@article{WeibelBasseLuesebrinkHessetal.2013, author = {Weibel, Stephanie and Basse-Luesebrink, Thomas Christian and Hess, Michael and Hofmann, Elisabeth and Seubert, Carolin and Langbein-Laugwitz, Johanna and Gentschev, Ivaylo and Sturm, Volker J{\"o}rg Friedrich and Ye, Yuxiang and Kampf, Thomas and Jakob, Peter Michael and Szalay, Aladar A.}, title = {Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by \(^{19}\)F-Magnetic Resonance Imaging (MRI)}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0056317}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130311}, pages = {e56317}, year = {2013}, abstract = {Background Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy. Methodology/Principal Findings The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors. Conclusions/Significance These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.}, language = {en} } @article{WangChenMinevetal.2013, author = {Wang, Huiqiang and Chen, Nanhai G. and Minev, Boris R. and Zimmermann, Martina and Aguilar, Richard J. and Zhang, Qian and Sturm, Julia B. and Fend, Falko and Yu, Yong A. and Cappello, Joseph and Lauer, Ulrich M. and Szalay, Aladar A.}, title = {Optical Detection and Virotherapy of Live Metastatic Tumor Cells in Body Fluids with Vaccinia Strains}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {9}, doi = {10.1371/journal.pone.0071105}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130059}, pages = {e71105}, year = {2013}, abstract = {Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.}, language = {en} } @article{DuggalGeissingerZhangetal.2013, author = {Duggal, Rohit and Geissinger, Ulrike and Zhang, Qian and Aguilar, Jason and Chen, Nanhai G. and Binda, Elena and Vescovi, Angelo L. and Szalay, Aladar A.}, title = {Vaccinia virus expressing bone morphogenetic protein-4 in novel glioblastoma orthotopic models facilitates enhanced tumor regression and long-term survival}, series = {Journal of Translational Medicine}, volume = {11}, journal = {Journal of Translational Medicine}, number = {155}, doi = {10.1186/1479-5876-11-155}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129626}, year = {2013}, abstract = {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.}, language = {en} } @article{EhrigKilincChenetal.2013, author = {Ehrig, Klaas and Kilinc, Mehmet O. and Chen, Nanhai G. and Stritzker, Jochen and Buckel, Lisa and Zhang, Qian and Szalay, Aladar A.}, title = {Growth inhibition of different human colorectal cancer xenografts after a single intravenous injection of oncolytic vaccinia virus GLV-1h68}, series = {Journal of Translational Medicine}, volume = {11}, journal = {Journal of Translational Medicine}, number = {79}, doi = {10.1186/1479-5876-11-79}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129619}, year = {2013}, abstract = {Background: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers. Methods: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models. Results: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke's type A-stage HCT-116 and Duke's type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls. Conclusion: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.}, language = {en} } @article{DeGiorgiBuonaguroWorschechetal.2013, author = {De Giorgi, Valeria and Buonaguro, Luigi and Worschech, Andrea and Tornesello, Maria Lina and Izzo, Francesco and Marincola, Francesco M. and Wang, Ena and Buonaguro, Franco M.}, title = {Molecular Signatures Associated with HCV-Induced Hepatocellular Carcinoma and Liver Metastasis}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {2}, doi = {10.1371/journal.pone.0056153}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131155}, pages = {e56153}, year = {2013}, abstract = {Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. In Italy, particularly Southern Italy, chronic hepatitis C virus (HCV) infection represents the main cause of HCC. Using high-density oligoarrays, we identified consistent differences in gene-expression between HCC and normal liver tissue. Expression patterns in HCC were also readily distinguishable from those associated with liver metastases. To characterize molecular events relevant to hepatocarcinogenesis and identify biomarkers for early HCC detection, gene expression profiling of 71 liver biopsies from HCV-related primary HCC and corresponding HCV-positive non-HCC hepatic tissue, as well as gastrointestinal liver metastases paired with the apparently normal peri-tumoral liver tissue, were compared to 6 liver biopsies from healthy individuals. Characteristic gene signatures were identified when normal tissue was compared with HCV-related primary HCC, corresponding HCV-positive non-HCC as well as gastrointestinal liver metastases. Pathway analysis classified the cellular and biological functions of the genes differentially expressed as related to regulation of gene expression and post-translational modification in HCV-related primary HCC; cellular Growth and Proliferation, and Cell-To-Cell Signaling and Interaction in HCV-related non HCC samples; Cellular Growth and Proliferation and Cell Cycle in metastasis. Also characteristic gene signatures were identified of HCV-HCC progression for early HCC diagnosis. Conclusions: A diagnostic molecular signature complementing conventional pathologic assessment was identified.}, language = {en} } @phdthesis{Nube2013, author = {Nube, Jacqueline Sui Lin}, title = {Comparative Analysis of Vaccinia Virus-Encoded Markers Reflecting Actual Viral Titres in Oncolytic Virotherapy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85689}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Using viruses to treat cancer is a novel approach to an age-old disease. Oncolytic viruses are native or recombinant viruses that have the innate or enhanced capability to infect tumour cells, replicate within the tumour microenvironment and subsequently lyse those cells. One representative, the vaccinia virus (VACV), belongs to the orthopoxvirus genus of the Poxviridae family. GLV-1h68, a recombinant and attenuated vaccinia virus devel- oped by the Genelux Corporation, is a member of this family currently being tested in various phase I/II clinical trials under the name GL-ONC1. It has been shown to specif- ically replicate in tumour cells while sparing healthy tissue and to metabolise prodrug at or transport immunological payloads to the site of affliction. Since imaging modalities offer little insight into viral replication deep within the body, and because oncolytic virotherapy is dependent on replication within the target tissue, the need for a monitoring system is evident. Pharmacokinetic analysis of this oncolytic agent was to give insight into the dynamics present in tumours during treatment. This, in turn, would give clinicians the opportunity to monitor the efficacy as early as possible after the onset of treatment, to observe treatment progression and possibly to gauge prognosis, without resorting to invasive procedures, e.g. biopsies. A criteria for viable biomarkers was that it had to be directly dependent on viral replica- tion. Ideally, a marker for treatment efficacy would be specific to the treatment modality, not necessarily the treatment type. Such a marker would be highly detectable (high sen- sitivity), specific for the treatment (high specificity), and present in an easily obtained specimen (blood). Taking this into consideration, the biomarkers were chosen for their potential to be indicators of viral replication. Thus, the biomarkers analysed in this thesis are: the native proteins expressed by the viral genes A27L and B5R, the virally encoded recombinant proteins β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), carboxypeptidase G2 (CPG2) and carcinoembryonic antigen (CEA). Each marker is under the control of one of five different promoters present. All recombinant viruses used in this thesis express A27L, B5R, GFP and β-glucuronidase and all are derived from the parental virus GLV-1h68. In addition to these markers, GLV-1h68 expresses β-galactosidase; GLV-1h181 expresses CPG2. [...]}, subject = {Onkolyse}, language = {en} } @article{BuchbergerBoehm2013, author = {Buchberger, Alexander and B{\"o}hm, Stephanie}, title = {The Budding Yeast Cdc48Shp1 Complex Promotes Cell Cycle Progression by Positive Regulation of Protein Phosphatase 1 (Glc7)}, series = {PLoS One}, journal = {PLoS One}, doi = {10.1371/journal.pone.0056486}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96073}, year = {2013}, abstract = {The conserved, ubiquitin-selective AAA ATPase Cdc48 regulates numerous cellular processes including protein quality control, DNA repair and the cell cycle. Cdc48 function is tightly controlled by a multitude of cofactors mediating substrate specificity and processing. The UBX domain protein Shp1 is a bona fide substrate-recruiting cofactor of Cdc48 in the budding yeast S. cerevisiae. Even though Shp1 has been proposed to be a positive regulator of Glc7, the catalytic subunit of protein phosphatase 1 in S. cerevisiae, its cellular functions in complex with Cdc48 remain largely unknown. Here we show that deletion of the SHP1 gene results in severe growth defects and a cell cycle delay at the metaphase to anaphase transition caused by reduced Glc7 activity. Using an engineered Cdc48 binding-deficient variant of Shp1, we establish the Cdc48Shp1 complex as a critical regulator of mitotic Glc7 activity. We demonstrate that shp1 mutants possess a perturbed balance of Glc7 phosphatase and Ipl1 (Aurora B) kinase activities and show that hyper-phosphorylation of the kinetochore protein Dam1, a key mitotic substrate of Glc7 and Ipl1, is a critical defect in shp1. We also show for the first time a physical interaction between Glc7 and Shp1 in vivo. Whereas loss of Shp1 does not significantly affect Glc7 protein levels or localization, it causes reduced binding of the activator protein Glc8 to Glc7. Our data suggest that the Cdc48Shp1 complex controls Glc7 activity by regulating its interaction with Glc8 and possibly further regulatory subunits.}, language = {en} } @article{SzalayWeibelHofmannetal.2013, author = {Szalay, Aladar A and Weibel, Stephanie and Hofmann, Elisabeth and Basse-Luesebrink, Thomas Christian and Donat, Ulrike and Seubert, Carolin and Adelfinger, Marion and Gnamlin, Prisca and Kober, Christina and Frentzen, Alexa and Gentschev, Ivaylo and Jakob, Peter Michael}, title = {Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer}, series = {Journal of Translational Medicine}, journal = {Journal of Translational Medicine}, doi = {doi:10.1186/1479-5876-11-106}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96016}, year = {2013}, abstract = {Background Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options. Methods In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma. Results We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment. Conclusions Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer.}, subject = {Lungenkrebs}, language = {en} } @phdthesis{Philipp2013, author = {Philipp, Carolin}, title = {Talglipide als Wirtsfaktoren f{\"u}r Mycobacterium leprae : Literaturrecherche und Entwicklung einer Methode zur Talganalyse}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85028}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Trotz weltweiter kostenlos zur Verf{\"u}gung stehender multi drug therapy und Eradikationsbem{\"u}hungen der WHO liegt die Leprainzidenz seit f{\"u}nf Jahren bei etwa 250 000 Neuerkrankungen pro Jahr. Der massive Abfall der Pr{\"a}valenz seit 1985 ist zum einen rechnerisch und durch die Art der Datenerhebung bedingt (s. 5.1.1), zum anderen l{\"a}sst sie keine R{\"u}ckschl{\"u}sse auf die Transmissionsereignisse, also die Aktivit{\"a}t der Krankheit zu. Dazu dient die Inzidenz, die nur unverh{\"a}ltnism{\"a}ßig gesunken ist (s. 2.4.1). F{\"u}r eine effektive Bek{\"a}mpfung der Lepra muss jedoch eine Reduktion der Neuerkrankungen erreicht werden. Bekannt ist, dass nur ein Bruchteil der mit M.leprae exponierten Individuen eine manifeste Krankheit entwickelt. Die Suche nach Wirtsfaktoren f{\"u}r eine Lepraerkrankung ist f{\"u}r gezielte Pr{\"a}ventions- und Prophylaxe-Maßnahmen, die Neuerkrankungen verhindern sollen, demnach von großer Bedeutung. In der vorliegenden Arbeit werden die Ergebnisse einer Literaturrecherche zum Thema "Talglipide als Wirtsfaktoren f{\"u}r M.leprae" pr{\"a}sentiert und eine Methode zur Talganalyse entwickelt. Anhand wissenschaftlicher Publikationen wurde der aktuelle Stand der Forschung in den verschiedenen Bereichen dieser Hypothese reviewartig dargestellt. Der aktuelle Erkenntnisstand hinsichtlich der Lepra{\"u}bertragung ist vereinbar mit mehreren {\"U}bertragungswegen, wobei die Tr{\"o}pfchen- und die Haut{\"u}bertragung favorisiert werden (s. 2.5.1). Die Interpretation der Genomanalysen von M.leprae hat in Kombination mit fr{\"u}heren biochemischen Erkenntnissen Aufschluss {\"u}ber die metabolischen F{\"a}higkeiten von M.leprae gegeben. M.leprae besitzt ein reduktives "Minimalgenom", das sich an die intrazellul{\"a}re Nische, in der es lebt, außerordentlich adaptiert hat. Der Verlust wichtiger Kohlenstoffquellen und eine stark eingeschr{\"a}nkte Atmungskette stehen im Gegensatz zu den fast vollst{\"a}ndig erhaltenen anabolen und katabolen Stoffwechselwegen der Lipide. Sowohl f{\"u}r die Zellwandsynthese als auch zur Energieproduktion ist M.leprae auf wirtsbezogene Lipide angewiesen. Freie Fetts{\"a}uren werden dabei veresterten Fetts{\"a}uren vorgezogen (s. 5.2.1.1). Epidemiologische Erkenntnisse stehen ebenfalls in Einklang mit unserer Lipidhypothese. Als Ursache f{\"u}r das Clustering von Lepraf{\"a}llen in Haushalten kommen sowohl genetische als auch zuf{\"a}llig verteilte Faktoren sowie ein auf den gesamten Haushalt wirkender "Haushaltsfaktor" in Frage. Als genetischer Faktor, der die Talglipidzusammensetzung beeinflusst, w{\"a}ren Enzymaktivit{\"a}ten f{\"u}r die Squalen- bzw. Sapiens{\"a}uresynthese denkbar (vgl. 5.2.1.3). Einen in der Bev{\"o}lkerung zuf{\"a}llig verteilten Suszeptibilit{\"a}tsfaktor k{\"o}nnte die residente Keimflora der Haut darstellen, die f{\"u}r die Menge der im Talg vorkommenden freien Fetts{\"a}uren verantwortlich ist (s. 5.2.1.2). Als m{\"o}glicher Haushaltsfaktor kommt "Armut" in Betracht, der nachgewiesenermaßen mit schlechter K{\"o}rper- und Kleidungshygiene einhergeht (s. 5.2.2.3). In Kombination mit einer vermutlich ebenfalls mit Armut assoziierten h{\"a}ufigeren kutanen Verletzung durch Parasiten und M{\"u}cken k{\"o}nnte dies eine kutane Schmierinfektion mit M.leprae beg{\"u}nstigen. F{\"u}r eine Schmierinfektion {\"u}ber kontaminierte Gegenst{\"a}nde spricht auch die gute extrakorporale {\"U}berlebensf{\"a}higkeit des Erregers und der Nachweis von M.leprae in Bodenproben (s. 5.2.2.2). Die epidemiologischen Daten zu Geschlechterverh{\"a}ltnis und Infektionszeitpunkt lassen ebenfalls eine Rolle der Talglipide als Wirtsfaktoren f{\"u}r eine Lepraerkrankung vermuten (s. 5.2.1.2). Auf Basis der Erkenntnis, dass Lipide und insbesondere Talglipide als Wirtsfaktoren f{\"u}r M.leprae in Betracht kommen, wurde zu diesbez{\"u}glich relevanten Talgkomponenten recherchiert. Als trophischer Wirtsfakor kommen die individuell in unterschiedlichen Mengen vorliegenden freien Fetts{\"a}uren des Sebums in Betracht. Die Menge an Squalen und Sapiens{\"a}ure im Talg hat nachgewiesenermaßen erheblichen Einfluss auf die Barrierefunktion der Haut gegen bakterielle Erreger (s. 5.2.1.3). Es wurde folglich eine auf D{\"u}nnschicht- und Gaschromatografie beruhende Methode zur Quantifizierung dieser Talgbestandteile entwickelt (s. 4.6). Die Probenentnahme erfolgt mittels Sebutape, was eine bew{\"a}hrte, reproduzierbare und auch unter tropischen Bedingungen leicht anwendbare Methode darstellt.}, subject = {Mycobacterium leprae}, language = {de} }