TY - JOUR A1 - Elias, Johannes A1 - Schouls, Leo M. A1 - van de Pol, Ingrid A1 - Keijzers, Wendy C. A1 - Martin, Diana R. A1 - Glennie, Anne A1 - Oster, Philipp A1 - Frosch, Matthias A1 - Vogel, Ulrich A1 - van der Ende, Arie T1 - Vaccine Preventability of Meningococcal Clone, Greater Aachen Region, Germany N2 - No abstract available KW - IMD Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68083 ER - TY - JOUR A1 - Duggal, Rohit A1 - Geissinger, Ulrike A1 - Zhang, Qian A1 - Aguilar, Jason A1 - Chen, Nanhai G. A1 - Binda, Elena A1 - Vescovi, Angelo L. A1 - Szalay, Aladar A. T1 - Vaccinia virus expressing bone morphogenetic protein-4 in novel glioblastoma orthotopic models facilitates enhanced tumor regression and long-term survival JF - Journal of Translational Medicine N2 - No abstract availableBackground: Glioblastoma multiforme (GBM) is one of the most aggressive forms of cancer with a high rate of recurrence. We propose a novel oncolytic vaccinia virus (VACV)-based therapy using expression of the bone morphogenetic protein (BMP)-4 for treating GBM and preventing recurrence. Methods: We have utilized clinically relevant, orthotopic xenograft models of GBM based on tumor-biopsy derived, primary cancer stem cell (CSC) lines. One of the cell lines, after being transduced with a cDNA encoding firefly luciferase, could be used for real time tumor imaging. A VACV that expresses BMP-4 was constructed and utilized for infecting several primary glioma cultures besides conventional serum-grown glioma cell lines. This virus was also delivered intracranially upon implantation of the GBM CSCs in mice to determine effects on tumor growth. Results: We found that the VACV that overexpresses BMP-4 demonstrated heightened replication and cytotoxic activity in GBM CSC cultures with a broad spectrum of activity across several different patient-biopsy cultures. Intracranial inoculation of mice with this virus resulted in a tumor size equal to or below that at the time of injection. This resulted in survival of 100% of the treated mice up to 84 days post inoculation, significantly superior to that of a VACV lacking BMP-4 expression. When mice with a higher tumor burden were injected with the VACV lacking BMP-4, 80% of the mice showed tumor recurrence. In contrast, no recurrence was seen when mice were injected with the VACV expressing BMP-4, possibly due to induction of differentiation in the CSC population and subsequently serving as a better host for VACV infection and oncolysis. This lack of recurrence resulted in superior survival in the BMP-4 VACV treated group. Conclusions: Based on these findings we propose a novel VACV therapy for treating GBM, which would allow tumor specific production of drugs in the future in combination with BMPs which would simultaneously control tumor maintenance and facilitate CSC differentiation, respectively, thereby causing sustained tumor regression without recurrence. KW - cancer stem cells (CSCs) and differentiation KW - glioblastoma multiforme (GBM) KW - vaccinia virus (VACV) KW - bone morphogenetic protein (BMP) Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129626 VL - 11 IS - 155 ER - TY - JOUR A1 - Gholami, Sepideh A1 - Chen, Chun-Hao A1 - Belin, Laurence J. A1 - Lou, Emil A1 - Fujisawa, Sho A1 - Antonacci, Caroline A1 - Carew, Amanda A1 - Chen, Nanhai G. A1 - De Brot, Marina A1 - Zanzonico, Pat B. A1 - Szalay, Aladar A. A1 - Fong, Yuman T1 - Vaccinia virus GLV-1h153 is a novel agent for detection and effective local control of positive surgical margins for breast cancer JF - Breast Cancer Research N2 - Introduction: Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model. Methods: GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 mu Ci of I-124-iodide. Results: Viral infectivity, measured by green fluorescent protein (GFP) expression, was time-and concentrationdependent. All cell lines showed less than 10% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( < 10,000-fold increase from the initial viral dose) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm(3) versus 168 mm(3) in untreated controls (P < 0.05). Conclusions: This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors. KW - conservation KW - carcinoma KW - mastectomy KW - metastases KW - stage-i KW - thyroid-cancer KW - radiation-therapy KW - conserving surgery KW - sodium-iodide symporter Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122140 VL - 15 IS - R26 ER - TY - JOUR A1 - Cecil, Alexander A1 - Gentschev, Ivaylo A1 - Adelfinger, Marion A1 - Dandekar, Thomas A1 - Szalay, Aladar A. T1 - Vaccinia virus injected human tumors: oncolytic virus efficiency predicted by antigen profiling analysis fitted boolean models JF - Bioengineered N2 - Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a promising approach for cancer therapy. Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a therapeutic potential in treating human prostate and hepatocellular carcinomas in xenografted mice. In this study, we describe the use of dynamic boolean modeling for tumor growth prediction of vaccinia virus-injected human tumors. Antigen profiling data of vaccinia virus GLV-1h68-injected human xenografted mice were obtained, analyzed and used to calculate differences in the tumor growth signaling network by tumor type and gender. Our model combines networks for apoptosis, MAPK, p53, WNT, Hedgehog, the T-killer cell mediated cell death, Interferon and Interleukin signaling networks. The in silico findings conform very well with in vivo findings of tumor growth. Similar to a previously published analysis of vaccinia virus-injected canine tumors, we were able to confirm the suitability of our boolean modeling for prediction of human tumor growth after virus infection in the current study as well. In summary, these findings indicate that our boolean models could be a useful tool for testing of the efficacy of VACV-mediated cancer therapy already before its use in human patients. KW - boolean modeling KW - oncolytic virus KW - human xenografted mouse models KW - cancer therapy Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200507 VL - 10 IS - 1 ER - TY - THES A1 - Nguyen, Hoang Duong T1 - Vaccinia virus mediated expression of human erythropoietin in colonized human tumor xenografts results in faster tumor regression and increased red blood cell biogenesis in mice T1 - Expression von humanem Erythropietin in Vaccinia Virus-kolonisierten Tumorxenograftmodellen fördert die Tumorregression und die Biogenese roter Blutzellen N2 - Cancer-related anemia is prevalent in cancer patients. Anemia negatively affects normal mental and physical function capacity with common symptoms s like fatigue, headache, or depression. Human erythropoietin (hEPO), a glycoprotein hormone regulating red blood cell formation, is approved for the treatment of cancer-related anemia. It has shown benefits in correcting anemia, and subsequently improving health-related quality of life and/or enhancing radio-, and chemotherapy. Several recent clinical trials have suggested that recombinant hEPO (rhEPO) may promote tumor growth that raises the questions concerning the safety of using rhEPO for cancer treatment. However in others, such effects were not indicated. As of today, the direct functional effect of rhEPO in tumor models remains controversial and needs to be further analyzed. Based on the GLV-1h68 backbone, the hEPO-expressing recombinant VACV strains (EPO-VACVs) GLV-1h210, GLV-1h211, GLV-1h212 and GLV-1h213 were generated by replacing the lacZ expression cassette at the J2R locus with hEPO under the control of different vaccinia promoters p7.5, pSE, pSEL, pSL, respectively. Also, GLV-1h209 was generated, which is similar to GLV-1h210 but expresses a mutated non-functinal EPO (R103A). The EPO-VACV strains were characterized for their oncolytic efficacy in lung (A549) cancer cells in culture and tumor xenografts. Concomitantly, the effects of locally expressed hEPO in tumors on virus replication, host immune infiltration, tumor vascularization and tumor growth were also evaluated. As expected, EPO-VACVs enhanced red blood cell (RBC) formation in xenograft model. The number of RBCs and hemoglobin (Hb) levels were significantly increased in EPO-VACVs-treated mice compared to GLV-1h68-treated or untreated control mice. However, the mean size of RBC or Hb content per RBC remained normal. Furthermore, over-expression of hEPO did not significantly affect numbers of lymphocytes, monocytes, leucocytes or platelets in the peripheral blood stream. The expression of hEPO in colonized tumors of mice treated with EPO-VACVs was demonstrated by immunohistological staining. Interestingly, there were 9 - 10 hEPO isoforms detected either in tumors, cells, or supernatant, while 3-4 basic isoforms were missing in blood serum, where only six hEPO isoforms were found. Tumor-bearing mice after treatment with EPO-VACVs showed enhanced tumor regression compared to GLV-1h68. The virus titers in tumors in EPO-VACVs-treated mice were 3-4 fold higher compared to GLV-1h68-treated mice. Nevertheless, no significant difference in virus titers among EPO-VACVs was found. The blood vessels in tumors were significantly enlarged while the blood vessel density remained unchanged compared to the GLV-1h68 treated mice, indicating that hEPO did not affect endothelial cell proliferation in this model. Meanwhile, rhEPO (Epoetin alfa) alone or in combination with GLV-1h68 did not show any signs of enhanced tumor growth when compared to untreated controls and GLV-1h68 groups, while doses used were clinical relevant (500 U/kg). These findings suggested that hEPO did not promote angiogenesis or tumor growth in the A549 tumor xenograft model. Human EPO has been reported to function as an immune modulator. In this study, however, we did not find any involvement of hEPO in immune cytokine and chemokine expression or innate immune cell infiltration (leucocytes, B cells, macrophages and dendritic cells) into infected tumors. The degree of immune infiltration and cytokine expression was directly correlated to the number of virus particles. Increased virus replication, led to more recruited immune cells and secreted cytokines/chemokines. It was proposed that tumor regression was at least partially mediated through activation of innate immune mechanisms. In conclusion, the novel EPO-VACVs were shown to significantly increase the number of RBCs, Hb levels, and virus replication in tumors as well as to enhance tumor regression in the A549 tumor xenograft model. Moreover, locally expressed hEPO did not promote tumor angiogenesis, tumor growth, and immune infiltration but was shown to causing enlarged tumoral microvessels which facilitated virus spreading. It is conceivable that in a possible clinical application, anemic cancer patients could benefit from the EPO-VACVs, where they could serve as “wellness pills” to decrease anemic symptoms, while simultaneously destroying tumors. N2 - Blutarmut stellt eine häufige Begleiterscheinung in Krebspatienten dar. Anämie beeinträchtigt die normale mentale und körperliche Funktionsfähigkeit. Menschliches Erythropoetin (hEPO), welches die Bildung roter Blutzellen reguliert, ist klinisch zur Behandlung von Krebs-induzierter Blutarmut zugelassen. Wenn es zur Behandlung von Anämie benutzt wird, verbessert es den Gesundheitszustand sowie Bestrahlungs- und Chemotherapie. Verschiedene klinische zeigten, dass rekombinantes hEPO (rhEPO) das Tumorwachstum anregen kann, was die Frage nach Sicherheit der Anwendung von rhEPO aufbringt. In anderen Studien hingegen, gab es keine Anzeichen für eine Tumorwachstum anregenden Wirkung oder für ein Eingreifen in krebsspezifische Signalwege. Verschiedene hEPO exprimierende rekombinante VACV Stämme (EPO-VACV) wurden hergestellt, GLV-1h210, GLV-1h211 und GLV-1h213, in welchen die lacZ Expressionskassette im J2R Lokus durch das hEPO Gen unter der Kontrolle von verschiedenen Promotoren, p7.5, pSE und pSL, ersetzt wurde. Ebenfalls wurde GLV-1h209 hergestellt, welches ähnlich zu GLV-1h210 ist, jedoch ein mutiertes und nicht-funktionelles EPO Protein (R103A) exprimiert. Alle EPO-VACV Stämme wurden bezüglich ihrer onkolytischen Funktion in Zellkulturexperimenten sowie in in vivo Tumormodellen charakterisiert. Die Expression von zwei Markergene war in Zellkultur sowie in Tumorxenograften für alle EPO-VACV vergleichbar mit der des parentalen GLV-1h68 Virus. Unterschiede in hEPO Transkription und Translation der EPO-VACV war deutlich abhängig von der Promotorstärke und stieg an von p7.5, über pSE und pSL zu pSEL 12 h nach Infektion von Zellen. Darüberhinaus hatte die Insertion von hEPO in das virale Genom keinen Einfluss auf Replikation oder Zytotoxizität aller EPO-VACV in A549 oder NCI-H1299 Zelllinien, obwohl zu frühen Zeitpunkten (24-48 hpi) die Replikation der EPO-VACV etwas höher war, als die des GLV-1h68 Virus. Die A549 Zellen war zugänglicher für virale Infektion durch alle untersuchten Viren als die NCI-H1299 Zellen. Von besonderem Interesse ist, dass hypoxische Bedingungen (2% O2) die Replikation und damit Expression des Markergens gusA, sowie Zytotoxizität für alle untersuchten VACV unabhängig von hEPO Expression verlangsamte. Alle EPO-VACV erhöhen die Bildung von roten Blutzellen (RBC) in Mausmodellen. Anzahl und RBCs sowie Hämoglobin (Hb) Level waren signifikant erhöht im Vergleich zu unbehandelten oder GLV-1h68 behandelten Mäusen. Die Durchschnittsgröße einer RBC sowie der Hämoglobinanteil hingegen waren unverändert. Darüberhinaus hatte die Expression von hEPO keinen signifikanten Einfluss auf Lymphozyten, Monozyten, Leukozyten oder Blutplättchen im peripheren Blut. Die Expression von hEPO in EPO-VACV kolonisierten Tumoren wurde durch immunohistologische Färbungen bestätigt. Interessanterweise konnten 9-10 EPO Isoformen in Tumoren, Zellen oder Zellüberständen gefunden werden, während im Blutserum 3-4 basische Isoformen fehlten und nur 6 Isoformen auftraten. Tumortragende Mäuse, die mit EPO-VACV behandelt wurden, wiesen im Vergleich zu GLV-1h68 behandelten Mäusen eine erhöhte Tumorregression auf. Ausserdem waren virale Titer in EPO-VACV behandleten Tumoren 3-4 fach höher also in denen, die mit GLV-1h68 behandelt wurden. Kein signifikanter Unterschied hingegen wurde zwischen viralen Titern der verschiedenen EPO-VACV in Tumoren gefunden. Tumorale Blutgefäße waren im Vergleich zu GLV-1h68 behandelten Mäusen deutlich vergrößert, wohingegen die Dichte an Blutgefäßen unverändert war, was andeuted, dass keine Proliferation von Endothelzellen angeregt wurde. Rekombinant hergestelltes Epoetin alfa in klinisch relevanten Dosen allein oder in Kombination mit GLV-1h68 hatte keinen Einfluss auf Verbesserung der Tumorregression verglichen mit unbehandelten oder GLV-1h68 behandelten Mäusen. Diese Ergbnisse legen nahe, dass weder Angiogenese noch Tumorwachstum durch hEPO im A549 Tumormodell angeregt wurde. In dieser Studie hingegen wurde kein Einfluss von hEPO im Bezug auf Zytokin- oder Chemokinexpression sowie Immunzellinfiltration in Tumore nachgewiesen. Das Ausmass an Immunzellinfiltratrion und Zytokinexpression konnte direkt mit der Anzahl an viralen Partikeln korreliert werden. Es wurde angenommen, dass Tumorregression zumindest teiweise durch eine Aktivierung des angeborenen Immunsystems bedingt ist. Zusammenfassend kann gesagt werden, dass durch die neuartigen EPO-VACV die Bildung von RBC, die Level an Hb und die virale Replikation signifikant angeregt wurden sowie eine erhöhte Tumorregression im Xenograftmodell auftrat. Darüberhinaus leitete lokal exprimiertes hEPO keine Tumorangiogenese oder Tumorwachstum ein, aber führte zu einer Vergrößerung von Tumorblutgefäßen, was die virale Ausbreitung erleichtern könnte. Es ist vorstellbar, dass anämische Patienten von einer möglichen klinischen Anwendung der EPO-Viren profitieren würden. KW - Erythropoietin KW - Lungenkrebs KW - Anämie KW - onkolytische Virotherapie KW - erythropoietin KW - lung cancer KW - anemia KW - oncolytic therapy KW - Onkolyse Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85383 ER - TY - THES A1 - Heß, Michael T1 - Vaccinia virus-encoded bacterial beta-glucuronidase as a diagnostic biomarker for oncolytic virotherapy T1 - Vaccinia Virus-codierte bakterielle Beta-Glucuronidase als diagnostischer Biomarker in der onkolytischen Virotherapie N2 - Oncolytic virotherapy represents a promising approach to revolutionize cancer therapy. Several preclinical and clinical trials display the safety of oncolytic viruses as wells as their efficiency against solid tumors. The development of complementary diagnosis and monitoring concepts as well as the optimization of anti-tumor activity are key points of current virotherapy research. Within the framework of this thesis, the diagnostic and therapeutic prospects of beta-glucuronidase expressed by the oncolytic vaccinia virus strain GLV-1h68 were evaluated. In this regard, a beta-glucuronidase-based, therapy-accompanying biomarker test was established which is currently under clinical validation. By using fluorescent substrates, the activity of virally expressed beta-glucuronidase could be detected and quantified. Thereby conclusions about the replication kinetics of oncolytic viruses in animal models and virus-induced cancer cell lysis could be drawn. These findings finally led to the elaboration and establishment of a versatile biomarker assay which allows statements regarding the replication of oncolytic viruses in mice based on serum samples. Besides the analysis of retrospective conditions, this test is able to serve as therapy-accompanying monitoring tool for virotherapy approaches with beta-glucuronidase-expressing viruses. The newly developed assay also served as complement to routinely used plaque assays as well as reference for virally expressed anti-angiogenic antibodies in additional preclinical studies. Further validation of this biomarker test is currently taking place in the context of clinical trials with GL-ONC1 (clinical grade GLV-1h68) and has already shown promising preliminary results. It was furthermore demonstrated that fluorogenic substrates in combination with beta-glucuronidase expressed by oncolytic viruses facilitated the optical detection of solid tumors in preclinical models. In addition to diagnostic purposes, virus-encoded enzymes could also be combined with prodrugs resulting in an improved therapeutic outcome of oncolytic virotherapy. In further studies, the visualization of virus-induced immune reactions as well as the establishment of innovative concepts to improve the therapeutic outcome of oncolytic virotherapy could be accomplished. In conclusion, the results of this thesis provide crucial findings about the influence of virally expressed beta-glucuronidase on various diagnostic concepts in the context of oncolytic virotherapy. In addition, innovative monitoring and therapeutic strategies could be established. Our preclinical findings have important clinical influence, particularly by the development of a therapy-associated biomarker assay which is currently used in different clinical trials. N2 - Onkolytische Viren stellen einen vielversprechenden Therapieansatz dar, der die Behandlung von Krebserkrankungen revolutionieren könnte. Intensive präklinische und klinische Studien zeigen sowohl die körperliche Verträglichkeit von onkolytischen Viren, als auch deren Wirksamkeit gegenüber soliden Tumoren. Die Entwicklung von therapiebegleitenden Diagnose- und Monitoringkonzepten sowie eine Optimierung der Antitumorwirkung onkolytischer Viren stellen Eckpunkte der aktuellen Forschung auf dem Gebiet der Virotherapie dar. Im Rahmen dieser Arbeit wurde untersucht, welche diagnostischen und therapeutischen Möglichkeiten die virale Expression von beta-Glucuronidase durch den onkolytischen Vaccinia-Virus-Stamm GLV-1h68 eröffnet. In diesem Zusammenhang wurde ein, auf beta-Glucuronidase basierender, therapiebegleitender Biomarkertest entwickelt, dessen klinische Validierung derzeit stattfindet. Mit Hilfe von fluorogenen Substraten konnte die Aktivität viral exprimierter beta-Glucuronidase detektiert und quantifiziert werden. Dies lies direkte Rückschlüsse auf das Replikationsverhalten von onkolytischen Viren im Tiermodell zu und ermöglichte zudem Aussagen über die Zelllyse Virus-infizierter Krebszellen. Diese Erkenntnisse führten letztendlich zur Ausarbeitung und Etablierung eines vielseitig anwendbaren Biomarker-Assays, der es ermöglicht anhand von Blutproben Aussagen über das Replikationsverhalten onkolytischer Viren in Mäusen zu machen. Neben retrospektiven Analysen erlaubt dieser Test auch ein therapiebegleitendes Monitoring der onkolytischen Virotherapie mit beta-Glucuronidase-exprimierenden Viren. In weiteren präklinischen Untersuchungen diente der entwickelte Assay zudem als Ergänzung zum viralen Plaque Assays sowie als Referenz für Virus-exprimierte anti-angiogene Antikörper. Eine fortführende Validierung dieses neuartigen Biomarkertests findet derzeit im Rahmen humaner Studien mit der klinischen Formulierung von GLV-1h68, GL-ONC1, statt und zeigte bereits erste positive Resultate. Weiterhin konnte im Rahmen dieser Arbeit gezeigt werden, dass die Expression von beta-Glucuronidase durch onkolytische Viren in Verbindung mit fluoreszierenden Substraten eine optische Detektion von Karzinomen im präklinischen Tiermodell ermöglicht. Neben diagnostischen Zwecken, konnten Virus-kodierte Enzyme in Kombination mit Prodrugs genutzt werden, um den Therapieerfolg der onkolytischen Virotherapie zu verbessern. In zusätzlichen Studien konnten zudem Methoden zur Visualisierung der Virus-induzierten Immunantwort sowie neuartige Konzepte zur Therapieverbesserung etabliert werden. Zusammenfassend liefern die Ergebnisse der vorliegenden Arbeit wichtige Erkenntnisse über den Einfluss Virus-exprimierter beta-Glucuronidase auf unterschiedliche Diagnosekonzepte im Rahmen der onkolytischen Virotherapie. Daneben konnten entscheidende Erkenntnisse über den möglichen Einsatz neuer Monitoring- und Therapieansätze erzielt werden. Insbesondere durch die Entwicklung eines therapiebegleitenden Biomarkertests haben diese Resultate erheblichen Einfluss auf die weitere klinische Anwendung von onkolytischen Vaccinia-Viren. KW - Vaccinia-Virus KW - Glucuronidase KW - Krebs KW - cancer KW - oncolytic virus KW - biomarker KW - beta-glucuronidase Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86789 ER - TY - JOUR A1 - Schäfer, Simon A1 - Weibel, Stephanie A1 - Donat, Ulrike A1 - Zhang, Quian A1 - Aguilar, Richard J. A1 - Chen, Nanhai G. A1 - Szalay, Aladar A. T1 - Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors JF - BMC Cancer N2 - Background Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. Methods For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. Results GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome. KW - microenvironment KW - angiogenesis KW - therapy KW - cancer KW - breast-tumors KW - matrix metalloproteinases KW - adenovirus KW - carcinoma KW - prostate KW - mice Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140800 VL - 12 IS - 366 ER - TY - JOUR A1 - Schäfer, Simon A1 - Weibel, Stephanie A1 - Donat, Ulrike A1 - Zhang, Qian A1 - Aguilar, Richard J. A1 - Chen, Nanhai G. A1 - Szalay, Aladar A. T1 - Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors N2 - Background: Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. Methods: For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. Results: GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions: Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome. KW - Biochemie Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-78220 ER - TY - THES A1 - Geissinger, Ulrike T1 - Vaccinia Virus-mediated MR Imaging of Tumors in Mice: Overexpression of Iron-binding Proteins in Colonized Xenografts T1 - Vaccinia Virus-vermittelte MR Bildgebung von Tumoren in Maeusen: Ueberexpression von Eisen-bindenden Proteinen in kolonisierten Heterotransplantaten N2 - Vaccinia virus plays an important role in human medicine and molecular biology ever since the 18th century after E. Jenner discovered its value as a vaccination virus against smallpox. After the successful eradication of smallpox, vaccinia virus, apart from its use as a vaccine carrier, is today mainly used as a viral vector in molecular biology and increasingly in cancer therapy. The capability to specifically target and destroy cancer cells makes it a perfect agent for oncolytic virotherapy. Furthermore, the virus can easily be modified by inserting genes encoding therapeutic or diagnostic proteins to be expressed within the tumor. The emphasis in this study was the diagnosis of tumors using different vaccinia virus strains. Viruses with metal-accumulating capabilities for tumor detection via MRI technology were generated and tested for their usefulness in cell culture and in vivo. The virus strains GLV-1h131, GLV-1h132, and GLV-1h133 carry the gene encoding the two subunits of the iron storage protein ferritin under the control of three different promoters. GLV-1h110, GLV-1h111, and GLV-1h112 encode the bacterial iron storage protein bacterioferritin, whereas GLV-1h113 encodes the codon-optimized version of bacterioferritin for more efficient expression in human cells. GLV-1h22 contains the transferrin receptor gene, which plays an important role in iron uptake, and GLV-1h114 and GLV-1h115 contain the murine transferrin receptor gene. For possibly better iron uptake the virus strains GLV-1h154, GLV-1h155, GLV-1h156, and GLV-1h157 were generated, each with a version of a ferritin gene and a transferrin receptor gene. GLV-1h154 carries the genes that encode bacterioferritin and human transferrin receptor, GLV-1h155 the human ferritin H-chain gene and the human transferrin receptor gene. GLV-1h156 and GLV-1h157 infected cells both express the mouse transferrin receptor and bacterioferritin or human ferritin H-chain, respectively. The virus strains GLV-1h186 and GLV-1h187 were generated to contain a mutated form of the ferritin light chain, which was shown to result in iron overload and the wildtype light chain gene, respectively. The gene encoding the Divalent Metal Transporter 1, which is a major protein in the uptake of iron, was inserted in the virus strain GLV-1h102. The virus strain GLV-1h184 contains the magA gene of the magnetotactic bacterium Magnetospirillum magnetotacticum, which produces magnetic nanoparticles for orientation in the earth’s magnetic field. Initially the infection and replication capability of all the virus strains were analyzed and compared to that of the parental virus strain GLV-1h68, revealing that all the viruses were able to infect cells of the human cancer cell lines A549 and GI-101A. All constructs exhibited a course of infection comparable to that of GLV-1h68. Next, to investigate the expression of the foreign proteins in GI-101A and A549 cells with protein analytical methods, SDS-gelelectrophoresis, Western blots and ELISAs were performed. The proteins, which were expressed under the control of the strong promoters, could be detected using these methods. To be able to successfully detect the protein expression of MagA and DMT1, which were expressed under the control of the weak promoter, the more sensitive method RT-PCR was used to at least confirm the transcription of the inserted genes. The determination of the iron content in infected GI-101A and A549 cells showed that infection with all used virus strains led to iron accumulation in comparison to uninfected cells, even infection with the parental virus strain GLV-1h68. The synthetic phytochelatin EC20 was also shown to enhance the accumulation of different heavy metals in bacterial cultures. In vivo experiments with A549 tumor-bearing athymic nude mice revealed that 24 days post infection virus particles were found mainly in the tumor. The virus-mediated expression of recombinant proteins in the tumors was detected successfully by Western blot. Iron accumulation in tumor lysates was investigated by using the ferrozine assay and led to the result that GLV-1h68-infected tumors had the highest iron content. Histological stainings confirmed the finding that iron accumulation was not a direct result of the insertion of genes encoding iron-accumulating proteins in the virus genome. Furthermore virus-injected tumorous mice were analyzed using MRI technology. Two different measurements were performed, the first scan being done with a seven Tesla small animal scanner seven days post infection whereas the second scan was performed using a three Tesla human scanner 21 days after virus injection. Tumors of mice injected with the virus strains GLV-1h113 and GLV-1h184 were shown to exhibit shortened T2 and T2* relaxation times, which indicates enhanced iron accumulation. In conclusion, the experiments in this study suggest that the bacterioferritin-encoding virus strain GLV-1h113 and the magA-encoding virus strain GLV-1h184 are promising candidates to be used for cancer imaging after further analyzation and optimization. N2 - Das Vaccinia Virus spielt in der Humanmedizin und Molekularbiologie eine wichtige Rolle seit E. Jenner im 18. Jahrhundert seinen Nutzen als Impfvirus entdeckt hat. Nach der erfolgreichen Ausrottung der Pocken, wird das Vaccinia Virus heutzutage neben der Anwendung als Impfstoffträger hauptsächlich als viraler Vektor in der Molekularbiologie und in zunehmendem Maße in der Krebstherapie verwendet. Die Fähigkeit Krebszellen gezielt zu zerstören, macht es zu einem perfekten Wirkstoff für die onkolytische Virotherapie. Des Weiteren kann das Virus durch das Inserieren von Genen, die für therapeutische oder diagnostische Proteine kodieren, und im Tumor exprimiert werden, modifiziert werden. Der Schwerpunkt dieser Arbeit war die Tumordiagnose mit Hilfe verschiedener Vaccinia Virusstämme. Viren mit der Fähigkeit, Metalle anzureichern wurden zur Tumordetektion mittels Kernspintomographie hergestellt und auf ihre Nutzbarkeit in Zellkultur und in vivo getestet. Die Virusstämme GLV-1h132, GLV-1h132 und GLV-1h133 tragen das Gen, welches für die zwei Untereinheiten des Eisenspeicherproteins Ferritin kodieren unter der Kontrolle von drei verschiedenen Promotoren. GLV-1h110, GLV-1h111, und GLV-1h112 tragen das Gen, welches für das bakterielle Eisenspeicherprotein Bacterioferritin kodiert, wohingegen das inserierte Gen in GLV-1h113 für die codon-optimierte Version dieses Proteins kodiert, die eine effizientere Expression in humanen Zellen ermöglichen soll. GLV-1h22 beinhaltet das Transferrin-Rezeptor-Gen, welches eine wichtige Rolle in der Eisenaufnahme spielt, und GLV-1h114 und GLV-1h115 beinhalten das murine Transferrin-Rezeptor-Gen. Für eine möglicherweise bessere Eisenaufnahme wurden die Virusstämme GLV-1h154, GLV-1h155, GLV-1h156 und GLV-1h157 mit je einer Version eines Ferritin-Gens und eines Transferrin-Rezeptor-Gens generiert. GLV-1h154 trägt die Gene, die für Bacterioferritin und den humanen Transferrin Rezeptor kodieren, GLV-1h155 trägt die Gene für die humane Ferritin H-Untereinheit und den humanen Transferrin Rezeptor. Zellen, die mit GLV-1h156 und GLV-1h157 infiziert wurden, exprimierten den Maus-Transferrin-Rezeptor und Bacterioferritin beziehungsweise die humane Ferritin-H-Untereinheit. Die Virusstämme GLV-1h186 und GLV-1h187 wurden mit einer mutierten Form der leichten Untereinheit von Ferritin, für die eine Überladung mit Eisen gezeigt wurde, beziehungsweise mit der leichten Untereinheit des wildtypischen Gens ausgestattet. Das Gen, das für den Divalenten Metal Transporter 1 kodiert, welches ein bedeutendes Protein für die Aufnahme von Eisen darstellt, wurde in den Virusstamm GLV-1h102 inseriert. Der Virusstamm GLV-1h184 trägt das magA Gen des magnetotaktischen Bakteriums Magnetospirillum magnetotacticum, welches magnetische Nanopartikel zur Orientierung im Erdmagnetfeld produziert. Zunächst wurde die Infektions- und Replikationsfähigkeit aller Viren analysiert und mit der des Ausgangsstammes GLV-1h68 verglichen, was zeigte, dass alle Viren in der Lage waren humane Krebszellen der Zelllinien GI-101A und A549 zu infizieren. Alle Konstrukte zeigten einen vergleichbaren Infektionsverlauf zu GLV-1h68. Als nächstes, um die Expression der fremden Proteine in GI-101A und A549 Zellen zu untersuchen, wurden SDS-Gelelektrophorese, Western Blots und ELISAs durchgeführt. Die Proteine, welche unter der Kontrolle von starken Promotoren exprimiert wurden, konnten mit diesen Methoden detektiert werden. Um die Expression von MagA und DMT1 zu detektieren, welche unter der Kontrolle des schwachen Promotors exprimiert wurden, wurde die sensitivere Methode RT-PCR angewendet, mit der zumindest die Transkription der Gene nachgewiesen werden konnte. Die Bestimmung des Eisengehaltes in infizierten GI-101A und A549 Zellen zeigte, dass die Infektion mit allen Viren im Vergleich zu uninfizierten Zellen zu einer Eisenanreicherung führte, sogar die Infektion mit dem Ausgangsstamm GLV-1h68. Für das synthetische Phytochelatin EC20 wurde auch eine Anhäufung von verschiedenen Schwermetallen in Bakterienkulturen gezeigt. In vivo Experimente mit A549 tumor-tragenden athymischen Nacktmäusen ergaben, dass Viruspartikel 24 Tage nach der Infektion hauptsächlich im Tumor gefunden wurden. Die von den Viren vermittelte Expression der rekombinanten Proteine in den Tumoren wurde erfolgreich mit Hilfe von Western Blots detektiert. Die Eisenansammlung in Tumorlysaten wurde mit dem Ferrozine Assay untersucht und führte zu dem Ergebnis, dass Tumore, die mit GLV-1h68 infiziert wurden, den höchsten Eisengehalt vorwiesen. Histologische Färbungen bestätigten die Erkenntnis, dass die Eisenansammlung nicht ein direktes Resultat der Insertion von eisenansammelnden Genen in das Virusgenom war. Darüberhinaus wurden tumortragende Mäuse, denen Virus injiziert wurde, mittels Kernspintomographie analysiert. Zwei verschiedene Messungen wurden durchgeführt, wobei die erste Messung sieben Tage nach Virusinjektion mit einem sieben Tesla Kleintier-Scanner durchgeführt wurde und die zweite Messung mit einem humanen drei Tesla Scanner 21 Tage nach Virusinjektion. Tumore von Mäusen, die mit den Virusstämmen GLV-1h113 und GLV-1h184 injiziert wurden, zeigten verkürzte T2- und T2*-Relaxationszeiten, was auf eine verbesserte Eisenakkumulation hinweist. Zusammenfassend deuten die Experimente dieser Studie darauf hin, dass der Virusstamm GLV-1h113, welcher für Bacterioferritin kodiert, und der Virusstamm GLV-1h184, welcher für MagA kodiert, nach weiterer Untersuchung und Optimierung vielversprechende Kandidaten für die Krebs Bildgebung sind. KW - Vaccinia-Virus KW - NMR-Tomographie KW - Tumor KW - Krebsbildgebung KW - Tumordetektion KW - Vaccinia Virus KW - Tumor detection KW - MRI Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-48099 ER - TY - THES A1 - Huang, Ting T1 - Vaccinia Virus-mediated Therapy of Solid Tumor Xenografts: Intra-tumoral Delivery of Therapeutic Antibodies T1 - Vaccini-Virus-vermittelte Therapie solider Tumoren: Intra-tumoraler Transport therapeutischer Antikörper N2 - Over the past 30 years, much effort and financial support have been invested in the fight against cancer, yet cancer still represents the leading cause of death in the world. Conventional therapies for treatment of cancer are predominantly directed against tumor cells. Recently however, new treatments options have paid more attention to exploiting the advantage of targeting the tumor stroma instead. Vaccinia virus (VACV) has played an important role in human medicine since the 18th century as a vaccination against smallpox. In our laboratory, the recombinant, replication-competent vaccinia virus, GLV-1h68, was shown to enter, colonize and destroy cancer cells both in cell culture, and in vivo, in xenograft models (Zhang, Yu et al. 2007). In addition, combined therapy of GLV-1h68 and anti-VEGF immunotherapy significantly enhanced antitumor therapy in vivo (Frentzen, Yu et al. 2009). In this study, we constructed several new recombinant VACVs carrying genes encoding different antibodies against fibroblast activation protein (FAP) in stroma (GLV-1h282), nanobody against the extracellular domain of epidermal growth factor receptor (EGFR, GLV-1h442) or antibodies targeting both vascular endothelial growth factor (VEGF) and EGFR (GLV-1h444) or targeting both VEGF and FAP (GLV-1h446). The expression of the recombinant proteins was first verified using protein analytical methods, SDS-gel electrophoresis, Western blot analysis, immunoprecipitation (IP) assays and ELISA assays. The proteins were detected after infection of the cells with the different VACVs and the recombinant proteins purified by affinity adsorption. The purified antibodies were shown to specifically bind to their respective antigens. Secondly, the infection and replication capability of all the virus strains was analyzed in cell culture using several human tumor cell lines (A549, FaDu or DU145), revealing that all the new recombinant VACVs were able to infect cancer cells with comparable efficiency to the parental viruses from which they were derived. Thirdly, the antitumor efficacy of the new recombinant VACVs was evaluated in vivo using several human cancer xenograft models in mice. In A549 and DU145 xenografts, the new recombinant VACVs exhibited an enhanced therapeutic efficacy compared to GLV-1h68 with no change in toxicity in mice. In the FaDu xenograft, treatment with GLV-1h282 (anti-FAP) significantly slowed down the speed of tumor growth compared to GLV-1h68. Additionally, treatment with the recombinant VACVs expressed the various antibodies achieved comparable or superior therapeutic effects compared to treatment with a combination of GLV-1h68 and the commercial therapeutic antibodies, Avastin, Erbitux or both. Next, the virus distribution in tumors and organs of treated mice was evaluated. For most of the viruses, the virus titer in tumors was not signficantly diffferent than GLV-1h68. However, for animals treated with GLV-1h282, the virus titer in tumors was significantly higher than with GLV-1h68. This may be the reason for enhanced antitumor efficacy of GLV-1h282 in vivo. Lastly, the underlying mechanisms of therapeutic antibody-enhanced antitumor effects were investigated by immunohistochemistry. Blood vessels density and cell proliferation in tumors were suppressed after treatment with the antibody-encoded VACVs. The results indicated that the suppression of angiogenesis or cell proliferation in tumors may cause the observed therapeutic effect. In conclusion, the results of the studies presented here support the hypothesis that the treatment of solid tumors with a combination of oncolytic virotherapy and immunotherapy has an additive effect over each treatment alone. Moreover, expression of the immunotherapeutic antibody by the oncolytic VACV locally in the tumor enhances the antitumor effect over systemic treatment with the same antibody. Combined, these results indicate that therapy with oncolytic VACVs expressing-therapeutic antibodies may be a promising approach for the treatment of cancer. N2 - In den letzten 30 Jahren wurde viel Aufwand und finanzielle Unterstützung in den Kampf gegen Krebs investiert, doch das Resultat ist limitiert, da Krebs immer noch die zweithöchste Todesursache in der Welt darstellt. Zusätzlich zu gegenwärtig verwendeten Therapien, die vorwiegend gegen Tumorzellen gerichtet sind, wird neuen Therapien mehr Aufmerksamkeit gewidmet, die stattdessen direkt auf das Tumorstroma zielen. Onkolytische Vaccinia Viren haben seit dem 18ten Jahrhundert als Impfstoff gegen Pocken in der Humanmedizin eine wichtige Rolle gespielt. In unserem Labor hat das rekombinante, replikationskompetente Vaccinia Virus GLV-1h68 gezeigt, dass es in Zellkultur und in Xenograft Modellen in Krebszellen eindringen sowie diese kolonisieren und zerstören kann (Zhang, Yu et al. 2007). Zusätzlich verbessert die kombinierte Therapie von GLV-1h68 und anti-VEGF Immunotherapy signifikant die Antitumortherapie in vivo (Frentzen, Yu et al. 2009). In dieser Studie haben wir mehrere neue rekombinante VACVs konstruiert, die die Gene für verschiedene Antikörper gegen das Fibroblasten Aktivierungs Protein (FAP) im Stroma (GLV-1h282) oder einen Nanobody gegen die extrazelluläre Domäne des Epidermalen Wachstumsfaktor (EGFR; GLV-1h442) kodieren. Ausserdem wurden Viren konstruiert, die eine Ko-Expression von Antikörpern gegen sowohl vaskulären Endothelwachstumsfaktor (VEGF) als auch EGFR (GLV-1h444) oder gegen sowohl VEGF als auch FAP (GLV-1h446) erlauben. Zunächst wurden SDS-Gelelektrophorese, Western Blot Analyse, Immunprezipitation (IP) und ELISA Assays durchgeführt, um die Expression der rekombinanten Proteine in Zellen mit proteinanalytischen Methoden zu untersuchen. Die Proteine waren nach Infektion der Zellen mit den verschiedenen VACVs nachweisbar und wurden mittles des FLAG Tags mit einem IP Kit aufgereinigt. Es konnte gezeigt werden, dass die aufgereinigten Antikörper spezifisch an ihr jeweiliges Antigen binden. Zweitens wurde die Infektion und Replikationsfähigkeit aller Virusstämme in Zellkultur untersucht (A549, FaDu oder DU145) und mit ihrem jeweiligen Ausgangsstamm GLV-1h68, GLV-1h164, GLV-1h282 oder GLV-1h442 verglichen. Die Ergebnisse zeigten, dass alle neuen rekombinanten VACVs Zellen mit vergleichbarer Effizienz infizieren konnten wie ihre Ausgangsstämme. Drittens, um die Antitumoreffizienz der neuen rekombinanten Stämme in vivo zu testen, wurden verschiedene humane Tumor Xenotransplantat-tragende Nacktmäuse mit verschiedenen VACVs behandelt. In A549 und DU145 Xenotransplantaten zeigten die neuen rekombinanten VACVs erhöhte therapeutische Effizienz verglichen mit dem Ausgangsstamm GLV-1h68, ohne Veränderung der Toxizität in Mäusen. Im FaDu Xenotransplantat verursachte die Behandlung mit GLV-1h68 keine Tumorregression, wohingegen die Behandlung mit GLV-1h282 (anti-FAP) die Geschwindigkeit des Tumorwachstums signifikant verlangsamte sowie das Überleben verlängerte. Zusätzlich haben wir herausgefunden, dass die Behandlung mit Antikörpern, die mittels Virus geliefert wurden, einen identischen oder sogar erhöhten inhibitorischen Effekt erzielen können, wie in einer Kombinationstherapie von GLV-1h68 und kommerziell erhältlichen Antikörpern, wie Avastin, Erbitux oder beidem. Um die virale Verteilung in vivo zu untersuchen, wurden Tumore und Organe von Mäusen seziert und homogenisiert, gefolgt von Titration der Virusmenge. Die Virus-Titer in Tumoren waren signifikant höher in Tieren, die mit GLV-1h282 behandelt wurden als solche, die mit GLV-1h68 behandelt wurden. Dies mag den Grund für die erhöhte Antitumoreffizienz von GLV-1h282 in vivo darstellen. Die Virus-Titer in allen anderen Gruppen zeigten keinen signifikanten Unterschied. Um den Mechanismus der durch therapeutische Antikörper erhöhten Antitumortherapie zu untersuchen, wurde Immunohistochemie durchgeführt. Nach Behandlung mit den Antikörper-kodierenden VACVs waren die Blutgefäβdichte und Zellproliferation in Tumoren reduziert, nachgewiesen durch die jeweilige CD31 and Ki67 Färbung. Die Resultate deuteten an, dass die Suppression der Angiogenese oder der Zellproliferation in Tumoren den beobachteten Effekt verursachen könnte. Zusammenfassend zeigen die hier präsentierten Daten dass die Kombination der Behandlung von onkolytischer Virotherapie mit Immunotherapie durch Virus-gelieferte Antikörper einen vielversprechenden Ansatz für Krebstherapie darstellt. KW - Vaccinia-Virus KW - therapeutic antibody KW - oncolytic virus KW - Krebs KW - Therapie KW - Antikörper KW - Tumor Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-91327 ER - TY - THES A1 - Camargo Molina, José Eliel T1 - Vacuum stability of models with many scalars T1 - Vakuumstabilität von Modellen mit vielen Skalaren N2 - One of the most popular extensions of the SM is Supersymmetry (SUSY). It is a symmetry relating fermions and bosons and also the only feasible extension to the symmetries of spacetime. With SUSY it is then possible to explain some of the open questions left by the SM while at the same time opening the possibility of gauge unification at a high scale. SUSY theories require the addition of new particles, in particular an extra Higgs doublet and at least as many new scalars as fermions in the SM. Much in the same way that the Higgs boson breaks SU (2)L symmetry, these new scalars can break any symmetry for which they carry a charge through spontaneous symmetry breaking. Let us assume there is a local minimum of the potential that reproduces the correct phenomenol- ogy for a parameter point of a given model. By exploring whether there are other deeper minima with VEVs that break symmetries we want to conserve, like SU (3)C or U (1)EM , it is possible to exclude regions of parameter space where that happens. The local minimum with the correct phenomenology might still be metastable, so it is also necessary to calculate the probability of tunneling between minima. In this work we propose and apply a framework to constrain the parameter space of models with many scalars through the minimization of the one-loop eff e potential and the calculation of tunneling times at zero and non zero temperature.After a brief discussion about the shortcomings of the SM and an introduction of the basics of SUSY, we introduce the theory and numerical methods needed for a successful vacuum stability analysis. We then present Vevacious, a public code where we have implemented our proposed framework. Afterwards we go on to analyze three interesting examples. For the constrained MSSM (CMSSM) we explore the existence of charge- and color- breaking (CCB) minima and see how it constraints the phenomenological relevant region of its parameter space at T = 0. We show that the regions reproducing the correct Higgs mass and the correct relic density for dark matter all overlap with regions suffering from deeper CCB minima. Inspired by the results for the CMSSM, we then consider the natural MSSM and check the region of parameter space consistent with the correct Higgs mass against CCB minima at T /= 0. We find that regions of parameter space with CCB minima overlap significantly with that reproducing the correct Higgs mass. When thermal eff are considered the majority of such points are then found to have a desired symmetry breaking minimum with very low survival probability. In both these studies we find that analytical conditions presented in the literature fail in dis- criminating regions of parameter space with CCB minima. We also present a way of adapting our framework so that it runs quickly enough for use with parameter fit studies. Lastly we show a different example of using vacuum stability in a phenomenological study. For the BLSSM we investigate the violation of R-parity through sneutrino VEVs and where in parameter space does this happen. We find that previous analyses in literature fail to identify regions with R-parity conservation by comparing their results to our full numerical analysis. N2 - Eine der populärsten Erweiterungen des SM ist die Supersymmetrie (SUSY). Dies ist eine Symmetrie, die Bosonen und Fermionen in Beziehung setzt und auch die einzige machbare Erweiterung der Raumzeitsymmetrien. SUSY kann einige offene Fragen des SM erklären und eröffnet die Möglichkeit einer Vereinheitlichung der Eichwechselwirkungen bei einer hohen Skala. Supersymmetrische Theorien erfordern das Hinzufügen neuer Teilchen, insbesondere eines zusätzlichen Higgs-Dubletts und zumindest eines Skalars für jedes Fermion im SM. So wie im SM das Higgs-Boson die SU (2)L-Symmetrie bricht, können diese neuen Skalare jede Symmetrie, deren Ladung sie tragen, spontan brechen. Angenommen, es gibt ein lokales Minimum des Potentials, das die korrekte Phänomenologie für einen Parameterraumpunkt eines Modells erzeugt: Durch die Suche nach anderen tieferen Minima mit Vakuumerwartungswerten, die gewünschte Symmetrien wie SU (3) oder U (1)EM brechen, ist es möglich Parameterraumpunkte, in denen dies passiert, auszuschliessen. Das lokale Minimum mit der korrekten Phänomenologie kann immernoch metastabil sein, weshalb es auch notwendig ist, die Tunnelwahrscheinlichkeit zwischen zwei Minima zu berechnen. In dieser Arbeit legen wir eine Prozedur vor und wenden sie an, um den Parameterraum von Modellen mit vielen Skalaren durch die Minimierung des effektiven Ein-Schleifen-Potentials und durch die Berechnung seiner Lebensdauer sowohl bei T = 0 und bei T /= 0 einzuschränken. Nach einer kurzen Diskussion der Unzulänglichkeiten des SM und Einführung der Grundlagen von SUSY erläutern wir die Theorie und die die nötigen numerischen Methoden für eine erfolgreiche Analyse der Vakuumstabilitaet. Danach präsentieren wir Vevacious, ein öffentliches Programmpaket, in das wir unsere Prozedur implementiert haben. Daraufhin analysieren wir drei interessante Beispiele. Für das Constrained MSSM (CMSSM) untersuchen wir die die Existenz von Minima, in denen die Farb- oder elektrische Ladung nicht erhalten ist (CCB-Minima), und wie dessen phänomenologisch relevante Region des Parameter- raums dadurch bei T = 0 eingeschränkt wird. Wir zeigen, dass die Regionen, die die korrekte Higgsmasse und die richtige Relikt-Dichte für die Dunkle Materie reproduzieren, mit Regionen, die tiefere CCB-Minima aufweisen, überlappen. Inspiriert durch die Ergebnisse für das CMSSM betrachten wir dann das Natural MSSM und prüfen die Parameterraumregion mit der korrekten Higgsmasse auf CCB-Minima bei T /= 0.Wir finden, dass die Region des Parameterraums mit CCB-Minima deutlich mit denen mit einer korrekten Higgsmasse überlappt. Bei Berücksichtigung von thermalen Effekten hat ein Großteil der bei T = 0 langlebigen Punkte ein gewünschtes symmetriebrechendes Minimum mit einer sehr geringen Überlebenswahrscheinlichkeit bei T /= 0. In beiden Studien finden wir, dass die analytischen Bedingungen, die bisher in der Literatur präsentiert wurden, nicht ausreichen, um Bereiche des Parameterraums mit CCB-Minima auszuweisen. Wir präsentieren einen Weg, unsere Prozedur für die Nutzung in Parameterraum-Fit-Studien zu beschleunigen. Zuletzt zeigen wir ein weiteres Beispiel. Für das BLSSM untersuchen wir die Verletzung der R-Parität durch Sneutrino- VEVs und in welchen Parameterraumbereichen dies geschieht. Wir stellen durch Vergleich mit unserer kompletten numerischen Analyse heraus, dass frühere Analysen in der Literatur darin fehlschlagen, diese Bereiche mit Erhaltung der R-Parität zu identifizieren. KW - Supersymmetry KW - Vacuum stability KW - Supersymmetry KW - Beyond Standard Model Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112755 ER - TY - JOUR A1 - Iotzov, Vassil A1 - Weiß, Martin A1 - Windmann, Sabine A1 - Hein, Grit T1 - Valence framing induces cognitive bias JF - Current Psychology N2 - Valence framing effects refer to inconsistent choice preferences in response to positive versus negative formulation of mathematically equivalent outcomes. Here, we manipulate valence framing in a two-alternative forced choice dictator game using gains and losses as frames to investigate the cognitive mechanisms underlying valence framing. We applied a Drift-Diffusion Model (DDM) to examine whether gain (i.e., “take” money) and loss (i.e., “give” money) frames evoke a cognitive bias as previous research did not consistently reveal framing effects using reaction times and response frequency as dependent variables. DDMs allow decomposing the decision process into separate cognitive mechanisms, whereby a cognitive bias was repeatedly associated with a shift in the starting point of the model. Conducting both a laboratory (N = 62) and an online study (N = 109), female participants allocated money between themselves and another person in a prosocial or selfish way. In each study, one group was instructed to give money (give frame), the other to take money (take frame). Consistent with previous studies, no differences were found in response times and response frequencies. However, in both studies, substantial bias towards the selfish option was found in the take frame groups, captured by the starting point of the DDM. Thus, our results suggest that valence framing induces a cognitive bias in decision processing in women, even when no behavioral differences are present. KW - valence framing KW - cognitive bias KW - decision making KW - drift-diffusion modeling KW - laboratory and online studies Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324824 SN - 1046-1310 VL - 42 IS - 34 ER - TY - THES A1 - Lang, Melanie T1 - Valence Shell Photoionization of Soot Precursors with Synchrotron Radiation T1 - Valenzschalen-Photoionisation von Rußvorläufern mit Synchrotron-Strahlung N2 - A series of combustion relevant species like radicals, carbenes and polycyclic aromatic hydrocarbons were characterized in the gas phase by vacuum UV synchrotron radiation and their ionization energies (IE) and further spectroscopic details of the respective cations were retrieved from threshold photoelectron spectra. The reactive intermediates were generated by flash vacuum pyrolysis from stable precursor molecules. Furthermore three polycyclic aromatic hydrocarbons were investigated by threshold photoelectron spectroscopy, too. The experiment was performed at the VUV beamline of the Swiss Light Source in Villigen/Switzerland and the iPEPICO (imaging photoelectron photoion coincidence) setup was applied to correlate ions and electrons from the same ionization event. From the threshold photoelectron spectra and from quantum chemical computations the vibrational structure of the molecule cations and the geometry changes upon ionization were assigned. The ionization energies of the two C4H5 isomers 2-butyn-1-yl and 1-butyn-3-yl were assigned to 7.94±0.02 eV and 7.97±0.02 eV, respectively. The isomerization between the two isomers was computed to have a barrier of 2.20 eV, so a rearrangement between the two radicals cannot be excluded. From the threshold photoelectron spectra of the two constitutional C4H7 isomers 1-methylallyl and 2-methylallyl the ionization energies were assigned to 7.48±0.02 eV and to 7.59±0.02 eV for 1-E-methylallyl and 1-Z-methylallyl, as well as to 7.88±0.01 eV for 2-methylallyl. The two radicals 9-fluorenyl, C13H9, and benzhydryl, C13H11, were observed to ionize at 7.01±0.02 eV and 6.7 eV. The threshold photoelectron spectrum of benzhydryl also incorporated the signal of the diphenylmethyl carbene, C13H10, which has an IE at 6.8 eV. In addition, the head-to-head dimers of 9-fluorenyl and benzhydryl were observed as products in the pyrolysis. C26H18 has an IE at 7.69±0.04 eV and C26H22 has an IE at 8.13±0.04 eV. The three polycyclic aromatic hydrocarbon DHP (C14H16) 1-PEN (C18H22) and THCT (C22H16) were investigated in an effusive beam. The ionization energies were determined to IE(DHP)= 7.38±0.02 eV, IE(1-PEN)=7.58±0.05 eV and IE(THCT)=6.40±0.02 eV. Furthermore the thermal decomposition and the dissociative photoionization of diazomeldrum’s acid was investigated. The pyrolysis products yielded beside several other products the two not yet (by photoelectron spectroscopy) characterized molecules E-formylketene, C3O2H2 and 2-diazoethenone, N2C2O. The dissociative photoionization showed the Wolff rearrangement to occur at higher internal energies. N2 - Die vorliegende Arbeit befasst sich mit VUV Valenz-Photoionisations-Experimenten, welche in der Gasphase an verschiedenen Kohlenwasserstoffradikalen und drei polyzyklischen aromatischen Kohlen- wasserstoffen (PAH) durchgeführt wurde. Des Weiteren wurden die Pyrolyseprodukte der Dia- zomeldrumsäure mit dem genannten Experiment untersucht. Die reaktiven Intermediate wurden im Vakuum mittels Flash-Pyrolyse aus stabilen Vorläufermolekülen erzeugt. Die meisten dieser waren kommerziell erhältlich, wobei auch einige Moleküle selbst im Würzburger Chemielabor synthetisiert wurden. Die erzeugten Radikale und Carbene wurden in einem kontinuierlichen Molekularstrahl ex- pandiert. Um den Vorläufer in die Gasphase zu überführen, wurden verschiedene Molekular-Quellen eingesetzt. Die Auswahl erfolgte dabei in Abhängigkeit des Dampfdrucks des Vorläufermoleküls. Die Polyzyklischen Aromaten (PAH) wurden in der Arbeitsgruppe von Prof. Dr. Anke Krüger im Insti- tut für Organische Chemie der Universität Würzburg synthetisiert. Die PAH wurden in einer Fest- stoffmolekularquelle geheizt und in einem effusiven Molekularstrahl expandiert. Die Ionisation aller Spezies erfolgte mit monochromatischem VUV-Synchrotronlicht, das an der Bending-Magnet Beam- line an der Swiss Light Source in Villigen/Schweiz erzeugt wird. Das Schwellenphotoelektronen- Photoionen-Koinzidenz (TPEPICO) Experiment wurde zur Detektion und Analyse der Ionisation- sprozesse angewendet. Dieses Experiment ermöglicht es massenselektierte Ionen und Schwellen- photoelektronen des selben Ionisationsereignisses zu korrelieren. Die Ionen wurden in einem Time- of-Flight Massenspektrometer detektiert. Durch Integration des Massensignals und anschließende Auswertung des zugeordneten Schwellenphotoelektronen-Signals erhält man das Schwellenphotoelek- tronen-Spektrum (TPES) des Moleküls bzw. Fragments. Aus den TPE-Spektren konnten Ion- isierungsenergien bestimmt werden und mit Hilfe von Franck-Condon-Simulationen sowohl die Schwin- gungsstruktur im Kation, als auch die Geometrieänderung, hervorgerufen durch die Ionisation, analysiert werden. Berechnete Ionisierungsenergien wurden zusätzlich mit den experimentellen Daten verglichen. Im Folgenden werden die einzelnen Ergebnisse aufgelistet. ... KW - Ultraviolett-Photoelektronenspektroskopie KW - photoelectron-photoion coincidence KW - Photoelektronen-Photoionen-Koinzidenz KW - reactive intermediates KW - pyrolysis KW - reaktive Intermediate KW - Pyrolyse KW - Fotoionisation Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117038 ER - TY - JOUR A1 - Wamser, Florian A1 - Seufert, Anika A1 - Hall, Andrew A1 - Wunderer, Stefan A1 - Hoßfeld, Tobias T1 - Valid statements by the crowd: statistical measures for precision in crowdsourced mobile measurements JF - Network N2 - Crowdsourced network measurements (CNMs) are becoming increasingly popular as they assess the performance of a mobile network from the end user's perspective on a large scale. Here, network measurements are performed directly on the end-users' devices, thus taking advantage of the real-world conditions end-users encounter. However, this type of uncontrolled measurement raises questions about its validity and reliability. The problem lies in the nature of this type of data collection. In CNMs, mobile network subscribers are involved to a large extent in the measurement process, and collect data themselves for the operator. The collection of data on user devices in arbitrary locations and at uncontrolled times requires means to ensure validity and reliability. To address this issue, our paper defines concepts and guidelines for analyzing the precision of CNMs; specifically, the number of measurements required to make valid statements. In addition to the formal definition of the aspect, we illustrate the problem and use an extensive sample data set to show possible assessment approaches. This data set consists of more than 20.4 million crowdsourced mobile measurements from across France, measured by a commercial data provider. KW - mobile networks KW - crowdsourced measurements KW - statistical validity Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284154 SN - 2673-8732 VL - 1 IS - 2 SP - 215 EP - 232 ER - TY - JOUR A1 - Ruf, Katharina C. A1 - Fehn, Sonja A1 - Bachmann, Michèle A1 - Moeller, Alexander A1 - Roht, Kristina A1 - Kriemler, Susi A1 - Hebestreit, Helge T1 - Validation of activity questionnaires in patients with cystic fibrosis by accelerometry and cycle ergometry N2 - Background: The objective of this study was to validate physical activity questionnaires for cystic fibrosis (CF) against accelerometry and cycle ergometry. Methods: 41 patients with CF (12-42 years) completed the Habitual Activity Estimation Scale (HAES), the 7-Day Physical Activity Recall questionnaire (7D-PAR) and the Lipid Research Clinics questionnaire (LRC) and performed an incremental exercise test according to the Godfrey protocol up to volitional fatigue. Time spent in moderate and vigorous physical activity (MVPA) assessed objectively by accelerometry was related to the time spent in the respective activity categories by correlation analyses and calculating intraclass correlation coefficients (ICC). Furthermore, the results of the exercise test were correlated with the results of the questionnaires. Results: Time spent in the categories ‘hard’,’very hard’ and ‘hard & very hard’ of the 7D-PAR (0.41 < r < 0.56) and ‘active’ (r = 0.33) of the HAES correlated significantly with MVPA. The activity levels of the LRC were not related to objectively determined physical activity. Significant ICCs were only observed between the 7D-PAR activitiy categories and MVPA (ICC = 0.40-0.44). Only the LRC showed moderate correlations with the exercise test (Wmax: r = 0.46, p = 0.002; VO2peak: r = 0.32, p = 0.041). Conclusions: In conclusion, the activity categories ‘hard’ and ‘very hard’ of the 7D-PAR best reflected objectively measured MVPA. Since the association was at most moderate, the 7D-PAR may be selected to describe physical activity within a population. None of the evaluated questionnaires was able to generate valid physical activity data exercise performance data at the individual level. Neither did any of the questionnaires provide a valid assessment of aerobic fitness on an invidual level. KW - Medizin Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-75083 ER - TY - JOUR A1 - Reiners, Philipp A1 - Asam, Sarah A1 - Frey, Corinne A1 - Holzwarth, Stefanie A1 - Bachmann, Martin A1 - Sobrino, Jose A1 - Göttsche, Frank-M. A1 - Bendix, Jörg A1 - Kuenzer, Claudia T1 - Validation of AVHRR Land Surface Temperature with MODIS and in situ LST — a TIMELINE thematic processor JF - Remote Sensing N2 - Land Surface Temperature (LST) is an important parameter for tracing the impact of changing climatic conditions on our environment. Describing the interface between long- and shortwave radiation fluxes, as well as between turbulent heat fluxes and the ground heat flux, LST plays a crucial role in the global heat balance. Satellite-derived LST is an indispensable tool for monitoring these changes consistently over large areas and for long time periods. Data from the AVHRR (Advanced Very High-Resolution Radiometer) sensors have been available since the early 1980s. In the TIMELINE project, LST is derived for the entire operating period of AVHRR sensors over Europe at a 1 km spatial resolution. In this study, we present the validation results for the TIMELINE AVHRR daytime LST. The validation approach consists of an assessment of the temporal consistency of the AVHRR LST time series, an inter-comparison between AVHRR LST and in situ LST, and a comparison of the AVHRR LST product with concurrent MODIS (Moderate Resolution Imaging Spectroradiometer) LST. The results indicate the successful derivation of stable LST time series from multi-decadal AVHRR data. The validation results were investigated regarding different LST, TCWV and VA, as well as land cover classes. The comparisons between the TIMELINE LST product and the reference datasets show seasonal and land cover-related patterns. The LST level was found to be the most determinative factor of the error. On average, an absolute deviation of the AVHRR LST by 1.83 K from in situ LST, as well as a difference of 2.34 K from the MODIS product, was observed. KW - Land Surface Temperature KW - AVHRR KW - MODIS KW - time series KW - Europe KW - validation KW - TIMELINE Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246051 SN - 2072-4292 VL - 13 IS - 17 ER - TY - JOUR A1 - Mayr, Stefan A1 - Kuenzer, Claudia A1 - Gessner, Ursula A1 - Klein, Igor A1 - Rutzinger, Martin T1 - Validation of earth observation time-series: a review for large-area and temporally dense land surface products JF - Remote Sensing N2 - Large-area remote sensing time-series offer unique features for the extensive investigation of our environment. Since various error sources in the acquisition chain of datasets exist, only properly validated results can be of value for research and downstream decision processes. This review presents an overview of validation approaches concerning temporally dense time-series of land surface geo-information products that cover the continental to global scale. Categorization according to utilized validation data revealed that product intercomparisons and comparison to reference data are the conventional validation methods. The reviewed studies are mainly based on optical sensors and orientated towards global coverage, with vegetation-related variables as the focus. Trends indicate an increase in remote sensing-based studies that feature long-term datasets of land surface variables. The hereby corresponding validation efforts show only minor methodological diversification in the past two decades. To sustain comprehensive and standardized validation efforts, the provision of spatiotemporally dense validation data in order to estimate actual differences between measurement and the true state has to be maintained. The promotion of novel approaches can, on the other hand, prove beneficial for various downstream applications, although typically only theoretical uncertainties are provided. KW - accuracy KW - error estimation KW - global KW - intercomparison KW - remote sensing KW - uncertainty Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-193202 SN - 2072-4292 VL - 11 IS - 22 ER - TY - JOUR A1 - Matthes, Niels A1 - Diers, Johannes A1 - Schlegel, Nicolas A1 - Hankir, Mohammed A1 - Haubitz, Imme A1 - Germer, Christoph-Thomas A1 - Wiegering, Armin T1 - Validation of MTL30 as a quality indicator for colorectal surgery JF - PLoS One N2 - Background Valid indicators are required to measure surgical quality. These ideally should be sensitive and selective while being easy to understand and adjust. We propose here the MTL30 quality indicator which takes into account 30-day mortality, transfer within 30 days, and a length of stay of 30 days as composite markers of an uneventful operative/postoperative course. Methods Patients documented in the StuDoQ|Colon and StuDoQ|Rectal carcinoma register of the German Society for General and Visceral Surgery (DGAV) were analyzed with regard to the effects of patient and tumor-related risk factors as well as postoperative complications on the MTL30. Results In univariate analysis, the MTL30 correlated significantly with patient and tumor-related risk factors such as ASA score (p<0.001), age (p<0.001), or UICC stage (p<0.001). There was a high sensitivity for the postoperative occurrence of complications such as re-operations (p<0.001) or subsequent bleeding (p<0.001), as well as a significant correlation with the CDC classification (p<0.001). In multivariate analysis, patient-related risk factors and postoperative complications significantly increased the odds ratio for a positive MTL30. A negative MTL30 showed a high specify for an uneventful operative and postoperative course. Conclusion The MTL30 is a valid indicator of colorectal surgical quality. KW - surgical care KW - discharge definition KW - definition KW - mortality KW - pancreatectomy KW - complications KW - superior KW - capture Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230530 VL - 15 IS - 8 ER - TY - BOOK A1 - Wang, Wen T1 - Validation of shRNA clones for gene silencing in 293FT cells N2 - ... N2 - The goal of the project was to establish knock down of mRNA in human mesenchymal stem cells. Since these cells are difficult to transfect, a viral approach is needed to achieve sufficient expression of e. g. shRNA in a high percentage of cells to allow for an efficient silencing of corresponding mRNAs. For this purpose for every gene product of interest, a number of shRNA clones have to be tested to detect an individual shRNA with sufficient efficacy. Lentiviral systems for shRNA approaches have recently become available. The principal advantage of the lentiviral system is that it allows gene silencing in nondividing cells and therefore expands the usefulness of the RNAi-based gene silencing system. Lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types. Since the viral transfection of MSCs is a time consuming process that involves transfection of 293 FT cells plus transduction of target cells, for this thesis the following approach was chosen: genes of interest were checked for expression in 293FT cells by RT-PCR. These gene products can be silenced in 293FT cells simply by transfection of shRNA clones and efficacy was subsequently tested by RT-PCR. Beyond this thesis then the project can proceed with effective clones to transduce primary MSCs with individual shRNA clones identified as effective silencing tool in this thesis. KW - shRNA KW - RNAi KW - .................................................................... KW - shRNA KW - RNAi Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-25955 N1 - Aus rechtlichen Gründen wurde der Zugriff auf den Volltext zu diesem Dokument gesperrt. ER - TY - JOUR A1 - Franke, Maximilian A1 - Bieber, Michael A1 - Stoll, Guido A1 - Schuhmann, Michael Klaus T1 - Validity and Efficacy of Methods to Define Blood Brain Barrier Integrity in Experimental Ischemic Strokes: A Comparison of Albumin Western Blot, IgG Western Blot and Albumin Immunofluorescence JF - Methods and Protocols N2 - The clinical and preclinical research of ischemic strokes (IS) is becoming increasingly comprehensive, especially with the emerging evidence of complex thrombotic and inflammatory interactions. Within these, the blood brain barrier (BBB) plays an important role in regulating the cellular interactions at the vascular interface and is therefore the object of many IS-related questions. Consequently, valid, economic and responsible methods to define BBB integrity are necessary. Therefore, we compared the three ex-vivo setups albumin Western blot (WB), IgG WB and albumin intensity measurement (AIM) with regard to validity as well as temporal and economic efficacy. While the informative value of the three methods correlated significantly, the efficacy of the IgG WB dominated. KW - IgG KW - albumin KW - immunohistochemistry KW - Western blot KW - stroke KW - tMCAO KW - blood brain barrier KW - neuroinflammation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234214 SN - 2409-9279 VL - 4 IS - 1 ER -