6 search hits
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Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A.
(2012)
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Stefanie D. Roth
Jörg Schüttrumpf
Peter Milanov
Daniela Abriss
Christopher Ungerer
Patricia Quade-Lyssy
Jeremy C. Simpson
Rainer Pepperkok
Erhard Seifried
Torsten Tonn
- nefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.
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The viral vector vaccine VSV-GP boosts immune response upon repeated applications
(2012)
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Reinhard Tober
Zoltan Banki
Asim Ejaz
Alex Muik
Lisa Mareike Egerer
Dorothee von Laer
Janine Kimpel
- Poster presentation AIDS Vaccine 2012 Boston, MA, USA. 9-12 September 2012
Background: Vesicular stomatitis virus (VSV) is a potent candidate vaccine vector for various viral diseases (e.g. HIV, HCV, RSV). The biggest limitation of VSV, however, is its neurotoxicity, which limits application in humans. The second drawback is that VSV induces neutralizing antibodies rapidly and is thus ineffective as a vaccine vector upon repeated applications. Our group has recently shown that VSV pseudotyped with the glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV), VSV-GP, is not neurotoxic. The aim of this project was to evaluate the potential of VSV-GP as a vaccine vector.
Methods: For this purpose, we used Ovalbumin (OVA) as a model antigen and analyzed immunogenicity of GP-pseudotyped and wildtype VSV containing OVA (VSV-GP-OVA and VSV-OVA) in vitro and in vivo in mouse models.
Results: We showed that both vectors infected murine bone marrow-derived dendritic cells (bmDCs) in vitro. These bmDCs were able to activate OVA specific CD8+ and CD4+ T cells. Immunization experiments in mice revealed that both VSV-OVA and VSV-GP-OVA induced functional OVA-specific cytotoxic T cells (CTLs) after a single immunization. In addition, with both viruses, mice generated antibodies against OVA. However, boosting with the same virus was only possible for the GP-pseudotyped virus but not for wild type VSV. The efficacy of repeated immunization with VSV-OVA was most likely limited by high levels of neutralizing antibodies, which we detected after the first immunization. In contrast, no neutralizing antibodies against VSV-GP were induced even after boosting.
Conclusion: Taken together, we showed that the non-neurotoxic VSV-GP is able to induce specific T cell and B cell responses against the model antigen OVA to the same level as the wild type VSV vector. However, in contrast to wild type VSV, VSV-GP-OVA boosted the immune response upon repeated applications. Thus, VSV-GP is a promising novel vaccine vector.
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Post-transcriptional regulation of 5-lipoxygenase mRNA expression via alternative splicing and nonsense-mediated mRNA decay
(2012)
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Meike Ochs
Bernd Lothar Sorg
Laura Pufahl
Manuel Grez
Beatrix Süß
Dieter Steinhilber
- 5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes (LT), a group of inflammatory lipid mediators derived from arachidonic acid. Here, we investigated the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD). In the present study, we report the identification of 2 truncated transcripts and 4 novel 5-LO splice variants containing premature termination codons (PTC). The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression.
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Einfluss des Transkriptionsfaktors Tal1 auf die Osteoklastogenese durch Regulation von DC-STAMP
(2012)
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Nadine Courtial
- Das menschliche Knochengewebe unterliegt einem ständigen Auf- und Abbau. Der
Knochenumbau, die so genannte Knochenremodellierung, findet stetig statt und etwa 10 %
des gesamten Knochengewebes werden innerhalb eines Jahres erneuert (Lerner UH, 2006).
Während der Knochenremodellierung befindet sich die Zellaktivität der Knochenaufbauenden
Osteoblasten und der Knochen-abbauenden Osteoklasten in einem
empfindlichen Gleichgewicht (Karsenty G und Wagner EF, 2002; Teitelbaum SL, 2000).
Durch Störung des Gleichgewichts zwischen Osteoblasten und Osteoklasten kann es zu
Knochen-assoziierten Krankheiten wie Osteoporose oder Osteopetrose kommen (Helfrich
MH, 2003; Sambrook P und Cooper C, 2006).
Osteoklasten sind multinukleäre Zellen, die in der Lage sind die Knochenmatrix zu
resorbieren (Teitelbaum SL, 2000). Sie entstehen aus pluripotenten, hämatopoetischen
Stammzellen durch Differenzierung und Zellfusion von Monozyten/Makrophagen-
Vorläuferzellen (Menaa C et al., 2000, Yavropoulou MP und Yovos JG, 2008). Die
Osteoklasten-Differenzierung wird hauptsächlich durch die Zytokine M-CSF (macrophage
colony stimulating factor) und RANKL (receptor activator of nuclear factor k b ligand)
induziert. Sie initiieren ein spezifisches Expressionsmuster Osteoklasten-spezifischer Gene
und aktivieren die Zellfusion in Osteoklasten-Vorläuferzellen zur Bildung reifer Osteoklasten
(Boyle WJ et al., 2003; Asagiri M und Takayanagi H, 2007). Die RANKL-vermittelte Induktion
der Osteoklastogenese beruht auf der Initiierung eines streng regulierten Netzwerks aus
Transkriptionsfaktoren (Yang X und Karsenty G, 2002). Einige Transkriptionsfaktoren, die
während der Osteoklasten-Differenzierung induziert und exprimiert werden, sind nicht auf
Osteoklasten beschränkt. Sie erfüllen auch Aufgaben in anderen hämatopoetischen
Differenzierungsprozessen (Engel I und Murre C et al., 1999), so dass vermutlich die
Kombination der Transkriptionsfaktoren entscheidend für die Osteoklastogenese ist.
Der basic helix-loop-helix-Transkriptionsfaktor Tal1 (T-cell acute lymphocytic
leukemia 1, auch Scl1, stem cell leukemia 1) ist ein entscheidender Faktor in der primitiven
und der definitiven Hämatopoese (Bloor AJ et al., 2002; Shivdasani RA et al., 1996). Die
Expression von Tal1 konnte bisher in verschiedenen hämatopoetischen Zelllinien gezeigt
werden, u.a. in monozytischen Zellen (Elefanty AG et al., 1998; Green AR et al., 1992;
Pulford K et al., 1995; Dey S et al., 2010).
In der vorliegenden Arbeit wurde der Einfluss des Transkriptionsfaktors Tal1 in
Monozyten und reifen Osteoklasten, vor allem in Bezug auf genregulatorische Prozesse
während der Osteoklasten-Differenzierung, untersucht. Der Transkriptionsfaktor Tal1 wird
in vitro und in vivo in Osteoklasten-Vorläuferzellen und reifen Osteoklasten exprimiert. Die
Proteinexpression von Tal1 wird durch die Inkubation der Zellen mit RANKL induziert, jedoch
wurde dies in Bezug auf die mRNA-Expression von Tal1 nicht beobachtet, so dass
vermutlich eine posttranskriptionelle Regulation von Tal1 vorliegt.
Die Überexpression von Tal1 sorgte für eine Blockade der Differenzierung von
Osteoklasten-Vorläuferzellen in reife Osteoklasten. Der Verlust von Tal1 in primären
Monozyten/Makrophagen-Zellen führte zur veränderten Expression von über 1200 Genen,
wobei jeweils etwa 600 Gene herauf- bzw. herabreguliert waren. Dies verdeutlicht, dass Tal1
sowohl an der Aktivierung als auch an der Reprimierung der Genexpression in Osteoklasten-
Vorläuferzellen beteiligt ist. Die Liste der herabregulierten Gene beinhaltete u.a. das
Osteoklasten-spezifische Enzym Acp5 (auch TRAP, tartrate resistant acid phosphatase), die
Liste der herauf regulierten Gene beinhaltete u.a. DC-STAMP (dendritic cell specific
transmembrane protein) und ATP6V0D2 (d2 isoform of vascuolar ATPase V0 domain), beide
werden im Zusammenhang mit der Zellfusion während der Osteoklasten-Differenzierung
beschrieben (Kim K et al., 2008; Kim T et al., 2010; Yagi M et al., 2005). Der Promotor von
DC-STAMP beinhaltet mehrere potentielle Bindestellen für Tal1 und Osteoklastenspezifische
Transkriptionsfaktoren. Es konnte gezeigt werden, dass Tal1, PU.1 und MITF im
Bereich um 343 bp vor dem Transkriptionsstartpunkt des DC-STAMP-Promotors binden und
dass Tal1 mit den Osteoklasten-spezifischen Transkriptionsfaktoren PU.1 und MITF
interagiert. Der inhibitorische Effekt von Tal1 auf die Osteoklasten-Differenzierung kommt
durch die Reprimierung der Aktivität der Osteoklasten-spezifischen Transkriptionsfaktoren
PU.1 und MITF auf dem DC-STAMP-Promotor in Osteoklasten-Vorläuferzellen zustande.
Während der Osteoklastogenese kommt es zu einer verringerten Tal1-Bindung auf dem DCSTAMP-
Promotor, wodurch die Tal1-vermittelte Inhibierung der Expression aufgehoben wird.
Die Bindung von PU.1 und MITF auf dem Promotor von DC-STAMP nimmt während der
Osteoklasten-Differenzierung zu. Die Expression von DC-STAMP wird im Verlauf der
Osteoklastogenese induziert, wodurch es zur Zell-Zell-Fusion kommt.
Die Analyse des transkriptionellen Netzwerks, das die Fusion mononukleärer Zellen
in reife Osteoklasten reguliert, vertieft das molekulare Verständnis der Osteoklasten-
Differenzierung und kann zur Entwicklung neuer therapeutischer Ansätze beitragen, die in
der Behandlung von Osteoporose, Riesenzelltumoren und anderen Osteoklastenassoziierten
Krankheiten verwendet werden können.
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Next generation of antiretroviral agents targeting the RNA binding site of the HIV-1 cellular cofactor DDX3: an innovative therapeutic approach
(2012)
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Giovanni Maga
Anna Garbelli
Marco Radi
Federico Falchi
Alberta Samuele
Stefania Paolucci
Fausto Baldanti
Fabrizio Manetti
Sandra Beermann
Ursula Dietrich
Maurizio Botta
- Introduction: Efficacy of currently approved anti-HIV drugs is hampered by mutations of the viral enzymes, leading invariably to drug resistance and chemotherapy failure. Recent data suggest that cellular co-factors also represent useful targets for anti-HIV therapy. We have recently provided evidence for the possibility to block HIV-1 replication by targeting its cellular cofactor DDX3.
Material and methods: Molecular modeling and in silico technologies were applied to rationally design small molecules specifically targeting the RNA binding site of human DDX3. Biochemical studies of mutated DDX3 enzymes were also used to identify additional potential drug binding sites.
Results
Optimization of compounds identified by application of a high-throughput docking approach afforded a promising lead compound which proved to inhibit both the helicase and ATPase activity of DDX3 and to reduce the viral load of peripheral blood mononuclear cells (PBMC) infected with HIV-1. A novel interaction site has been also identified in DDX3, which, when blocked, can reduce viral replication, representing an additional target for small molecules inhibitors.
Conclusions: We have identified the first inhibitors of HIV-1 replication targeting the RNA binding site of the cellular cofactor human DDX3. These compounds may offer superior selectivity over the ATP-competitive inhibitors previously developed. In addition, a novel RNA interacting motif specific to DDX3 has been identified, opening new venues for HIV-1 drug development.
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Cytotoxic capacity of IL-15-stimulated cytokine-induced killer cells against human acute myeloid leukemia and rhabdomyosarcoma in humanized preclinical mouse models
(2012)
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Eva Rettinger
Vida Meyer
Hermann Kreyenberg
Andreas Volk
Selim Kuçi
Andre Willasch
Ewa Koscielniak
Simone Fulda
Winfried S. Wels
Halvard Bönig
Thomas Klingebiel
Peter Bader
- Allogeneic stem cell transplantation (allo-SCT) has become an important treatment modality for patients with high-risk acute myeloid leukemia (AML) and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD) status ultimately leading to patients’ relapse. Adoptive donor lymphocyte infusions based on MRD status using IL-15-expanded cytokine-induced killer (CIK) cells may prevent relapse without causing graft-versus-host-disease (GvHD). To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL-2Rγc−, NSG) were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS) cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow followed by liver, lung, spleen, peripheral blood (PB), and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at equal amounts were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells 250 times more CIK than THP-1 cells were needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliable 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells have potent cytotoxic capacity against AML and RMS cells without causing GvHD.
