TY - JOUR A1 - Subramanian, Hariharan A1 - Döring, Frank A1 - Kollert, Sina A1 - Rukoyatkina, Natalia A1 - Sturm, Julia A1 - Gambaryan, Stepan A1 - Stellzig-Eisenhauer, Angelika A1 - Meyer-Marcotty, Philipp A1 - Eigenthaler, Martin A1 - Wischmeyer, Erhard T1 - PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling JF - PLoS One N2 - Aim Primary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet. Materials and Methods In HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in Xenopus laevis oocytes. Results In HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished. Conclusions PTH1R mutations affect intracellular PTH-regulated signaling in vitro. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement. KW - phosphorylation KW - xenopus oocytes KW - calcium signaling KW - intracellular receptors KW - mutation KW - teeth KW - tooth eruption KW - transfection Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147967 VL - 11 IS - 11 ER - TY - THES A1 - Sturm, Julia T1 - Effekte von Hyper-IL-6 in der Vaccinia-Virus-vermittelten Krebstherapie T1 - Effects of Hyper-IL-6 in vaccinia virus-mediated cancer therapy N2 - In der vorliegenden Arbeit wurde ein onkolytisches Vaccinia-Virus unter Ausnutzung seiner Eigenschaft als Vektorsystem mit dem Designer-Zytokin Hyper-IL-6 ausgestattet (GLV 1h90). Bei Hyper IL 6 handelt es sich um ein Fusionsprotein bestehend aus humanem Interleukin-6 und der Liganden-Bindungsdomäne des löslichen Interleukin-6-Rezeptors, welche kovalent über einen flexiblen Linker miteinander verbunden sind. Dieses chimäre Designer-Zytokin erlaubt die Untersuchung von IL-6-Effekten, welche über das IL-6-Trans-Signaling vermittelt werden. Daraus ergibt sich einerseits eine beträchtliche Erweiterung des Wirkspektrums und darüber hinaus weist Hyper-IL-6 sowohl in vitro als auch in vivo eine 100-1000fach verstärkte biologische Aktivität auf. Aufgrund der Tatsache, dass Hyper-IL-6, neben seiner Tumor-inhibierenden Wirkung, eine Vielzahl weiterer Effekte zugeschrieben wird, wurde in dieser Arbeit durch die Kombination des Designer-Zytokins mit einem onkolytischen Vaccinia-Virus nicht nur additive Effekte auf die Tumorregression, sondern darüber hinaus auch mögliche systemisch-vermittelte Hyper-IL-6-Effekte untersucht. Nach intravenöser Injektion von GLV-1h90 in DU-145-Tumor-tragende Mäuse konnte neben der intratumoralen Replikation des Virus und der Expression des Markerproteins Ruc-GFP zusätzlich die Expression des integrierten Designer-Zytokins Hyper-IL-6 im Tumor nachgewiesen werden. Von entscheidender Bedeutung war der zusätzliche Nachweis des Designer-Zytokins in Serum-Proben von GLV-1h90-injizierten Mäusen. Nach einer aktiven Hyper-IL-6-Sekretion von infizierten Tumorzellen, bildet der Transport in die Blutbahn die Voraussetzung für systemisch-vermittelte Hyper-IL-6-Effekte. In dieser Arbeit wurde untersucht, ob sich durch die Überexpression von Hyper-IL-6 im Tumor, zusätzlich zu den onkolytischen Eigenschaften des Vaccinia-Virus, additive anti-Tumor-Effekte ergeben. Eine systemische Injektion von GLV 1h90 bzw. GLV 1h68 in DU-145-Tumor-tragende Mäuse führte zu einer signifikanten Reduktion des Tumorvolumens im Vergleich zu PBS-injizierten Mäusen. Neben Effekten, welche mit Entzündungsprozessen assoziiert sind, wie eine Rotfärbung der Haut, eine signifikanten Vergrößerung der Leber sowie eine massive Stimulation der Akute-Phase-Antwort in der Leber, konnte in GLV-1h90-injizierten Mäusen ein verbesserter Gesundheitszustand auf der Basis einer signifikanten Gewichtszunahme, verbunden mit einer beschleunigten Wundheilung Virus-induzierter Schwanzläsionen, beobachtet werden. Darüber hinaus konnte für Hyper-IL-6 eine Stimulierung der Megakaryopoese im Knochenmark nachgewiesen werden, welche zu einer signifikanten Erhöhung der Thrombozyten-Zahl im Blutkreislauf von GLV-1h90-injizierten Mäusen führte. Es ist von entscheidender Bedeutung anzumerken, dass alle beobachteten systemischen Hyper-IL-6-Effekte eine zeitliche Limitierung aufwiesen, welche sich höchstwahrscheinlich auf die Virus-bedingte Zerstörung Hyper IL 6-produzierender Tumorzellen zurückführen lässt. Dies impliziert zudem, dass eventuelle Komplikationen, welche durch die Überexpression des Designer-Zytokins hervorgerufen werden können, ebenfalls selbstlimitierend sind. Es konnte bereits mehrfach gezeigt werden, dass eine Kombinationstherapie aus onkolytischen Viren und Chemotherapie über synergistische Effekte zu einer signifikant verbesserten Tumorregression führt. Allerdings kommt es in Folge einer Chemotherapie oft zu einer Vielzahl von gefährlichen Nebenwirkungen, da alle schnell proliferierenden Zellen des Körpers betroffen sind. Thrombozytopenie ist eine der am häufigsten vorkommenden Nebenwirkung und beschreibt eine massive Reduktion der Thrombozyten-Zahl im Blut. Im Hinblick auf eine mögliche klinische Anwendung von GLV 1h90 wurde deshalb untersucht, ob in einer Kombinationstherapie mit Mitomycin C, neben einer Verstärkung der therapeutischen Effekte des Virus, basierend auf den beobachteten Hyper-IL-6-Effekten, zusätzlich der Gesundheitszustand der behandelten Mäuse verbessert werden kann. Die Experimente belegen, dass eine Kombination onkolytischer Vaccinia-Virus-Konstrukte mit Mitomycin C zu einer signifikant verbesserten Tumorregression im Vergleich zu den jeweiligen Monotherapien führt. Von bedeutender Relevanz war die Beobachtung, dass in einer Kombinationstherapie von Mitomycin C und GLV-1h90, im Gegensatz zu GLV-1h68, eine signifikante zeitliche Verkürzung der auftretenden Thrombozytopenie erreicht wird. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass eine systemische Injektion von GLV-1h90 zu einer funktionellen Expression des Designer-Zytokins Hyper-IL-6 führte, welches in der Lage ist eine erfolgreiche Kombinationstherapie aus einem onkolytischen Vaccinia-Virus und dem Chemotherapeutikum Mitomycin C durch eine Reduktion der Nebenwirkungen zusätzlich zu optimieren. N2 - In this thesis, an oncolytic vaccinia virus was armed with the designer cytokine Hyper-IL-6 by recombinant integration (GLV-1h90), exploiting its features as a vector system. Hyper IL-6 is composed of human interleukin-6 (IL-6) and the cytokine-binding domain of its soluble receptor sIL-6R which are bond covalently by a flexible peptide linker. Hyper-IL-6 is a multifunctional cytokine which exhibits not only anti-tumor activity, but also a variety of other effects. For this reason, the combination of the designer cytokine and an oncolytic vaccinia virus was used to study possible improvements regarding tumor regression and more importantly additional systemically mediated Hyper IL-6 effects. In addition to intratumoral replication and visualization of the marker gene ruc-gfp, intratumoral expression of the inserted designer cytokine Hyper-IL-6 could be detected after systemic administration of GLV-1h90 into DU-145-tumor-bearing mice. Of special interest was the presence of hyper-IL-6 in blood serum samples of GLV-1h90-injected mice. Following active hyper-IL-6 secretion of infected tumor cells, the transport into the blood circulation is essential for its ability to induce signal transduction pathways outside the tumor. IL-6 is a pro-inflammatory cytokine which is postulated to exhibit both, tumor promoting as well as tumor inhibiting effects. However, growth or proliferation inhibition of tumors could only be observed after addition of soluble IL-6 receptor and is consequently associated with the IL 6-trans-signaling pathway. Therefore, the thesis deals with the question of whether overexpression of hyper-IL-6 can further enhance the pre-existing oncolytic effects of vaccinia virus. Systemic administration of either GLV-1h90 or GLV-1h68 led to significant tumor regression compared to PBS-treated mice. Comparison of the two viral constructs demonstrated a slightly increased oncolytic activity of GLV-1h90. However, further studies have to clarify to which extend this improvement is resulting from an intratumoral overexpression of hyper IL 6. Following the detection of hyper-IL-6 in the blood circulation as a consequence of GLV 1h90-mediated overexpression in the tumor, functionality of the designer cytokine was analyzed regarding systemically mediated effects. Besides effects which can be associated with inflammatory processes, such as red skin, significant enlargement of the liver as well as enormous stimulation of the acute-phase-response, GLV-1h90-injected mice showed improved healthiness. Health status was assessed by significant gain in body weight associated with accelerated epithelial barrier repair of virus-induced tail lesions. Moreover, it could be demonstrated that Hyper-IL-6 stimulates megakaryopoiesis in the bone marrow, which in turn leads to significantly elevated levels of blood platelets in GLV-1h90-injected mice. It is particularly important to note that all observed systemic Hyper-IL-6 effects occurred only temporarily, which could be explained by virus-mediated oncolysis, reducing the amount of viable Hyper-IL-6 producing tumor cells. The results also implicate that potential complications associated with the overexpression of the designer cytokine can be self-limiting due to the destruction of the virus replication site. Recently, we and others demonstrated that the combination of oncolytic virotherapy and chemotherapy could lead to synergistic interactions that ultimately result in enhanced tumor regression. On the other hand, chemotherapy is often associated with serious side effects, since all fast proliferating cells are affected. Among the most frequently observed adverse effects is thrombocytopenia, which is characterized by a massive reduction of blood platelets. With regard to a possible clinical application of GLV 1h90, combination therapy of the hyper IL 6 encoding vaccinia-virus strain and the chemotherapeutic agent mitomycin C was investigated. Besides therapeutic effects of the virus, the issue was addressed, whether the health status of mice can be improved based on the observed hyper-IL-6 effects. Experimental results clearly demonstrated that combination therapy of mitomycin C and oncolytic vaccinia viruses led to a significantly improved DU-145 tumor regression compared to the respective monotherapies. Of particular importance was the finding that as compared to GLV-1h68, a combination of GLV-1h90 and mitomycin C reduced the time interval during which treated mice suffered from thrombocytopenia significantly. Taken together, this thesis revealed that systemic injection of GLV-1h90 leads to functional expression of the designer cytokine hyper-IL-6, which is able to further optimize the already effective combination therapy of the oncolytic virus GLV-1h90 and the chemotherapeutic agent mitomycin C by reducing of serious adverse effects. KW - Prostatakrebs KW - Vaccinia-Virus KW - Interleukin 6 KW - Chemotherapie KW - Mitomycin C KW - Onkolytische Virotherapie KW - Hyper-IL-6 KW - Thrombozytopenie KW - oncolytic virotherapy KW - Hyper-IL-6 Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-66831 ER - TY - JOUR A1 - Hess, Michael A1 - Stritzker, Jochen A1 - Härtl, Barbara A1 - Sturm, Julia A1 - Gentschev, Ivaylo A1 - Szalay, Aladar T1 - Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies N2 - Background: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. Methods: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. Results: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) rena l excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. Conclusion: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells KW - Virologie KW - beta-glucuronidase KW - oncolytic virus KW - cancer KW - reporter KW - fluorescent probe Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69163 ER -