA level.
- Published
- 2006
- Full Text
- View/download PDF
46. The cytotoxic activity of the bacteriophage lambda-holin protein reduces tumour growth rates in mammary cancer cell xenograft models.
- Author
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Agu CA, Klein R, Schwab S, König-Schuster M, Kodajova P, Ausserlechner M, Binishofer B, Bläsi U, Salmons B, Günzburg WH, and Hohenadl C
- Subjects
- Animals, Bacteriophage lambda metabolism, Cell Survival, HeLa Cells, Humans, Immunohistochemistry, Mice, Mice, Inbred BALB C, Mice, SCID, Transplantation, Heterologous, Viral Proteins metabolism, Bacteriophage lambda genetics, Breast Neoplasms therapy, Genetic Therapy, Viral Proteins genetics
- Abstract
Background: The potential use of gene therapy for cancer treatment is being intensively studied. One approach utilises the expression of genes encoding cytotoxic proteins. Such proteins can affect cellular viability, for example by inhibiting the translation machinery or disturbing membrane integrity. The bacteriophage Lambda (lambda)-holin protein is known to form a lesion in the cytoplasmic membrane of E. coli, triggering bacterial cell lysis and thereby enabling the release of new bacteriophage particles. The aim of this study was to evaluate whether the lambda-holin protein has a cytotoxic impact on eukaryotic cells and whether it holds potential as a new therapeutic protein for cancer gene therapy., Methods: To explore this possibility, stably transfected human cell lines were established that harbour a tetracycline (Tet)-inducible system for controlled expression of the lambda-holin gene. The effect of the lambda-holin protein on eukaryotic cells was studied in vitro by applying several viability assays. We also investigated the effect of lambda-holin gene expression in vivo using a human breast cancer cell tumour xenograft as well as a syngeneic mammary adenocarcinoma mouse model., Results: The lambda-holin-encoding gene was inducibly expressed in eukaryotic cells in vitro. Expression led to a substantial reduction of cell viability of more than 98%. In mouse models, lambda-holin-expressing tumour cell xenografts revealed significantly reduced growth rates in comparison to xenografts not expressing the lambda-holin gene., Conclusions: The lambda-holin protein is cytotoxic for eukaryotic cells in vitro and inhibits tumour growth in vivo suggesting potential therapeutic use in cancer gene therapy., (Copyright 2005 John Wiley & Sons, Ltd.)
- Published
- 2006
- Full Text
- View/download PDF
47. HIV-1 Rev can specifically interact with MMTV RNA and upregulate gene expression.
- Author
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Dangerfield JA, Hohenadl C, Egerbacher M, Kodajova P, Salmons B, and Günzburg WH
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Electrophoretic Mobility Shift Assay, Gene Products, rev genetics, HIV-1 genetics, Humans, Mammary Tumor Virus, Mouse genetics, Mice, Protein Binding, RNA, Viral genetics, Regulatory Sequences, Nucleic Acid physiology, rev Gene Products, Human Immunodeficiency Virus, Gene Expression Regulation, Viral physiology, Gene Products, rev metabolism, HIV-1 metabolism, Mammary Tumor Virus, Mouse metabolism, RNA, Viral metabolism, Up-Regulation physiology
- Abstract
We present evidence that the HIV-1 Rev protein can heterologously regulate expression of the simple beta retrovirus mouse mammary tumour virus (MMTV). Up to 10-fold upregulation was seen in a functional assay system when specific MMTV sequences were substituted for the HIV-1 Rev responsive element (RRE). RNA gel shift analysis showed that purified recombinant Rev could specifically bind to MMTV unique region 3 prime (U3) RNA and that these sequences could compete for wild-type Rev-RRE binding approximately 20-fold more efficiently than a non-specific competitor RNA. Using a combination of in silico and deletion mutation analyses, it was not possible to define any single specific secondary structure responsive to Rev, suggesting that a structure or combination of structures that only form in the context of the complete U3 transcript is/are required to interact with Rev. Taken together, these results suggest that HIV-1 Rev can directly bind to MMTV RNA as well as mediate upregulation of MMTV gene expression.
- Published
- 2005
- Full Text
- View/download PDF
48. A 470 bp WAP-promoter fragment confers lactation independent, progesterone regulated mammary-specific gene expression in transgenic mice.
- Author
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Lipnik K, Petznek H, Renner-Müller I, Egerbacher M, Url A, Salmons B, Günzburg WH, and Hohenadl C
- Subjects
- Animals, Epithelial Cells physiology, Estrus physiology, Female, Genetic Vectors, Lactation, Mice, Mice, Inbred C57BL, Mice, Transgenic, Pregnancy, Progesterone physiology, Promoter Regions, Genetic, Retroviridae, Tumor Cells, Cultured, Breast Neoplasms genetics, Breast Neoplasms pathology, Carcinoma genetics, Carcinoma pathology, Gene Expression Regulation, Mammary Glands, Animal physiology, Milk Proteins genetics
- Abstract
The ability of a 470 bp sub-fragment of the murine whey acidic protein (WAP) promoter in the context of a retroviral expression plasmid to direct gene expression to mammary epithelial cells was analysed in a number of independent transgenic mouse lines. In contrast to previous findings with the genuine 2.5 kb promoter fragment, our studies revealed a highly mammary gland-specific expression detectable only in non-lactating animals. This suggested a mainly progesterone-regulated activity of the short fragment. Therefore, transgene expression was examined in the progesterone-determined estrous cycle and during pregnancy. In accordance with in vitro data from stably transfected cell lines, in both situations expression was upregulated at stages associated with high progesterone levels. Taken together these data provide deeper insight into WAP-promoter regulation and stress the usefulness of the shortened fragment for a lactation independent mammary-targeted expression.
- Published
- 2005
- Full Text
- View/download PDF
49. Generation and characterization of immortalized rat pancreatic stellate cells.
- Author
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Sparmann G, Hohenadl C, Tornøe J, Jaster R, Fitzner B, Koczan D, Thiesen HJ, Glass A, Winder D, Liebe S, and Emmrich J
- Subjects
- Animals, Antigens, Polyomavirus Transforming genetics, Cell Division, Collagen biosynthesis, Gene Expression Profiling, Immunochemistry, Lipid Metabolism, Male, Oligonucleotide Array Sequence Analysis, Pancreas metabolism, Rats, Rats, Inbred Lew, Retroviridae genetics, Reverse Transcriptase Polymerase Chain Reaction, Cell Line, Transformed, Cell Transformation, Viral, Pancreas cytology
- Abstract
Pancreatic stellate cells (PSCs) are involved in, among other things, the pathogenesis of pancreatic fibrosis. Here, we present the generation of immortalized PSCs 7 and 14 days after isolation by retroviral gene transfer of the SV40 large T antigen encoding region. Propagated cell lines [large T immortalized cells (LTC)-7, LTC-14] retained characteristics of primary cells in terms of morphology, responsiveness to mediators regulating cellular functions such as proliferation, and expression profile of a number of investigated genes. Whereas LTC-14 kept the morphological features of the differentiation status of the primary cells they were made of, LTC-7 appeared similar to an earlier stage. Thus the established cell lines represent a versatile tool to investigate various aspects of PSC biology.
- Published
- 2004
- Full Text
- View/download PDF
50. Specific packaging of spliced retroviral vector transcripts lacking the Psi-region.
- Author
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Pambalk K, Hohenadl C, Salmons B, Günzburg WH, and Renner M
- Subjects
- Animals, Base Sequence, Binding Sites, Cell Line, DNA Primers, Green Fluorescent Proteins, HeLa Cells, Humans, Leukemia Virus, Murine genetics, Luminescent Proteins genetics, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Genetic Vectors, RNA Splicing, Retroviridae genetics, Transcription, Genetic
- Abstract
The effect of a cryptic splice acceptor (cSA) site located at the end of the extended packaging signal in murine leukemia virus (MLV)-based vectors was investigated. Although this cSA is also present in wild type MLV, it was found to result in a smaller transcript in which the packaging signal (Psi) had been removed by splicing only in MLV-derived vectors. Splicing occurs both in packaging cells producing the MLV-vectors as well as in the infected target cells. Transcripts lacking the Psi-sequence (Psi(-)) are packaged relatively efficiently into virus particles, even in the presence of wild type Psi(+)-vector transcripts. The Psi(-)-viral RNA is reverse transcribed in vector transduced cells as is any other retroviral genome. The titer obtained from the Psi(-)-vector was only 1000-fold reduced in comparison to the same Psi(+)-vector. These results suggest that Psi(-)-transcripts may be packaged more frequently than previously supposed and that splicing patterns should be carefully analysed on an individual basis for retroviral vectors used in gene therapy.
- Published
- 2002
- Full Text
- View/download PDF
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