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Brenndorfer E.D.,Karolinska University Hospital | Brass A.,Karolinska University Hospital | Soderholm J.,Inovio Biomedical Corporation | Soderholm J.,Gothenburg University | And 4 more authors.
Gut | Year: 2012

Background: The non-structural (NS) 3/4A protease/ helicase of the hepatitis C virus is known to modulate signalling pathways in the infected hepatocyte by cleaving CARD adaptor inducing IFNb (Cardif), T-cell protein tyrosine phosphatase (TC-PTP) and TIR domain-containing adaptor inducing IFNb (TRIF), but the effects of NS3/4A in vivo still remain unclear. Aim: To investigate the influence of NS3/4A on intracellular and intercellular signalling in vivo by analysing the intrahepatic inflammatory response of naïve, lipopolysaccharide (LPS)/D-galactosamine (D-galN) or tumour necrosis factor-α (TNFα)/D-galN-treated NS3/4A-transgenic (Tg) mice. Methods: The intrahepatic immunity of nä?ve and LPS/D-galN- or TNFα/D-galN-treated NS3/4A-Tg mice was determined using western blot, ELISA, real-time PCR, flow cytometry and survival monitoring. The injection of cytokines or antibodies against signalling components was performed to analyse the relevance of the respective pathways for the investigated issues. A Tg mouse lineage expressing an inactivated NS3/4A protease (NS3/4A Ile1073Ala- Tgs) was generated to examine if protective effects were NS3/4A protease dependent. Results: The activation of hepatic signal transducer and activator of transcription 1 and 2 was impaired in NS3/4A-Tg mice after treatment with LPS/D-galN or TNFα/D-galN. This was paralleled by a reduction in hepatic interferon-γ (IFNγ). Reconstitution of IFNγ reverted the resistance to LPS/TNFα in NS3/4A-Tg mice. Subsequently, blocking IFNγ in vivo rendered wild-type mice resistant against treatment with LPS/TNFα. A new Tg mouse expressing an inactivated NS3/4A protease had the same phenotype as wild-type mice with respect to hepatic IFNγ levels and sensitivity to LPS/D-galN. Finally, the chemokine profile was altered in the NS3/4A-Tg mice towards an anti-inflammatory state, which helps to explain the altered immune cell subsets and reduction in hepatic IFNγ production. Conclusions: Our data demonstrate that the NS3/4A protease reduces the intrahepatic production of IFNγ and alters TNFα-mediated effects, thereby impairing the hepatic inflammatory response. This may contribute to viral persistence.

Muthumani K.,University of Pennsylvania | Lambert V.M.,University of South Florida | Kawalekar O.,University of Pennsylvania | Heller R.,Old Dominion University | And 3 more authors.
Vaccine | Year: 2010

By virtue of its ability to induce cell cycle arrest and apoptosis, the HIV accessory protein Vpr (viral protein R) has been evaluated by us and others as an anti-proliferative/anti-cancer agent. We have demonstrated that Vpr, when delivered to established experimental B16.F10 melanoma tumors in mice as a DNA expression plasmid through in vivo electroporation, can result in complete regression of the established tumors. We have also demonstrated that Vpr peptides from the carboxy region of the protein can inhibit in vitro growth of both B16.F10 melanoma as well as human HeLa cervical carcinoma tumor cells. These findings, summarized in this report, underscore the potential of Vpr as an anti-cancer agent and warrants, we believe, further experimental as well as clinical evaluation. © 2009 Elsevier Ltd. All rights reserved.

Nystrom J.,Karolinska University Hospital | Chen A.,Karolinska University Hospital | Frelin L.,Karolinska University Hospital | Ahlen G.,Karolinska University Hospital | And 8 more authors.
Journal of Infectious Diseases | Year: 2010

Hepatitis B virus core antigen (HBcAg) is thought to be a major target for specific cytotoxic T cells (CTLs) in hepatitis B virus infections. A single dose of hepatitis C virus nonstructural 3/4A DNA (<5 μg) effectively primes functional specific CTLs, independently of CD4+ T helper cells and by different routes of immunization. In contrast, HBcAg-specific CTL priming was T helper cell dependent and highly sensitive to the dose and route of delivery. Although CTL priming was improved 10-fold by codon optimization and in vivo electroporation, low levels of DNA still failed to prime CTLs effectively. Only high doses (≥5μg) of codon-optimized HBcAg delivered by in vivo electroporation primed in vivo lytic and polyfunctional CTLs. The ability of endogenous HBcAg to prime CTLs is surprisingly inefficient and differs from that of nonstructural 3/4A. This has important implications for the design of HBcAg-based therapeutic vaccines in humans. © 2010 by the Infectious Diseases Society of America. All rights reserved.

Bodles-Brakhop A.M.,Inovio Biomedical Corporation
Methods in molecular biology (Clifton, N.J.) | Year: 2011

For plasmid-mediated gene therapy applications, a major limitation to scale up from rodents to large animals is the low expression level of injected plasmid DNA. The electroporation technique, which results in the passage of foreign material through the cell membrane, is one method that has been shown to be effective at improving local plasmid uptake and consequently, expression levels. Previous studies have determined that optimized electroporation parameters (such as electric field intensity, number of pulses, lag time between plasmid injections and electroporations, and optimal plasmid formulation conditions) are dependent on the target muscle type and individual species. Here, we provide a detailed protocol to optimize conditions for the successful intramuscular electroporation of plasmid DNA to swine, a large animal model. Our results suggest that the technique is safe and effective for veterinary applications. Furthermore, these results provide evidence for the feasibility of upcoming human applications.

Inovio Biomedical Corporation | Date: 2006-03-02

Medical apparatus for use in delivering drugs and genes to cellular structures by means of electroporation; and instructional manuals sold as a unit therewith.

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