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Sample records for adenoviral sub-unit proteins

  1. Radiolabeled Adenoviral Sub-unit Proteins for Molecular Imaging and Therapeutic Applications in Oncology

    SciTech Connect

    Srivastava, S.; Meinken, G.; Springer, K. Awasthi, V.; Freimuth, P.

    2004-10-06

    The objective of this project was to develop and optimize new ligand systems, based on adenoviral vectors (intact adenovirus, adeno-viral fiber protein, and the knob protein), for delivering suitable radionuclides into tumor cells for molecular imaging and combined gene/radionuclide therapy of cancer.

  2. Adenoviral gene transfer of macrophage inflammatory protein-2 in rat lung.

    PubMed Central

    Foley, R.; Driscoll, K.; Wan, Y.; Braciak, T.; Howard, B.; Xing, Z.; Graham, F.; Gauldie, J.

    1996-01-01

    Replication-defective adenoviral vectors are capable of localized transfer and expression of incorporated gene product in lung tissue. We have constructed an adenoviral vector that expresses rat macrophage inflammatory protein (MIP)-2, a C-X-C chemokine specifically chemotactic for neutrophils, Supernatants from 293 cells, infected with the adenoviral MIP-2 (ADMIP-2) construct, showed potent chemotactic activity and the ability of the ADMIP-2 vector to transcribe and make functional protein was confirmed. In vivo analysis of bronchoalveolar lavage fluid from rats after intratracheal instillation of ADMIP-2 (10(9) plaque-forming units) showed a 10-fold increase in the absolute number of neutrophils in bronchoalveolar lavage fluid as opposed to rats treated with an equal titer of an E1-disabled control virus expressing firefly luciferase (ADCA-18). Neutrophils constituted 65% of total BAL cells with alveolar macrophages being the other major cell type recovered. Rat MIP-2 protein was increased (nanograms per milliliter) in bronchoalveolar lavage fluid over a period of 7 days in ADMIP-2-treated animals. MIP-2 mRNA was demonstrated by Northern blot analysis in lung tissue, and histological analysis confirmed the presence of massive localized tissue neutrophilia. Evidence of chronic tissue injury and repair (ie, fibrosis) was not detected up to 2 weeks after the neutrophil infiltrate had resolved, subsequent to decreased chemokine presence. Adenoviral gene transfer proved an effective tool for the assessment of lung tissue expression of this chemokine in vivo and is useful in developing rodent models of tissue neutrophilia. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 PMID:8863686

  3. Sequence dependent interaction of hnRNP proteins with late adenoviral transcripts.

    PubMed Central

    van Eekelen, C; Ohlsson, R; Philipson, L; Mariman, E; van Beek, R; van Venrooij, W

    1982-01-01

    Irradiation with ultraviolet light was used to induce covalent linkage between hnRNA and its associated proteins in intact HeLa cells, late after infection with adenovirus type 2. Covalently linked hnRNA-protein complexes, containing polyadenylated adenoviral RNA, were isolated and their protein moiety characterized. Host 42,000 Mr hnRNP proteins proved to be the major proteins crosslinked to viral hnRNA. To investigate their possible involvement in RNA processing, the localization of these cross-linked polypeptides on adenoviral late transcripts was determined. Sequences of RNA around the attachment sites of the protein were isolated. After in vitro labeling they were hybridized to Southern blots of adeno DNA fragments. The hybridization patterns revealed that the 42,000 Mr polypeptides can be linked to adenoviral transcripts over the entire length of the RNA, corresponding to 16.2-91.5 m.u. of the viral genome. Fine mapping within the Hind III B region (16.8-31.5 m.u.) established, however, that the localization of the cross-linked polypeptides was not random in all parts of the transcript. Sequences around the third leader and the 3' part of the i-leader were overrepresented, whereas the regions encoding VA I and VA II RNA and the late region 1 mRNA bodies were underrepresented in the cross-linked RNA. Using genomic DNA fragments and a cDNA clone containing the tripartite leader it appeared that leader and intervening sequences were represented about equally in cross-linked RNA fragments. Although these results do not support the notion that introns or exons are specifically interacting with one RNP protein, they demonstrate that the 42,000 hnRNP proteins are non randomly positioned on the RNA sequence. Images PMID:6296766

  4. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    NASA Astrophysics Data System (ADS)

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-06-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

  5. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness.

    PubMed

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  6. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    PubMed Central

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  7. Adenoviral expression of murine serum amyloid A proteins to study amyloid fibrillogenesis.

    PubMed

    Kindy, M S; King, A R; Yu, J; Gerardot, C; Whitley, J; de Beer, F C

    1998-06-15

    Serum amyloid A (SAA) proteins are one of the most inducible acute-phase reactants and are precursors of secondary amyloidosis. In the mouse, SAA1 and SAA2 are induced in approximately equal quantities in response to amyloid induction models. These two isotypes differ in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited into amyloid fibrils. SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2. However, even though it is more closely related to SAA2 it is not deposited as amyloid fibrils. We have developed an adenoviral vector system to overexpress SAA proteins in cell culture to determine the ability of these proteins to form amyloid fibrils, and to study the structural features in relation to amyloid formation. Both the SAA2 and CE/J SAA proteins were synthesized in large quantities and purified to homogeneity. Electron microscopic analysis of the SAA proteins revealed that the SAA2 protein was capable of forming amyloid fibrils, whereas the CE/J SAA was incapable. Radiolabelled SAAs were associated with normal or acute-phase high-density lipoproteins (HDLs); we examined them for their clearance from the circulation. In normal mice, SAA2 had a half-life of 70 min and CE/J SAA had a half-life of 120 min; however, in amyloid mice 50% of the SAA2 cleared in 55 min, compared with 135 min for the CE/J protein. When the SAA proteins were associated with acute-phase HDLs, SAA2 clearance was decreased to 60 min in normal mice compared with 30 min in amyloidogenic mice. Both normal and acute-phase HDLs were capable of depositing SAA2 into preformed amyloid fibrils, whereas the CE/J protein did not become associated with amyloid fibrils. This established approach opens the doors for large-scale SAA production and for the examination of specific amino acids involved in the fibrillogenic capability of the SAA2 molecule in vitro

  8. Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

    SciTech Connect

    Ugai, Hideyo; Dobbins, George C.; Wang, Minghui; Le, Long P.; Matthews, David A.; Curiel, David T.

    2012-10-25

    Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.

  9. Specific interaction between adenoviral 55-kDa E1B protein and in vivo produced p53 fusion proteins.

    PubMed

    Chumakov, A; Koeffler, H P

    1993-09-15

    Several protein fusion systems have been used in recent years to study protein-protein and DNA-protein interactions. Most of them use bacterially produced proteins which have several inherent disadvantages, notably, the absence of correct post-translational modifications and the frequent insolubility of recombinant proteins. We sought to develop a system to study proteins interacting with the nuclear phosphoprotein p53, which is believed to be a tumor suppressor. To prepare fusions of p53, we developed a convenient system that permits both in vivo and in vitro production and easy affinity purification of peptides and protein fragments as glutathione-transferase fusions. We placed the coding sequence of the Schistosoma japonica glutathione S-transferase (GST) under the control of the strong CMV/T7 promoter and SV40 splice and polyadenylation signals. An extensive polylinker (MCS) at the 3' end of the GST gene is preceded by the sequence encoding the cleavage site of the site-specific protease. We cloned the complete coding sequences of human wild-type p53, as well as p53 mutants representing all four mutational hotspots (codons 141, 175, 248, and 273), into our expression vector. In vitro transcription using the upstream T7 promoter and translation in reticulocyte lysates form an easy way to produce hybrid proteins; affinity purification on a glutathione-agarose column removes proteins that are present in reticulocyte lysates. We have also studied specific in vivo interactions of human p53 with the adenoviral 55-kDa E1B protein by transfecting expression constructs of GST-p53 fusions into human Ad5-transformed 293 cells. PMID:8406015

  10. Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

    SciTech Connect

    Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

    2011-11-25

    We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: > We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. > Cre/loxP recombination was used to modify the adenovirus genome. > A targeting ligand present on capsid protein IX was removed or replaced using recombination. > Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

  11. Adenoviral vector which delivers FasL-GFP fusion protein regulated by the tet-inducible expression system.

    PubMed

    Rubinchik, S; Ding, R; Qiu, A J; Zhang, F; Dong, J

    2000-05-01

    Fas ligand (FasL) is a member of the tumor necrosis family and when bound to its receptor, Fas, induces apoptosis. It plays important roles in immune response, degenerative and lymphoproliferative diseases, development and tumorigenesis. It is also involved in generation of immune privilege sites in the eye and testis. Harnessing the power of this molecule is expected to lead to a powerful chemotherapeutic. We describe the construction and characterization of replication-deficient adenoviral vectors that express a fusion of murine FasL and green fluorescent protein (GFP). FasL-GFP retains full activity of wild-type FasL, at the same time allowing for easy visualization and quantification in both living and fixed cells. The fusion protein is under the control of a tetracycline-regulated gene expression system. Tight control of expression is achieved by creating a novel 'double recombinant' Ad vector, in which the tet-responsive element and the transactivator element are built into the opposite ends of the same vector to avoid enhancer interference. Expression can be conveniently regulated by tetracycline or its derivatives in a dose-dependent manner. The vector was able to deliver FasL-GFP gene to cells in vitro efficiently, and the expression level and function of the fusion protein was modulated by the concentration of doxycycline. This regulation allows us to produce high titers of the vector by inhibiting FasL expression in an apoptosis-resistant cell line. Induction of apoptosis was demonstrated in all cell lines tested. These results indicate that our vector is a potentially valuable tool for FasL-based gene therapy of cancer and for the study of FasL/Fas-mediated apoptosis and immune privilege. PMID:10845726

  12. Adenoviral E4orf3 and E4orf6 Proteins, But Not E1B55K, Increase Killing of Cancer Cells by Radiotherapy in vivo

    SciTech Connect

    Liikanen, Ilkka; Dias, Joao D.; Nokisalmi, Petri; Sloniecka, Marta; Kangasniemi, Lotta; Rajecki, Mari; Dobner, Thomas; Tenhunen, Mikko; Kanerva, Anna; Pesonen, Sari; Ahtiainen, Laura Ph.D.; Hemminki, Akseli

    2010-11-15

    Purpose: Radiotherapy is widely used for treatment of many tumor types, but it can damage normal tissues. It has been proposed that cancer cells can be selectively sensitized to radiation by adenovirus replication or by using radiosensitizing transgenes. Adenoviral proteins E1B55K, E4orf3, and E4orf6 play a role in radiosensitization, by targeting the Mre11, Rad50, and NBS1 complex (MRN) and inhibiting DNA double-strand break (DSB) repair. We hypothesize that combined with irradiation, these adenoviral proteins increase cell killing through the impairment of DSB repair. Methods and Materials: We assessed the radiosensitizing/additive potential of replication-deficient adenoviruses expressing E1B55K, E4orf3, and E4orf6 proteins. Combination treatments with low-dose external photon beam radiotherapy were studied in prostate cancer (PC-3MM2 and DU-145), breast cancer (M4A4-LM3), and head and neck cancer (UT-SCC8) cell lines. We further demonstrated radiosensitizing or additive effects in mice with PC-3MM2 tumors. Results: We show enhanced cell killing with adenovirus and radiation combination treatment. Co-infection with several of the viruses did not further increase cell killing, suggesting that both E4orf6 and E4orf3 are potent in MRN inhibition. Our results show that adenoviral proteins E4orf3 and E4orf6, but not E1B55K, are effective also in vivo. Enhanced cell killing was due to inhibition of DSB repair resulting in persistent double-strand DNA damage, indicated by elevated phospho-H2AX levels at 24 h after irradiation. Conclusions: This knowledge can be applied for improving the treatment of malignant tumors, such as prostate cancer, for development of more effective combination therapies and minimizing radiation doses and reducing side effects.

  13. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  14. [Construction of recombinant adenoviral vector expressing genes of the conservative influenza proteins M2 and nucleoprotein].

    PubMed

    Esmagambetov, I B; Sedova, E S; Shcherbinin, D N; Lysenko, A A; Garas, M N; Shmarov, M M; Logunov, D Iu

    2014-01-01

    Influenza is a highly contagious and one of the most massive infection diseases. General epidemiological significance has a strain, which belongs to subtype A. A high degree of genetic variety leads to the permanent changes in the antigenic structure of the influenza virus. Therefore, the current influenza vaccines require periodic updating of the composition of strains. Presently, it is important to develop a universal vaccine that can protect against different strains of influenza A virus at the same time and is based on the conserved antigens of the influenza virus. The recombinant adenovirus vectors expressing genes of conserved viral antigenes may be a promising candidate vaccine against influenza A. Using the method of the homologous recombination, we developed in this study recombinant adenovirus of fifth serotype that expresses genes of the ion channel M2 and nucleoprotein NP of the influenza virus A. Genes of the consensus protein M2 and NP of human influenza A virus were included into the structure of the viral genome. The expression of the antigens M2 and NP using recombinant adenovirus vector was detected by a Western blot assay. The immunogenicity of the developed recombinant adenovirus vector was demonstrated by the intranasal immunization of laboratory mice. PMID:25080815

  15. Heterologous Prime-Boost Regimens with a Recombinant Chimpanzee Adenoviral Vector and Adjuvanted F4 Protein Elicit Polyfunctional HIV-1-Specific T-Cell Responses in Macaques

    PubMed Central

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN (‘A’), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 (‘P’), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques

  16. Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.

    PubMed

    Souza, Sandra C; Muliro, Kizito V; Liscum, Laura; Lien, Ping; Yamamoto, Mia T; Schaffer, Jean E; Dallal, Gerard E; Wang, Xinzhong; Kraemer, Fredric B; Obin, Martin; Greenberg, Andrew S

    2002-03-01

    Perilipin (Peri) A is a phosphoprotein located at the surface of intracellular lipid droplets in adipocytes. Activation of cyclic AMP-dependent protein kinase (PKA) results in the phosphorylation of Peri A and hormone-sensitive lipase (HSL), the predominant lipase in adipocytes, with concurrent stimulation of adipocyte lipolysis. To investigate the relative contributions of Peri A and HSL in basal and PKA-mediated lipolysis, we utilized NIH 3T3 fibroblasts lacking Peri A and HSL but stably overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport protein 1 (FATP1). When incubated with exogenous fatty acids, ACS1/FATP1 cells accumulated 5 times more triacylglycerol (TG) as compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of Peri A in ACS1/FATP1 cells enhanced TG accumulation and inhibited lipolysis, whereas expression of HSL fused to green fluorescent protein (GFPHSL) reduced TG accumulation and enhanced lipolysis. Forskolin treatment induced Peri A hyperphosphorylation and abrogated the inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri A Delta 3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222, and Ser-276) reduced Peri A hyperphosphorylation and blocked constitutive and forskolin-stimulated lipolysis. Thus, perilipin expression and phosphorylation state are critical regulators of lipid storage and hydrolysis in ACS1/FATP1 cells. PMID:11751901

  17. A peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kDa protein but not export of viral late mRNAs

    SciTech Connect

    Flint, S.J. . E-mail: sjflint@molbio.princeton.edu; Huang, Wenying; Goodhouse, Joseph; Kyin, Saw

    2005-06-20

    The human subgroup C adenoviral E1B 55 kDa and E4 Orf6 proteins are required for efficient nuclear export of viral late mRNAs, but the cellular pathway that mediates such export has not been identified. As a first step to develop a general approach to address this issue, we have assessed the utility of cell-permeable peptide inhibitors of cellular export receptors. As both E1B and E4 proteins have been reported to contain a leucine-rich nuclear export signal (NES), we synthesized a cell-permeable peptide containing such an NES. This peptide induced substantial inhibition of export of the E1B protein, whereas a control, non-functional peptide did not. However, under the same conditions, the NES peptide had no effect on export of viral late mRNAs. These observations establish that viral late mRNAs are not exported by exportin1, as well as the value of peptide inhibitors in investigation of mRNA export regulation in adenovirus-infected cells.

  18. STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

    PubMed

    Yang, Xuechao; van der Kraan, Peter M; van den Dolder, Juliette; Walboomers, X Frank; Bian, Zhuan; Fan, Mingwen; Jansen, John A

    2007-11-01

    Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium. PMID:17824831

  19. Enhancement of Protective Efficacy through Adenoviral Vectored Vaccine Priming and Protein Boosting Strategy Encoding Triosephosphate Isomerase (SjTPI) against Schistosoma japonicum in Mice

    PubMed Central

    Dai, Yang; Wang, Xiaoting; Tang, Jianxia; Zhao, Song; Xing, Yuntian; Dai, Jianrong; Jin, Xiaolin; Zhu, Yinchang

    2015-01-01

    Background Schistosomiasis japonica is a zoonotic parasitic disease; developing transmission blocking veterinary vaccines are urgently needed for the prevention and control of schistosomiasis in China. Heterologous prime-boost strategy, a novel vaccination approach, is more effective in enhancing vaccine efficacy against multiple pathogens. In the present study, we established a novel heterologous prime-boost vaccination strategy, the rAdV-SjTPI.opt intramuscular priming and rSjTPI subcutaneous boosting strategy, and evaluated its protective efficacy against Schistosoma japonicum in mice. Methodology/Principal Findings Adenoviral vectored vaccine (rAdV-SjTPI.opt) and recombinant protein vaccine (rSjTPI) were prepared and used in different combinations as vaccines in a mouse model. The specific immune responses and protective efficacies were evaluated. Furthermore, the longevity of protective efficacy was also determined. Results showed that the rAdV-SjTPI.opt priming-rSjTPI boosting strategy elicited higher levels of specific IgG responses and broad-spectrum specific cellular immune responses. The protective efficacy could reach up to nearly 70% and 50% of protection could be observed at 10 weeks after the last immunization in mice. Conclusions/Significance The rAdV-SjTPI.opt intramuscular priming-rSjTPI subcutaneous boosting vaccination strategy is a novel, highly efficient, and stable approach to developing vaccines against Schistosoma japonicum infections in China. PMID:25793406

  20. Inhibition of proteolytic processing of adenoviral proteins by epsilon-aminocaproic acid and ambenum in adenovirus-infected cells.

    PubMed

    Nosach, Lidiya; Dyachenko, Nataliya; Zhovnovataya, Valentina; Lozinskiy, Miron; Lozitsky, Victor

    2002-01-01

    Maturation of adenovirus particles is markedly affected by proteolytic processing. The possibility for blocking the conversion of precursor structural core protein (preVII) into mature structure protein VII by officinal drugs epsilon-aminocaproic acid and ambenum has been demonstrated in Hep-2 cells infected with adenovirus. Proteolytic processing may be regarded as one of the targets for inhibiting adenovirus reproduction. PMID:12545207

  1. Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2

    PubMed Central

    Neumann, Alexander J.; Gardner, Oliver F. W.; Williams, Rebecca; Alini, Mauro; Archer, Charles W.; Stoddart, Martin J.

    2015-01-01

    Articular cartilage progenitor cells (ACPCs) represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs). This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs) are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2). hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1) concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase). To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs represent a

  2. Impact of the Adenoviral E4 Orf3 Protein on the Activity and Posttranslational Modification of p53

    PubMed Central

    DeHart, Caroline J.; Perlman, David H.

    2015-01-01

    ABSTRACT Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1–17, 2014, http://dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076–1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells. IMPORTANCE The tumor suppressor p53, a master regulator of cellular responses to stress, is inactivated and destroyed in cells infected by species C human adenoviruses, such as type 5. It is targeted for proteasomal degradation by the action of a virus-specific E3

  3. Capsid modification strategies for detargeting adenoviral vectors.

    PubMed

    Parker, Alan L; Bradshaw, Angela C; Alba, Raul; Nicklin, Stuart A; Baker, Andrew H

    2014-01-01

    Adenoviral vectors hold immense potential for a wide variety of gene therapy based applications; however, their efficacy and toxicity is dictated by "off target" interactions that preclude cell specific targeting to sites of disease. A number of "off target" interactions have been described in the literature that occur between the three major capsid proteins (hexon, penton, and fiber) and components of the circulatory system, including cells such as erythrocytes, white blood cells, and platelets, as well as circulatory proteins including complement proteins, coagulation factors, von Willebrand Factor, p-selectin as well as neutralizing antibodies. Thus, to improve efficacious targeting to sites of disease and limit nonspecific uptake of virus to non-target tissues, specifically the liver and the spleen, it is necessary to develop suitable strategies for genetically modifying the capsid proteins to preclude these interactions. To this end we have developed versatile systems based on homologous recombination for modification of each of the major capsid proteins, which are described herein. PMID:24132476

  4. Targeting different types of human meningioma and glioma cells using a novel adenoviral vector expressing GFP-TRAIL fusion protein from hTERT promoter

    PubMed Central

    2011-01-01

    Objective The objective of this study was to evaluate the anti-tumor effects of Ad/gTRAIL (an adenoviral vector in which expression of GFP and TRAIL is driven by a human telomerase reverse transcriptase promoter, hTERT) on malignant meningiomas and gliomas. Background Gliomas and meningiomas are the two most common types of human brain tumors. Currently there is no effective cure for recurrent malignant meningiomas or for gliomas. Ad/gTRAIL has been shown to be effective in killing selected lung, colon and breast cancer cells, but there have been no studies reporting its antitumor effects on malignant meningiomas. Therefore, we tested the antitumor effect of Ad/gTRAIL for the first time in human malignant meningioma and glioma cell lines, and in intracranial M6 and U87 xenografts. Methods Materials and Methods: Human malignant meningioma and glioma cells were infected with adenoviruses, Ad/gTRAIL and Ad/CMV-GFP. Cell viability was determined by proliferation assay. FACS analysis and quantification of TRAIL were used to measure apoptosis in these cells. We injected Ad/gTRAIL viruses in intracranial M6 and U87 xenografts, and measured the brain tumor volume, quantified apoptosis by TUNEL assay in the brain tumor tissue. Results Our studies demonstrate that in vitro/in vivo treatment with Ad/gTRAIL virus resulted in significant increase of TRAIL activity, and elicited a greater tumor cell apoptosis in malignant brain tumor cells as compared to treatment with the control, Ad/CMV-GFP virus without TRAIL activity. Conclusions We showed for the first time that adenovirus Ad/gTRAIL had significant antitumor effects against high grade malignant meningiomas as well as gliomas. Although more work needs to be done, our data suggests that Ad/gTRAIL has the potential to be useful as a tool against malignant brain tumors. PMID:22035360

  5. Helper-Dependent Adenoviral Vectors

    PubMed Central

    Rosewell, Amanda; Vetrini, Francesco; Ng, Philip

    2012-01-01

    Helper-dependent adenoviral vectors are devoid of all viral coding sequences, possess a large cloning capacity, and can efficiently transduce a wide variety of cell types from various species independent of the cell cycle to mediate long-term transgene expression without chronic toxicity. These non-integrating vectors hold tremendous potential for a variety of gene transfer and gene therapy applications. Here, we review the production technologies, applications, obstacles to clinical translation and their potential resolutions, and the future challenges and unanswered questions regarding this promising gene transfer technology. PMID:24533227

  6. Capsid-Modified Adenoviral Vectors for Improved Muscle-Directed Gene Therapy

    PubMed Central

    Guse, Kilian; Suzuki, Masataka; Sule, Gautam; Bertin, Terry K.; Tyynismaa, Henna; Ahola-Erkkilä, Sofia; Palmer, Donna; Suomalainen, Anu; Ng, Philip; Cerullo, Vincenzo; Hemminki, Akseli

    2012-01-01

    Abstract Skeletal muscle represents an attractive target tissue for adenoviral gene therapy to treat muscle disorders and as a production platform for systemic expression of therapeutic proteins. However, adenovirus serotype 5 vectors do not efficiently transduce adult muscle tissue. Here we evaluated whether capsid modifications on adenoviral vectors could improve transduction in mature murine muscle tissue. First-generation and helper-dependent serotype 5 adenoviral vectors featuring the serotype 3 knob (5/3) showed significantly increased transduction of skeletal muscle after intramuscular injection in adult mice. Furthermore, we showed that full-length dystrophin could be more efficiently transferred to muscles of mdx mice using a 5/3-modified helper-dependent adenoviral vector. In contrast to first-generation vectors, helper-dependent adenoviral vectors mediated stable marker gene expression for at least 1 year after intramuscular injection. In conclusion, 5/3 capsid-modified helper-dependent adenoviral vectors show enhanced transduction in adult murine muscle tissue and mediate long-term gene expression, suggesting the suitability of these vectors for muscle-directed gene therapy. PMID:22888960

  7. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    SciTech Connect

    Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie; Soliymani, Rabah; Tenhunen, Mikko; Ahtiainen, Laura; Hemminki, Akseli

    2012-05-01

    Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

  8. Adenoviral virotherapy for malignant brain tumors

    PubMed Central

    Nandi, Suvobroto; Lesniak, Maciej S

    2009-01-01

    Glioblastoma multiforme (GBM) is the most common form of primary brain cancer. In the past decade, virotherapy of tumors has gained credence, particularly in glioma management, as these tumors are not completely resectable and tend to micro-metastasize. Adenoviral vectors have an advantage over other viral vectors in that they are relatively non-toxic and do not integrate in the genome. However, the lack of coxsackie and adenovirus receptors (CAR) on surface of gliomas provides for inefficient transduction of wild-type adenoviral vectors in these tumors. By targeting receptors that are over-expressed in gliomas, modified adenoviral constructs have been shown to efficiently infect glioma cells. In addition, by taking advantage of tumor specific promoter (TSP) elements, oncolytic adenoviral vectors offer the promise of selective tumor-specific replication. This dual targeting strategy has enabled specificity in both laboratory and pre-clinical settings. This review looks at current trends in adenoviral virotherapy of gliomas, with an emphasis on targeting modalities and future clinical applications. PMID:19456208

  9. Adenoviral virotherapy for malignant brain tumors.

    PubMed

    Nandi, Suvobroto; Lesniak, Maciej S

    2009-06-01

    Glioblastoma multiforme is the most common form of primary brain cancer. In the past decade, virotherapy of tumors has gained credence, particularly in glioma management, as these tumors are not completely resectable and tend to micro-metastasize. Adenoviral vectors have an advantage over other viral vectors in that they are relatively non-toxic and do not integrate in the genome. However, the lack of coxsackie and adenovirus receptors on surface of gliomas provides for inefficient transduction of wild-type adenoviral vectors in these tumors. By targeting receptors that are overexpressed in gliomas, modified adenoviral constructs have been shown to efficiently infect glioma cells. In addition, by taking advantage of tumor-specific promoter elements, oncolytic adenoviral vectors offer the promise of selective tumor-specific replication. This dual targeting strategy has enabled specificity in both laboratory and pre-clinical settings. This review examines current trends in adenoviral virotherapy of gliomas, with an emphasis on targeting modalities and future clinical applications. PMID:19456208

  10. Actin-binding protein alpha-actinin 4 (ACTN4) is a transcriptional co-activator of RelA/p65 sub-unit of NF-kB

    PubMed Central

    Aksenova, Vasilisa; Turoverova, Lidia; Khotin, Mikhail; Magnusson, Karl-Eric; Tulchinsky, Eugene; Melino, Gerry; Pinaev, George P.; Barlev, Nickolai; Tentler, Dmitri

    2013-01-01

    ACTN4 is an actin-binding protein that participates in cytoskeleton organisation. It resides both in the cytoplasm and nucleus and physically associates with various transcription factors. Here, we describe an effect of ACTN4 expression on transcriptional activity of the RelA/p65 subunit of NF-kB. We demonstrate that ACTN4 enhances RelA/p65-dependant expression of c-fos, MMP-3 and MMP-1 genes, but it does not affect TNC, ICAM1 and FN1 expression. Importantly, actin-binding domains of ACTN4 are not critical for the nuclear translocation and co-activation of RelA/p65-dependent transcription. Collectively, our data suggest that in the nucleus, ACTN4 functions as a selective transcriptional co-activator of RelA/p65. PMID:23482348

  11. [Immunogenicity and heterologous protection in mice with a recombinant adenoviral-based vaccine carrying a hepatitis C virus truncated NS3 and core fusion protein].

    PubMed

    Guan, Jie; Deng, Yao; Chen, Hong; Yang, Yang; Wen, Bo; Tan, Wenjie

    2015-01-01

    To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine. PMID:25997323

  12. Amplification of inflammation in emphysema and its association with latent adenoviral infection.

    PubMed

    Retamales, I; Elliott, W M; Meshi, B; Coxson, H O; Pare, P D; Sciurba, F C; Rogers, R M; Hayashi, S; Hogg, J C

    2001-08-01

    This study examines the hypothesis that the cigarette smoke-induced inflammatory process is amplified in severe emphysema and explores the association of this response with latent adenoviral infection. Lung tissue from patients with similar smoking histories and either no (n = 7), mild (n = 7), or severe emphysema (n = 7) was obtained by lung resection. Numbers of polymorphonuclear cells (PMN), macrophages, B cells, CD4, CD8 lymphocytes, and eosinophils present in tissue and airspaces and of epithelial cells expressing adenoviral E1A protein were determined using quantitative techniques. Severe emphysema was associated with an absolute increase in the total number of inflammatory cells in the lung tissue and airspaces. The computed tomography (CT) determined extent of lung destruction was related to the number of cells/m(2) surface area by R(2) values that ranged from 0.858 (CD8 cells) to 0.483 (B cells) in the tissue and 0.630 (CD4 cells) to 0.198 (B cells) in the airspaces. These changes were associated with a 5- to 40-fold increase in the number of alveolar epithelial cells expressing adenoviral E1A protein in mild and severe disease, respectively. We conclude that cigarette smoke-induced lung inflammation is amplified in severe emphysema and that latent expression of the adenoviral E1A protein expressed by alveolar epithelial cells influenced this amplification process. PMID:11500352

  13. Immunocompromised Children with Severe Adenoviral Respiratory Infection

    PubMed Central

    Tylka, Joanna C.; McCrory, Michael C.; Gertz, Shira J.; Custer, Jason W.; Spaeder, Michael C.

    2016-01-01

    Purpose. To investigate the impact of severe respiratory adenoviral infection on morbidity and case fatality in immunocompromised children. Methods. Combined retrospective-prospective cohort study of patients admitted to the intensive care unit (ICU) in four children's hospitals with severe adenoviral respiratory infection and an immunocompromised state between August 2009 and October 2013. We performed a secondary case control analysis, matching our cohort 1 : 1 by age and severity of illness score with immunocompetent patients also with severe respiratory adenoviral infection. Results. Nineteen immunocompromised patients were included in our analysis. Eleven patients (58%) did not survive to hospital discharge. Case fatality was associated with cause of immunocompromised state (p = 0.015), multiple organ dysfunction syndrome (p = 0.001), requirement of renal replacement therapy (p = 0.01), ICU admission severity of illness score (p = 0.011), and treatment with cidofovir (p = 0.005). Immunocompromised patients were more likely than matched controls to have multiple organ dysfunction syndrome (p = 0.01), require renal replacement therapy (p = 0.02), and not survive to hospital discharge (p = 0.004). One year after infection, 43% of immunocompromised survivors required chronic mechanical ventilator support. Conclusions. There is substantial case fatality as well as short- and long-term morbidity associated with severe adenoviral respiratory infection in immunocompromised children. PMID:27242924

  14. Genetically engineering adenoviral vectors for gene therapy.

    PubMed

    Coughlan, Lynda

    2014-01-01

    Adenoviral (Ad) vectors are commonly used for various gene therapy applications. Significant advances in the genetic engineering of Ad vectors in recent years has highlighted their potential for the treatment of metastatic disease. There are several methods to genetically modify the Ad genome to incorporate retargeting peptides which will redirect the natural tropism of the viruses, including homologous recombination in bacteria or yeast. However, homologous recombination in yeast is highly efficient and can be achieved without the need for extensive cloning strategies. In addition, the method does not rely on the presence of unique restriction sites within the Ad genome and the reagents required for this method are widely available and inexpensive. Large plasmids containing the entire adenoviral genome (~36 kbp) can be modified within Saccharomyces cerevisiae yeast and genomes easily rescued in Escherichia coli hosts for analysis or amplification. A method for two-step homologous recombination in yeast is described in this chapter. PMID:24243238

  15. Adenoviral Vectors for Hemophilia Gene Therapy

    PubMed Central

    Brunetti-Pierri, N; Ng, Philip

    2013-01-01

    Hemophilia is an inherited blood clotting disorder resulting from deficiency of blood coagulation factors. Current standard of care for hemophilia patients is frequent intravenous infusions of the missing coagulation factor. Gene therapy for hemophilia involves the introduction of a normal copy of the deficient coagulation factor gene thereby potentially offering a definitive cure for the bleeding disorder. A variety of approaches have been pursued for hemophilia gene therapy and this review article focuses on those that use adenoviral vectors. PMID:24883229

  16. Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

    PubMed Central

    Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

    2013-01-01

    Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

  17. Specific NFκB subunit activation and kinetics of cytokine induction in adenoviral keratitis

    PubMed Central

    Rajaiya, Jaya; Sadeghi, Neda

    2009-01-01

    Purpose Corneal inflammation associated with ocular adenoviral infection is caused by leukocytic infiltration of the subepithelial stroma in response to expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected corneal cells. We have shown that these two chemokines are activated by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase (JNK) for MCP-1. It is also well established that transcription of each of these chemokines is tightly controlled by the nuclear factor kappa B (NFκB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFκB in adenoviral infection of the cornea. Methods Primary keratocytes derived from human donor corneas were treated with signaling inhibitors and small interfering RNA specific to MAPKs, and infected with adenovirus for different time periods before analysis. Activation of specific NFκB subunits was analyzed by western blot, confocal microscopy, electromobility shift assay, and chromatin immunoprecipitation, and chemokine expression was quantified by enzyme-linked immunosorbent assay. Results Upon adenoviral infection, NFκB p65, p50, and cREL subunits translocate to the nucleus. This translocation is blocked by inhibitors of specific MAPK signaling pathways. Confocal microscopy showed that inhibitors of the p38, JNK, and ERK pathways differentially inhibited NFκB nuclear translocation, while PP2, an inhibitor of Src family kinases, completely inhibited NFκB nuclear translocation. Western blot analysis revealed that activation of specific NFκB subunits was time dependent following infection. Chromatin immunoprecipitation experiments indicated that binding of NFκB p65 and p50 subunits to the IL-8 promoter upon viral infection was differentially reduced by chemical inhibitors of MAPKs. Electromobility shift assay and luciferase assay analysis

  18. Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells.

    PubMed

    Blum, Jeremy S; Parrott, M Brandon; Mikos, Antonios G; Barry, Michael A

    2004-03-01

    We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone. PMID:15013104

  19. Transfection of Primary Hepatocytes with Liver-Enriched Transcription Factors Using Adenoviral Vectors.

    PubMed

    Benet, Marta; Jover, Ramiro; Bort, Roque

    2015-01-01

    Primary cultured hepatocytes are probably the best model to study endogenous metabolic pathways, toxicity, or drug metabolism. Many of these studies require expression of ectopic genes. It would be desirable to use a method of transfection that allows dose-response studies, high efficiency of transfection, and the possibility to express several genes at the same time. Adenoviral vectors fulfill these requirements, becoming a valuable tool for primary hepatocyte transfection. Moreover, they are easy to generate and do not require a high level of biocontainment. In the present chapter, we describe the generation, cloning, amplification, and purification of an adenoviral vector capable of infecting primary cultured hepatocytes. This recombinant adenovirus induces robust expression of the protein of interest in hepatocytes within a wide range of doses. PMID:26272145

  20. Inhibition of apoptosis reduces immunogeneic potential of adenoviral-treated syngeneic liver grafts.

    PubMed

    Puellmann, Kerstin; Beham, Alexander; Kienle, Klaus; Vogel, Mandy; Schlitt, Hans Juergen; Jauch, Karl Walter; Rentsch, Markus

    2006-11-27

    Effects of adenoviral therapy and reduced apoptosis on immune response were investigated in a rat liver transplantation model after prolonged ischemia-reperfusion. Liver donors were treated i.v. either with an adenoviral construct, expressing bcl-2, green-fluorescent-protein, or doxycyclin. Intrahepatic apoptosis was assessed by terminal transferase dUTP nick end labeling assay. The intrahepatic presence of CD4, CD8a, CD163, immunoglobulin (Ig)beta, tumor necrosis factor (TNF)-alpha and myeloperoxidase (MPO) was quantified by realtime polymerase chain reaction at 24 hours and seven days after transplantation. Bcl-2 expression abrogated the TNF-alpha elevation and reduced apoptosis of hepatocytes and sinusoidal endothelial cells as compared to advCMV green fluorescent protein. No effects on CD4, CD8a, CD163 and MPO expression were noticed in bcl-2 pretreated livers, whereas Igbeta was slightly enhanced compared to controls. Adenoviral infected liver grafts trigger an immune response but reduced apoptosis resulted in down-regulation of TNF-alpha. Thus, bcl-2 transfer might simultaneously reduce graft ischemia reperfusion injury and immunogenicity. PMID:17130789

  1. An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

    SciTech Connect

    Cardoso, F.M.; Kato, Sayuri E.M.; Huang Wenying; Flint, S. Jane; Gonzalez, Ramon A.

    2008-09-01

    It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells.

  2. CREB (cAMP response element binding protein) and C/EBPalpha (CCAAT/enhancer binding protein) are required for the superstimulation of phosphoenolpyruvate carboxykinase gene transcription by adenoviral E1a and cAMP.

    PubMed Central

    Routes, J M; Colton, L A; Ryan, S; Klemm, D J

    2000-01-01

    In the present study, we observed superstimulated levels of cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter in cells infected with wild-type adenovirus expressing 12 S and 13 S E1a proteins, or in cells expressing 13 S E1a alone. cAMP-stimulated transcription was inhibited in cells expressing only 12 S E1a, but slightly elevated in cells expressing E1a proteins with mutations in conserved regions 1 or 2, leading us to conclude that the superstimulation was mediated by conserved region 3 of 13 S E1a. E1a failed to enhance cAMP-stimulated transcription from promoters containing mutations that abolish binding by cAMP response element binding protein (CREB) or CCAAT/enhancer binding proteins (C/EBPs). This result was supported by experiments in which expression of dominant-negative CREB and/or C/EBP proteins repressed E1a- and cAMP-stimulated transcription from the PEPCK gene promoter. In reconstitution experiments using a Gal4-responsive promoter, E1a enhanced cAMP-stimulated transcription when chimaeric Gal4-CREB and Gal4-C/EBPalpha were co-expressed. Phosphorylation of CREB on serine-133 was stimulated in cells treated with dibutyryl cAMP, whereas phosphorylation of C/EBPalpha was increased by E1a expression. Our data support a model in which cAMP agonists increase CREB activity and stimulate PEPCK gene transcription, a process that is enhanced by E1a through the phosphorylation of C/EBPalpha. PMID:11085926

  3. Magnetofection Enhances Adenoviral Vector-based Gene Delivery in Skeletal Muscle Cells

    PubMed Central

    Pereyra, Andrea Soledad; Mykhaylyk, Olga; Lockhart, Eugenia Falomir; Taylor, Jackson Richard; Delbono, Osvaldo; Goya, Rodolfo Gustavo; Plank, Christian; Hereñu, Claudia Beatriz

    2016-01-01

    The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models. PMID:27274908

  4. Recombinant adenoviral microRNA-206 induces myogenesis in C2C12 cells

    PubMed Central

    Zhang, Weiwei; Wang, Tao; Su, Yongping; Li, Wang; Frame, Lynn T.; Ai, Guoping

    2011-01-01

    Summary Background The expression of microRNA-206 (miR-206) is high in skeletal muscle but low in most other tissues. The expression of miR-206 is increased in muscular dystrophy, suggesting its involvement in the pathogenesis of muscle diseases. To determine the role of miR-206 in muscle cell differentiation and explore a possible gene therapy vector, we constructed a miR-206 adenoviral expression vector (AdvmiR-206) and tested for transfection into C2C12 stem cells. Material/Methods A 355-bp PCR amplicon from C57B6 mouse skeletal muscle genomic DNA was inserted into the adenoviral shuttle vector pAdTrack-CMV, which was then co-transformed with the adenoviral backbone plasmid pAdEasy-1 into competent E. coli BJ5183 bacteria. The specificity and function of this recombinant adenoviral MiR-206 were studied in C2C12 cells by Northern blot, immunofluorescence, Western blot, and flow cytometry. Results Increased expression of miR-206 in AdvmiR-206 transfected C2C12 cells (P<0.001) and resulted in morphological and biochemical changes over time that were similar to serum deprivation, including elongated cells and increased myosin heavy chain proteins. Even in the absence of serum deprivation, miR-206 overexpression accounted for a 50% reduction of S-phase cells (P<0.01). Moreover, in untransfected C2C12 cells, the introduction of miR-206-specific antisense oligoribonucleotides inhibited the normal response to serum deprivation. Twenty-four hours after lipofection of antisense oligoribonucleotides, the number of elongated cells was reduced by half (P<0.01). Conclusions Collectively, these data support a role for miR-206 in myoblast differentiation. We foresee potential applications for the AdvmiR-206 vector in research and therapy. PMID:22129894

  5. Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers

    SciTech Connect

    Oestberg, Sara; Toermaenen Persson, Heidi; Akusjaervi, Goeran

    2012-11-25

    The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3 Prime splice sites with a weak sequence context. Here we show that L4-33K expressed from a plasmid is localized at the nuclear margin of uninfected cells. During an infection L4-33K is relocalized to the periphery of E2A-72K containing viral replication centers. We also show that serine 192 in the tiny RS repeat of the conserved carboxy-terminus of L4-33K, which is critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of the protein to viral replication sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to the periphery of viral replication centers.

  6. A cost-effective method to enhance adenoviral transduction of primary murine osteoblasts and bone marrow stromal cells

    PubMed Central

    Buo, Atum M; Williams, Mark S; Kerr, Jaclyn P; Stains, Joseph P

    2016-01-01

    We report here a method for the use of poly-l-lysine (PLL) to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells (BMSCs) in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein (GFP)-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research. PMID:27547486

  7. Extensions to the D-Cam sub-unit architecture

    NASA Astrophysics Data System (ADS)

    Ryan, Padraig; Connell, Joseph

    2005-06-01

    Multispectral imaging produces large amounts of data which extend processing, transmission and storage systems to their upper limits. Although there are several interface standards specific to image data acquisition, such as CameraLink, it is Firewire which provides a high-speed data bus, integrated control capability, without loss of flexibility, and which is commonly available as a low cost solution. The class of multispectral imaging requires a different treatment of the processing principals than standard imaging. The same spatial region is captured multiple times using different optical wavelengths. This technique finds application in such diverse areas as coastal monitoring, fruit sorting and automated agriculture. Modifications and additional features to the camera operating and configuration parameters are therefore required which are not generally present with conventional imaging sensors. This paper describes extensions to the IIDC Digital Camera (D-Cam) specification in the development of a Firewire technology platform for transmitting the data structures described and for providing real-time, online control of spectral information acquisition. Additionally, it describes how a set of registers in the sub-unit architecture of the Firewire protocol is augmented to accommodate the demands of a multispectral system. The extensions are specification conformant and do not alter underlining compliance with the base standard. The paper also describes the implementation of the extended D-Cam in the Firewire subsystem of a smart multispectral camera used in commercial applications.

  8. Improved Gene Delivery to Intestinal Mucosa by Adenoviral Vectors Bearing Subgroup B and D Fibers

    PubMed Central

    Lecollinet, S.; Gavard, F.; Havenga, M. J. E.; Spiller, O. B.; Lemckert, A.; Goudsmit, J.; Eloit, M.; Richardson, J.

    2006-01-01

    A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses. PMID:16501084

  9. Molecular epidemiology of adenoviral keratoconjunctivitis in Saudi Arabia

    PubMed Central

    Omar, Nazri; Hammouda, Ehab; Akanuma, Masataka; Ohguchi, Takeshi; Ariga, Toshihide; Tagawa, Yoshitsugu; Kitaichi, Nobuyoshi; Ishida, Susumu; Aoki, Koki; Ishiko, Hiroaki; Ohno, Shigeaki

    2010-01-01

    Purpose Adenoviral keratoconjunctivitis is a major cause of ocular morbidity and may lead to visual loss. Adenovirus types 8, 19, and 37 may cause epidemic keratoconjunctivitis. The main objective of this study was to determine the types of adenoviruses causing keratoconjunctivitis in Saudi Arabia. Methods We conducted a non-interventional observational clinical study. Seventy three eyes from 65 patients who presented to The Eye Center in Riyadh, Saudi Arabia with clinical features of acute adenoviral keratoconjunctivitis were included. Each patient underwent complete clinical examination and features such as membranous reaction, conjunctival hemorrhage, subepithelial corneal infiltrates, and preauricular lymph node enlargement were recorded. Conjunctival swabs were obtained from patients with presumed acute viral conjunctivitis. Immunochromatography (IC) and restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) were performed on the conjunctival swabs obtained from each eye. Serotype identification was performed using direct sequencing technique. Results Forty-nine (67.1%) were adenovirus type 8, 8 (11.0%) were adenovirus type 3, 6 (8.2%) type 37, 5 (6.8%) were adenovirus type 4, and 2 (2.3%) type 19. The remaining 5 were types 14, 19, and 22. The prevalence of membranous conjunctivitis was highest (83%) among eyes with adenovirus type 37 while subepithelial corneal opacities were most commonly seen among eyes with adenovirus type 8 (47%). Immunochromatography tests were positive for adenovirus in 48 (65.7%) out of 73 eyes. Conclusions This study determined the types of adenoviruses causing keratoconjunctivitis at one center in Saudi Arabia. Direct sequencing techniques is an efficient, accurate, and rapid means of diagnosing adenoviral keratoconjunctivitis. The most common causes of adenoviral keratoconjunctivitis in Saudi Arabia were adenovirus types 8, 3, and 37. Membranous conjunctivitis and subepithelial opacities had the highest

  10. Regulated Expression of Adenoviral Vectors-Based Gene Therapies

    PubMed Central

    Curtin, James F.; Candolfi, Marianela; Puntel, Mariana; Xiong, Weidong; Muhammad, A. K. M.; Kroeger, Kurt; Mondkar, Sonali; Liu, Chunyan; Bondale, Niyati; Lowenstein, Pedro R.; Castro, Maria G.

    2008-01-01

    Summary Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the “OFF” state, and expression should quickly reach therapeutic levels in the “ON” state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA2S-M2 inducer and tTSKid silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo. PMID:18470649

  11. Immunization with Recombinant Adenoviral Vectors Expressing HCV Core or F Proteins Leads to T Cells with Reduced Effector Molecules Granzyme B and IFN-γ: A Potential New Strategy for Immune Evasion in HCV Infection.

    PubMed

    Samrat, Subodh Kumar; Vedi, Satish; Singh, Shakti; Li, Wen; Kumar, Rakesh; Agrawal, Babita

    2015-01-01

    Multispecific, broad, and potent T cell responses have been correlated with viral clearance in hepatitis C virus (HCV) infection. However, the majority of infected patients develop chronic infection, suggesting that natural infection mostly leads to development of inefficient T cell immunity. Multiple mechanisms of immune modulation and evasion have been shown in HCV infection through various investigations. This study examined the generation and modulation of T cell responses against core and frameshift (F) proteins of HCV. A single immunization of mice with replication incompetent recombinant adenovirus vectors encoding for F or core antigens induces poor T cell responses and leads to generation of CD4+ and CD8+ T cells with low granzyme B (GrB) expression. These T cells have impaired GrB enzyme activity and are unable to kill peptide loaded target cells. The low intracellular expression of GrB is not due to degranulation of cytotoxic granules containing cytotoxic T cells. Addition of exogenous IL-2 in in vitro cultures leads to partial recovery of GrB production, whereas immunization with the Toll-like receptor (TLR) agonist poly I:C leads to complete restoration of GrB expression in both CD4+ and CD8+ T cells. Thus, a possible new strategy of T cell modulation is recognized wherein effector T cells are caused to be dysfunctional by HCV-derived antigens F or core, and strategies are also delineated to overcome this dysfunction. These studies are important in the investigation of prophylactic vaccine and immunotherapy strategies for HCV infection. PMID:26133045

  12. Accelerated evolution and coevolution drove the evolutionary history of AGPase sub-units during angiosperm radiation

    PubMed Central

    Corbi, Jonathan; Dutheil, Julien Y.; Damerval, Catherine; Tenaillon, Maud I.; Manicacci, Domenica

    2012-01-01

    Background and Aims ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch biosynthesis. In the green plant lineage, it is composed of two large (LSU) and two small (SSU) sub-units encoded by paralogous genes, as a consequence of several rounds of duplication. First, our aim was to detect specific patterns of molecular evolution following duplication events and the divergence between monocotyledons and dicotyledons. Secondly, we investigated coevolution between amino acids both within and between sub-units. Methods A phylogeny of each AGPase sub-unit was built using all gymnosperm and angiosperm sequences available in databases. Accelerated evolution along specific branches was tested using the ratio of the non-synonymous to the synonymous substitution rate. Coevolution between amino acids was investigated taking into account compensatory changes between co-substitutions. Key Results We showed that SSU paralogues evolved under high functional constraints during angiosperm radiation, with a significant level of coevolution between amino acids that participate in SSU major functions. In contrast, in the LSU paralogues, we identified residues under positive selection (1) following the first LSU duplication that gave rise to two paralogues mainly expressed in angiosperm source and sink tissues, respectively; and (2) following the emergence of grass-specific paralogues expressed in the endosperm. Finally, we found coevolution between residues that belong to the interaction domains of both sub-units. Conclusions Our results support the view that coevolution among amino acid residues, especially those lying in the interaction domain of each sub-unit, played an important role in AGPase evolution. First, within SSU, coevolution allowed compensating mutations in a highly constrained context. Secondly, the LSU paralogues probably acquired tissue-specific expression and regulatory properties via the coevolution between sub-unit interacting domains. Finally, the

  13. Retrograde adenoviral vector targeting of nociresponsive pontospinal noradrenergic neurons in the rat in vivo

    PubMed Central

    Howorth, Patrick W; Teschemacher, Anja G; Pickering, Anthony E

    2009-01-01

    The spinal dorsal horn receives a dense innervation of noradrenaline-containing fibers that originate from pontine neurons in the A5, locus coeruleus (LC), and A7 cell groups. These pontospinal neurons are believed to constitute a component of the endogenous analgesic system. We used an adenoviral vector with a catecholaminergic-selective promoter (AVV-PRS) to retrogradely label the noradrenergic neurons projecting to the lumbar (L4–L5) dorsal horn with enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein (mRFP). Retrogradely labeled neurons (145 ± 12, n = 14) were found in A5-12%, LC-80% and A7-8% after injection of AVV-PRS-EGFP to the dorsal horn of L4–L5. These neurons were immunopositive for dopamine β-hydroxylase, indicating that they were catecholaminergic. Retrograde labeling was optimal 7 days after injection, persisted for over 4 weeks, and was dependent on viral vector titer. The spinal topography of the noradrenergic projection was examined using EGFP- and mRFP-expressing adenoviral vectors. Pontospinal neurons provide bilateral innervation of the cord and there was little overlap in the distribution of neurons projecting to the cervical and lumbar regions. The axonal arbor of the pontospinal neurons was visualized with GFP immunocytochemistry to show projections to the inferior olive, cerebellum, thalamus, and cortex but not to the hippocampus or caudate putamen. Formalin testing evoked c-fos expression in these pontospinal neurons, suggesting that they were nociresponsive (A5-21%, LC-16%, and A7-26%, n = 8). Thus, we have developed a viral vector-based strategy to selectively, retrogradely target the pontospinal noradrenergic neurons that are likely to be involved in the descending control of nociception. PMID:19003793

  14. Development of Adenoviral Delivery Systems to Target Hepatic Stellate Cells In Vivo

    PubMed Central

    Meier, Claudia; Kowtharapu, Bhavani S.; Timm, Franziska; Vollmar, Brigitte; Herchenröder, Ottmar; Abshagen, Kerstin; Pützer, Brigitte M.

    2013-01-01

    Hepatic stellate cells (HSCs) are known as initiator cells that induce liver fibrosis upon intoxication or other noxes. Deactivation of this ongoing remodeling process of liver parenchyma into fibrotic tissue induced by HSCs is an interesting goal to be achieved by targeted genetic modification of HSCs. The most widely applied approach in gene therapy is the utilization of specifically targeted vectors based on Adenovirus (Ad) serotype 5. To narrow down the otherwise ubiquitous tropism of parental Ad, two modifications are required: a) ablating the native tropism and b) redirecting the vector particles towards a specific entity solely present on the cells of interest. Therefore, we designed a peptide of the nerve growth factor (NGFp) with specific affinity for the p75 neurotrophin receptor (p75NTR) present on HSCs. Coupling of this NGFp to vector particles was done either via chemical conjugation using bifunctional polyethylene glycol (PEG) or, alternatively, by molecular bridging with a fusion protein specific for viral fiber knob and p75NTR. Both Ad vectors transmit the gene for the green fluorescent protein (GFP). GFP expression was monitored in vitro on primary murine HSCs as well as after systemic administration in mice with healthy and fibrotic livers using intravital fluorescence microscopy. Coupling of NGFp to Ad via S11 and/or PEGylation resulted in markedly reduced liver tropism and an enhanced adenoviral-mediated gene transfer to HSCs. Transduction efficiency of both specific Ads was uniformly higher in fibrotic livers, whereas Ad.GFP-S11-NGFp transduce activated HSCs better than Ad.GFP-PEG-NGFp. These experiments contribute to the development of a targeted gene transfer system to specifically deliver antifibrotic compounds into activated HSCs by systemically applied adenoviral vector modified with NGFp. PMID:23825626

  15. 7 CFR 275.7 - Selection of sub-units for review.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AGRICULTURE FOOD STAMP AND FOOD DISTRIBUTION PROGRAM PERFORMANCE REPORTING SYSTEM Management Evaluation (ME... eligibility, maintaining (or having easy access to) casefiles, and transmitting information to the data... review system. (c) Combined responsibilities. (1) When a sub-unit has more than one of the areas...

  16. 7 CFR 275.7 - Selection of sub-units for review.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AGRICULTURE FOOD STAMP AND FOOD DISTRIBUTION PROGRAM PERFORMANCE REPORTING SYSTEM Management Evaluation (ME... eligibility, maintaining (or having easy access to) casefiles, and transmitting information to the data... review system. (c) Combined responsibilities. (1) When a sub-unit has more than one of the areas...

  17. 7 CFR 275.7 - Selection of sub-units for review.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... AGRICULTURE FOOD STAMP AND FOOD DISTRIBUTION PROGRAM PERFORMANCE REPORTING SYSTEM Management Evaluation (ME... eligibility, maintaining (or having easy access to) casefiles, and transmitting information to the data... review system. (c) Combined responsibilities. (1) When a sub-unit has more than one of the areas...

  18. 7 CFR 275.7 - Selection of sub-units for review.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Agriculture Regulations of the Department of Agriculture (Continued) FOOD AND NUTRITION SERVICE, DEPARTMENT OF AGRICULTURE FOOD STAMP AND FOOD DISTRIBUTION PROGRAM PERFORMANCE REPORTING SYSTEM Management Evaluation (ME... Food Stamp Program, exclusive of Post Offices which may issue coupons. Sub-units shall be...

  19. 7 CFR 275.7 - Selection of sub-units for review.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 4 2011-01-01 2011-01-01 false Selection of sub-units for review. 275.7 Section 275.7 Agriculture Regulations of the Department of Agriculture (Continued) FOOD AND NUTRITION SERVICE, DEPARTMENT OF AGRICULTURE FOOD STAMP AND FOOD DISTRIBUTION PROGRAM PERFORMANCE REPORTING SYSTEM Management Evaluation (ME) Reviews § 275.7 Selection...

  20. Efficacy of Topical Immunoglobulins against Experimental Adenoviral Ocular Infection

    PubMed Central

    Nwanegbo, Edward C.; Romanowski, Eric G.; Gordon, Y. Jerold; Gambotto, Andrea

    2007-01-01

    Purpose Presently, there is no U.S. Federal Drug Administration (FDA)–approved antiviral therapy for the treatment of adenoviral (Ad) ocular infections. The goal of the present study was to determine the antiviral efficacy of human immunoglobulin (Ig), a preparation of highly purified and concentrated immunoglobulin (IgG) antibodies isolated from a large pool of human plasma donors, in vitro and on acute Ad replication in the Ad5 New Zealand White (NZW) rabbit ocular model. Methods The antiviral activity of human Ig against multiple wild-type and human ocular isolates of adenovirus serotypes was investigated in vitro by using neutralizing assays in different human epithelial cell lines. In vivo bilateral topical ocular toxicity and antiviral efficacy were evaluated with established Ad5/NZW rabbit ocular models. In vivo Ig antiviral results were compared with those obtained with topical 0.5% cidofovir and saline. Results In three different epithelial cell lines, ≤6.25 mg/mL of the Ig neutralized several wild-type adenoviral serotypes that cause ocular infections. A dose of ≤10 mg/mL neutralized 88% of ocular isolates of the adenovirus serotypes. After treatment of infected animals, adenovirus-positive cultures per total cultures (days 1–14; P = 0.021), the duration of Ad5 shedding, (P = 0.008), and the mean combined ocular viral titer during the early (days 1–5; P = 0.0001) and the late (days 7–14; P = 0.013) phases of infection were significantly lower in Ig-treated animals than in saline-treated animals and were similar to those in cidofovir-treated animals. Conclusions Ig demonstrated antiviral properties against multiple adenoviral serotypes in vitro and in the Ad5/NZW rabbit ocular model. Further studies are needed to advance topical immunoglobulin for treatment and prophylaxis of ocular infections. PMID:17724203

  1. Adenoviral vector-based strategies against infectious disease and cancer

    PubMed Central

    Zhang, Chao; Zhou, Dongming

    2016-01-01

    ABSTRACT Adenoviral vectors are widely employed against infectious diseases or cancers, as they can elicit specific antibody responses and T cell responses when they are armed with foreign genes as vaccine carriers, and induce apoptosis of the cancer cells when they are genetically modified for cancer therapy. In this review, we summarize the biological characteristics of adenovirus (Ad) and the latest development of Ad vector-based strategies for the prevention and control of emerging infectious diseases or cancers. Strategies to circumvent the pre-existing neutralizing antibodies which dampen the immunogenicity of Ad-based vaccines are also discussed. PMID:27105067

  2. Chromatography purification of canine adenoviral vectors.

    PubMed

    Segura, María Mercedes; Puig, Meritxell; Monfar, Mercè; Chillón, Miguel

    2012-06-01

    Canine adenovirus vectors (CAV2) are currently being evaluated for gene therapy, oncolytic virotherapy, and as vectors for recombinant vaccines. Despite the need for increasing volumes of purified CAV2 preparations for preclinical and clinical testing, their purification still relies on the use of conventional, scale-limited CsCl ultracentrifugation techniques. A complete downstream processing strategy for CAV2 vectors based on membrane filtration and chromatography is reported here. Microfiltration and ultra/diafiltration are selected for clarification and concentration of crude viral stocks containing both intracellular and extracellular CAV2 particles. A DNase digestion step is introduced between ultrafiltration and diafiltration operations. At these early stages, concentration of vector stocks with good recovery of viral particles (above 80%) and removal of a substantial amount of protein and nucleic acid contaminants is achieved. The ability of various chromatography techniques to isolate CAV2 particles was evaluated. Hydrophobic interaction chromatography using a Fractogel propyl tentacle resin was selected as a first chromatography step, because it allows removal of the bulk of contaminating proteins with high CAV2 yields (88%). An anion-exchange chromatography step using monolithic supports is further introduced to remove the remaining contaminants with good recovery of CAV2 particles (58-69%). The main CAV2 viral structural components are visualized in purified preparations by electrophoresis analyses. Purified vector stocks contained intact icosahedral viral particles, low contamination with empty viral capsids (10%), and an acceptable total-to-infectious particle ratio (below 30). The downstream processing strategy that was developed allows preparation of large volumes of high-quality CAV2 stocks. PMID:22799886

  3. Production of human epidermal growth factor using adenoviral based system

    PubMed Central

    Negahdari, Babak; Shahosseini, Zahra; Baniasadi, Vahid

    2016-01-01

    Epidermal growth factor (EGF), a growth factor involved in cell growth and differentiation, is a small polypeptide with molecular weight of approximately 6 kDa known to be present in a number of different mammalian species. Experimental studies in animals and humans have demonstrated that the topical application of EGF accelerates the rate of epidermal regeneration of partial-thickness wounds and second-degree burns. Due to its commercial applications, Human EGF (hEGF) has been cloned in several forms. In the present study, adenoviral based expression system was used to produce biologically active recombinant hEGF. The presence of secreted recombinant hEGF was confirmed by a dot blot and its expression level was determined by enzyme-linked immuno-sorbent assay. Moreover, biological activity of secreted hEGF was evaluated by a proliferation assay performed on A549 cells. For production of hEGF in a secretory form, a chimeric gene coding for the hEGF fused to the signal peptide was expressed using adenoviral based method. This method enables the production of hEGF at the site of interest and moreover it could be used for cell proliferation and differentiation assays in tissue engineering research experiments instead of using commercially available EGF. PMID:27051431

  4. Adenoviral-mediated gene transfer to rabbit synovium in vivo.

    PubMed Central

    Roessler, B J; Allen, E D; Wilson, J M; Hartman, J W; Davidson, B L

    1993-01-01

    Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium. Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes). Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions. In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques. Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits. Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy. High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection. Images PMID:8349791

  5. Gene Transfer into Rat Brain Using Adenoviral Vectors

    PubMed Central

    Puntel, Mariana; Kroeger, Kurt M.; Sanderson, Nicholas S.R.; Thomas, Clare E.; Castro, Maria G.; Lowenstein, Pedro R.

    2010-01-01

    Viral vector–mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose—efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described. Curr. Protoc. Neurosci. 50:4.24.1–4.24.49. © 2010 by John Wiley & Sons, Inc. PMID:20066657

  6. A formalism for scattering of complex composite structures. I. Applications to branched structures of asymmetric sub-units

    NASA Astrophysics Data System (ADS)

    Svaneborg, Carsten; Pedersen, Jan Skov

    2012-03-01

    We present a formalism for the scattering of an arbitrary linear or acyclic branched structure build by joining mutually non-interacting arbitrary functional sub-units. The formalism consists of three equations expressing the structural scattering in terms of three equations expressing the sub-unit scattering. The structural scattering expressions allow composite structures to be used as sub-units within the formalism itself. This allows the scattering expressions for complex hierarchical structures to be derived with great ease. The formalism is generic in the sense that the scattering due to structural connectivity is completely decoupled from internal structure of the sub-units. This allows sub-units to be replaced by more complex structures. We illustrate the physical interpretation of the formalism diagrammatically. By applying a self-consistency requirement, we derive the pair distributions of an ideal flexible polymer sub-unit. We illustrate the formalism by deriving generic scattering expressions for branched structures such as stars, pom-poms, bottle-brushes, and dendrimers build out of asymmetric two-functional sub-units.

  7. A formalism for scattering of complex composite structures. I. Applications to branched structures of asymmetric sub-units.

    PubMed

    Svaneborg, Carsten; Pedersen, Jan Skov

    2012-03-14

    We present a formalism for the scattering of an arbitrary linear or acyclic branched structure build by joining mutually non-interacting arbitrary functional sub-units. The formalism consists of three equations expressing the structural scattering in terms of three equations expressing the sub-unit scattering. The structural scattering expressions allow composite structures to be used as sub-units within the formalism itself. This allows the scattering expressions for complex hierarchical structures to be derived with great ease. The formalism is generic in the sense that the scattering due to structural connectivity is completely decoupled from internal structure of the sub-units. This allows sub-units to be replaced by more complex structures. We illustrate the physical interpretation of the formalism diagrammatically. By applying a self-consistency requirement, we derive the pair distributions of an ideal flexible polymer sub-unit. We illustrate the formalism by deriving generic scattering expressions for branched structures such as stars, pom-poms, bottle-brushes, and dendrimers build out of asymmetric two-functional sub-units. PMID:22423826

  8. An Adenoviral Vector Based Vaccine for Rhodococcus equi

    PubMed Central

    Giles, Carla; Ndi, Olasumbo; Barton, Mary D.; Vanniasinkam, Thiru

    2016-01-01

    Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals. PMID:27008624

  9. An Adenoviral Vector Based Vaccine for Rhodococcus equi.

    PubMed

    Giles, Carla; Ndi, Olasumbo; Barton, Mary D; Vanniasinkam, Thiru

    2016-01-01

    Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals. PMID:27008624

  10. GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells.

    PubMed

    Honda, Tsuyoshi; Coppola, Simona; Ghibelli, Lina; Cho, Song H; Kagawa, Shunsuke; Spurgers, Kevin B; Brisbay, Shawn M; Roth, Jack A; Meyn, Raymond E; Fang, Bingliang; McDonnell, Timothy J

    2004-04-01

    The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes. PMID:15002033

  11. Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways.

    PubMed

    Kushwah, R; Oliver, J R; Cao, H; Hu, J

    2007-08-01

    Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors. PMID:17525704

  12. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes

    PubMed Central

    Ruan, Merry ZC; Cerullo, Vincenzo; Cela, Racel; Clarke, Chris; Lundgren-Akerlund, Evy; Barry, Michael A; Lee, Brendan HL

    2016-01-01

    Osteoarthritis (OA) is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs). Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab). We show that a10mab-conjugated HDV (a10mabHDV)-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4) into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting. PMID:27626040

  13. Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes.

    PubMed

    Ruan, Merry Zc; Cerullo, Vincenzo; Cela, Racel; Clarke, Chris; Lundgren-Akerlund, Evy; Barry, Michael A; Lee, Brendan Hl

    2016-01-01

    Osteoarthritis (OA) is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs). Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab). We show that a10mab-conjugated HDV (a10mabHDV)-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4) into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting. PMID:27626040

  14. Restoration of β -Adrenergic Signaling in Failing Cardiac Ventricular Myocytes via Adenoviral-Mediated Gene Transfer

    NASA Astrophysics Data System (ADS)

    Akhter, Shahab A.; Skaer, Christine A.; Kypson, Alan P.; McDonald, Patricia H.; Peppel, Karsten C.; Glower, Donald D.; Lefkowitz, Robert J.; Koch, Walter J.

    1997-10-01

    Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β -adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β -adrenergic signaling defects including down-regulation of myocardial β -adrenergic receptors (β -ARs), functional β -AR uncoupling, and an upregulation of the β -AR kinase (β ARK1). Adenoviral-mediated gene transfer of the human β 2-AR or an inhibitor of β ARK1 to these failing myocytes led to the restoration of β -AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of β ARK1 activity in the heart.

  15. The sodium pump α1 sub-unit: a disease progression–related target for metastatic melanoma treatment

    PubMed Central

    Mathieu, Véronique; Pirker, Christine; Martin de Lassalle, Elisabeth; Vernier, Mathieu; Mijatovic, Tatjana; DeNeve, Nancy; Gaussin, Jean-François; Dehoux, Mischael; Lefranc, Florence; Berger, Walter; Kiss, Robert

    2009-01-01

    Melanomas remain associated with dismal prognosis because they are naturally resistant to apoptosis and they markedly metastasize. Up-regulated expression of sodium pump α sub-units has previously been demonstrated when comparing metastatic to non-metastatic melanomas. Our previous data revealed that impairing sodium pump α1 activity by means of selective ligands, that are cardiotonic steroids, markedly impairs cell migration and kills apoptosis-resistant cancer cells. The objective of this study was to determine the expression levels of sodium pump α sub-units in melanoma clinical samples and cell lines and also to characterize the role of α1 sub-units in melanoma cell biology. Quantitative RT-PCR, Western blotting and immunohistochemistry were used to determine the expression levels of sodium pump α sub-units. In vitro cytotoxicity of various cardenolides and of an anti-α1 siRNA was evaluated by means of MTT assay, quantitative videomicroscopy and through apoptosis assays. The in vivo activity of a novel cardenolide UNBS1450 was evaluated in a melanoma brain metastasis model. Our data show that all investigated human melanoma cell lines expressed high levels of the α1 sub-unit, and 33% of human melanomas displayed significant α1 sub-unit expression in correlation with the Breslow index. Furthermore, cardenolides (notably UNBS1450; currently in Phase I clinical trials) displayed marked anti-tumour effects against melanomas in vitro. This activity was closely paralleled by decreases in cMyc expression and by increases in apoptotic features. UNBS1450 also displayed marked anti-tumour activity in the aggressive human metastatic brain melanoma model in vivo. The α1 sodium pump sub-unit could represent a potential novel target for combating melanoma. PMID:19243476

  16. Isolation and Characterization of Anti-Adenoviral Secondary Metabolites from Marine Actinobacteria

    PubMed Central

    Strand, Mårten; Carlsson, Marcus; Uvell, Hanna; Islam, Koushikul; Edlund, Karin; Cullman, Inger; Altermark, Björn; Mei, Ya-Fang; Elofsson, Mikael; Willassen, Nils-Peder; Wadell, Göran; Almqvist, Fredrik

    2014-01-01

    Adenovirus infections in immunocompromised patients are associated with high mortality rates. Currently, there are no effective anti-adenoviral therapies available. It is well known that actinobacteria can produce secondary metabolites that are attractive in drug discovery due to their structural diversity and their evolved interaction with biomolecules. Here, we have established an extract library derived from actinobacteria isolated from Vestfjorden, Norway, and performed a screening campaign to discover anti-adenoviral compounds. One extract with anti-adenoviral activity was found to contain a diastereomeric 1:1 mixture of the butenolide secondary alcohols 1a and 1b. By further cultivation and analysis, we could isolate 1a and 1b in different diastereomeric ratio. In addition, three more anti-adenoviral butenolides 2, 3 and 4 with differences in their side-chains were isolated. In this study, the anti-adenoviral activity of these compounds was characterized and substantial differences in the cytotoxic potential between the butenolide analogs were observed. The most potent butenolide analog 3 displayed an EC50 value of 91 μM and no prominent cytotoxicity at 2 mM. Furthermore, we propose a biosynthetic pathway for these compounds based on their relative time of appearance and structure. PMID:24477283

  17. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    PubMed Central

    Coughlan, Lynda; Alba, Raul; Parker, Alan L.; Bradshaw, Angela C.; McNeish, Iain A.; Nicklin, Stuart A.; Baker, Andrew H.

    2010-01-01

    Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated

  18. The Evolution of Adenoviral Vectors through Genetic and Chemical Surface Modifications

    PubMed Central

    Capasso, Cristian; Garofalo, Mariangela; Hirvinen, Mari; Cerullo, Vincenzo

    2014-01-01

    A long time has passed since the first clinical trial with adenoviral (Ad) vectors. Despite being very promising, Ad vectors soon revealed their limitations in human clinical trials. The pre-existing immunity, the marked liver tropism and the high toxicity of first generation Ad (FG-Ad) vectors have been the main challenges for the development of new approaches. Significant effort toward the development of genetically and chemically modified adenoviral vectors has enabled researchers to create more sophisticated vectors for gene therapy, with an improved safety profile and a higher transduction ability of different tissues. In this review, we will describe the latest findings in the high-speed, evolving field of genetic and chemical modifications of adenoviral vectors, a field in which different disciplines, such as biomaterial research, virology and immunology, co-operate synergistically to create better gene therapy tools for modern challenges. PMID:24549268

  19. Intranasal vaccination with a helper-dependent adenoviral vector enhances transgene-specific immune responses in BALB/c mice.

    PubMed

    Fu, Yuan-hui; He, Jin-sheng; Zheng, Xian-xian; Wang, Xiao-bo; Xie, Can; Shi, Chang-xin; Zhang, Mei; Tang, Qian; Wei, Wei; Qu, Jian-guo; Hong, Tao

    2010-01-01

    Helper-dependent adenoviral (HDAd) vectors were developed primarily for genetic disease therapy by deleting all coding regions for attenuating the host cellular immune response to adenovirus (Ad) and long-lasting gene expression. Recently Harui et al. reported that HDAd vaccine could stimulate superior transgene-specific cytotoxic T lymphocyte (CTL) and antibody responses via the intraperitoneal route, compared to first-generation adenoviral (FGAd) vaccine. This prompted us to explore the potential of HDAd as a vaccine vector administrated intranasally. In this study, we prepared HDAd and FGAd vectors expressing enhanced green fluorescent protein (EGFP), respectively, and compared their efficacy in mice. Mice were immunized intranasally with 5x10(9) vp HDAd or FGAd vector particles. Despite stimulating similar anti-Ad antibody responses with FGAd vaccine in the prime/boost strategy, HDAd vector expressing EGFP displayed superior transgene-specific serum IgG, mucosal IgA and cellular immune response, with the characterization of balanced or mixed Th1/Th2 CD4+ T-cell responses. Meanwhile, a single dose of intranasal (i.n.) vaccine of HDAd-EGFP induced a serum IgG response with more efficacy than FGAd-EGFP. In addition, i.n. boost immunization enhanced transgene-specific humoral and cellular responses, compared to single i.n. HDAd-EGFP immunization. Our results suggest that HDAd has potential for a mucosal vaccine vector via i.n. route, which will be useful for the development of vaccines against respiratory viruses, such as respiratory syncytial virus and influenza virus. PMID:19945423

  20. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation

    PubMed Central

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo. PMID:26808122

  1. Combination recombinant simian or chimpanzee adenoviral vectors for vaccine development.

    PubMed

    Cheng, Cheng; Wang, Lingshu; Ko, Sung-Youl; Kong, Wing-Pui; Schmidt, Stephen D; Gall, Jason G D; Colloca, Stefano; Seder, Robert A; Mascola, John R; Nabel, Gary J

    2015-12-16

    Recombinant adenoviral vector (rAd)-based vaccines are currently being developed for several infectious diseases and cancer therapy, but pre-existing seroprevalence to such vectors may prevent their use in broad human populations. In this study, we investigated the potential of low seroprevalence non-human primate rAd vectors to stimulate cellular and humoral responses using HIV/SIV Env glycoprotein (gp) as the representative antigen. Mice were immunized with novel simian or chimpanzee rAd (rSAV or rChAd) vectors encoding HIV gp or SIV gp by single immunization or in heterologous prime/boost combinations (DNA/rAd; rAd/rAd; rAd/NYVAC or rAd/rLCM), and adaptive immunity was assessed. Among the rSAV and rChAd tested, rSAV16 or rChAd3 vector alone generated the most potent immune responses. The DNA/rSAV regimen also generated immune responses similar to the DNA/rAd5 regimen. rChAd63/rChAd3 and rChAd3 /NYVAC induced similar or even higher levels of CD4+ and CD8+ T-cell and IgG responses as compared to rAd28/rAd5, one of the most potent combinations of human rAds. The optimized vaccine regimen stimulated improved cellular immune responses and neutralizing antibodies against HIV compared to the DNA/rAd5 regimen. Based on these results, this type of novel rAd vector and its prime/boost combination regimens represent promising candidates for vaccine development. PMID:26514419

  2. Effects of fatigue on the chemical and mechanical degradation of model stent sub-units.

    PubMed

    Dreher, Maureen L; Nagaraja, Srinidhi; Batchelor, Benjamin

    2016-06-01

    Understanding the fatigue and durability performance of implantable cardiovascular stents is critical for assessing their performance. When the stent is manufactured from an absorbable material, however, this durability assessment is complicated by the transient nature of the device. Methodologies for evaluating the fatigue performance of absorbable stents while accurately simulating the degradation are limited and little is known about the interaction between fatigue and degradation. In this study, we investigated the fatigue behavior and effect of fatigue on the degradation rate for a model absorbable cardiovascular stent. Custom v-shaped stent sub-units manufactured from poly(L-lactide), i.e., PLLA, were subjected to a simultaneous fatigue and degradation study with cycle counts representative of one year of expected in vivo use. Fatigue loading was carried out such that the polymer degraded at a rate that was aligned with a modest degree of fatigue acceleration. Control, un-loaded specimens were also degraded under static immersion conditions representative of simulated degradation without fatigue. The study identified that fatigue loading during degradation significantly increased specimen stiffness and lowered the force at break. Fatigue loading also significantly increased the degree of molecular weight decline highlighting an interaction between mechanical loading and chemical degradation. This study demonstrates that fatigue loading during degradation can affect both the mechanical properties and the chemical degradation rate. The results are important for defining appropriate in vitro degradation conditions for absorbable stent preclinical evaluation. PMID:26759973

  3. INSM1 promoter-driven adenoviral herpes simplex virus thymidine kinase cancer gene therapy for the treatment of primitive neuroectodermal tumors.

    PubMed

    Wang, Hong-Wei; Breslin, Mary B; Chen, Chiachen; Akerstrom, Victoria; Zhong, Qiu; Lan, Michael S

    2009-11-01

    The INSM1 gene encodes a developmentally regulated zinc finger transcription factor. INSM1 expression is normally absent in adult tissues, but is reactivated in neuroendocrine tumor cells. In the present study, we analyzed the therapeutic potential of an adenoviral INSM1 promoter-driven herpes simplex virus thymidine kinase (HSV-tk) construct in primitive neuroectodermal tumors (PNETs). We constructed an adenoviral INSM1 promoter-driven HSV-tk gene for therapy in PNETs. The PNET-specific adeno-INSM1 promoter HSV-tk construct was tested both in vitro and in vivo in a nude mouse tumor model. Northern blot analysis and transient transfection of an INSM1 promoter-driven luciferase reporter gene indicated that the INSM1 promoter was active in neuroblastoma (IMR-32), retinoblastoma (Y79), and medulloblastoma (D283 Med) cells, but not in glioblastoma (U-87 MG) cells. After Ad-INSM1p-HSV-tk infection, the levels of HSV-tk protein expression were consistent with INSM1 promoter activities. Furthermore, in vitro multiplicity of infection and ganciclovir (GCV) sensitivity studies indicated that the INSM1 promoter could mediate specific expression of the HSV-tk gene and selective killing of INSM1-positive PNETs. In vivo intratumoral adenoviral delivery demonstrated that the INSM1 promoter could direct HSV-tk gene expression in a nude mouse tumor model and effectively repressed tumor growth in response to GCV treatment. Taken together, our data show that the INSM1 promoter is specific and effective for targeted cancer gene therapy in PNETs. PMID:19604042

  4. Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells.

    PubMed

    Lowe, S L; Rubinchik, S; Honda, T; McDonnell, T J; Dong, J Y; Norris, J S

    2001-09-01

    In prostate carcinoma, overexpression of the anti-apoptotic gene Bcl-2 has been found to be associated with resistance to therapies including radiation and androgen ablation. Restoring the balance of Bcl-2 family members may result in the induction of apoptosis in prostate cancer cells previously resistant to treatment. To accomplish this, a strategy involving overexpression of the pro-apoptotic gene Bax was executed. The use of cytotoxic genes such as Bax require selective expression of the gene. In this study, we examined the ability of selective expression of Bax protein directed by a prostate-specific promoter to induce apoptosis in human prostate carcinoma. A second-generation adenoviral vector was constructed with the modified prostate-specific probasin promoter, ARR2PB, directing expression of an HA-tagged Bax gene and a green fluorescent protein reporter translated from an internal ribosome entry site (ARR2PB.Bax.GFP). ARR2PB promoter activity is tightly regulated and highly prostate specific and is responsive to androgens and glucocorticoids. The prostate-specific promoter-Bax-GFP transgene cassette was inserted into a cloning site near the right inverted terminal repeat of the adenoviral vector to retain specificity of the promoter. LNCaP cells infected with Ad/ARR(2)PB.Bax.GFP showed high levels of Bax expression 48 h after infection resulting in an 85% reduction in cell viability. Importantly, LNCaP cells stably transfected to overexpress Bcl-2 showed similar patterns of cell death when infected with Ad/ARR(2)PB.Bax.GFP, an 82% reduction in cell viability seen 48 h after infection. Apoptosis was confirmed by measuring caspase activation and using the TUNEL assay. Tissue specificity was evaluated using A549 cells (lung adenocarcinoma), SK-Hep-1 (liver cancer) cells, and Hela (cervical cancer) cells which did not show detectable expression of virally delivered Bax protein or any increase in cell death. Systemic administration of Ad/ARR2PB. Bax.GFP in nude

  5. Effect of 6-azacytidine on the course of experimental adenoviral infection in newborn Syrian hamsters.

    PubMed

    Zarubalev, V V; Slita, A V; Sukhinin, V P; Nosach, L N; Dyachenko, N S; Povnitsa, O Y; Zhovnovataya, V L; Alexeeva, I V; Palchikovskaya, L I

    2007-02-01

    Adenoviral infection is a serious human pathology leading to respiratory, gastrointestinal and ocular disorders and epidemic outbreaks, especially in children's groups. Here we present the results from an investigation of anti- adenoviral effect of 6-azacytidine (6-AC) both in vitro and in vivo. The selectivity index of 6-AC for adenovirus type 5 in HEp-2 cells was 374, the 50% effective concentration was 0.5 mg/ml. For in vivo investigations we developed a model of disseminated adenoviral infection in newborn Syrian hamsters. The infectious virus was recovered from the liver, kidney, lungs and heart. Application of 6-AC led to a reduced period of the virus presence (7 days in the liver and 4 days in the kidney and heart) and lowered virus titers on day 3 post-inoculation (p.i.) (liver - 2.7 and 4.1, heart - 0 and 3.2, kidney - 0 and 2.4 log(10 )CPD(50)/mg tissue weight, in the presence and absence of 6-AC, respectively). Application of 6-AC to newborn Syrian hamsters led to partial destruction of their splenocytes. The results obtained suggest that 6-AC or 6-ACbased drugs with lower toxicity or applied topically may be suitable for therapy and prevention of adenoviral infection in humans. PMID:17309850

  6. AN UPDATE OF ADENOVIRAL HEMORRHAGIC DISEASE IN MULE DEER IN CALIFORNIA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the summer and fall of 1993, a newly recognized disease, adenoviral hemorrhagic disease, caused widespread mortality in black-tailed (Odocoileus hemionus columbianus) and California mule deer (Odocoileus hemionus californicus) in northern California. Greater than a thousand deer were estimated t...

  7. Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

    PubMed Central

    2011-01-01

    Background Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. Methods In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector. Results The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. Conclusion The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin. PMID:21255430

  8. The prevalence of adenoviral conjunctivitis at the Clinical Hospital of the State University of Campinas, Brazil

    PubMed Central

    Pinto, Roberto Damian Pacheco; Lira, Rodrigo Pessoa Cavalcanti; Arieta, Carlos Eduardo Leite; de Castro, Rosane Silvestre; Bonon, Sandra Helena Alves

    2015-01-01

    OBJECTIVES: Viral conjunctivitis is a common, highly contagious disease that is often caused by an adenovirus. The aim of this study was to evaluate the prevalence of adenoviral conjunctivitis by analyzing data from a prospective clinical study of 122 consecutively enrolled patients who were treated at the Clinical Hospital of the State University of Campinas (UNICAMP) after a clinical diagnosis of infectious conjunctivitis between November 2011 and June 2012. METHODS: Polymerase chain reaction was used to evaluate all cases of clinically diagnosed infectious conjunctivitis and based on the laboratory findings, the prevalence of adenoviral infections was determined. The incidence of subepithelial corneal infiltrates was also investigated. RESULTS: Of the 122 patients with acute infectious conjunctivitis included, 72 had positive polymerase chain reaction results for adenoviruses and 17 patients developed subepithelial corneal infiltrates (13.93%). CONCLUSIONS: The polymerase chain reaction revealed that the prevalence of adenoviral conjunctivitis was 59% in all patients who presented with a clinical diagnosis of infectious conjunctivitis from November 2011 to June 2012. The prevalence of adenoviral conjunctivitis in the study population was similar to its prevalence in other regions of the world. PMID:26602522

  9. Replication-deficient adenoviral vector for gene transfer potentiates airway neurogenic inflammation.

    PubMed

    Piedimonte, G; Pickles, R J; Lehmann, J R; McCarty, D; Costa, D L; Boucher, R C

    1997-03-01

    Human trials for the treatment of cystic fibrosis lung disease with adenoviral vectors have been complicated by acute inflammatory reactions of unknown etiology. Because replicating respiratory viruses can potentiate tachykinin-mediated neurogenic inflammatory responses in airways, we studied whether the endotracheal administration of a replication-deficient adenoviral vector potentiated this response. The vector Ad5CMVLacZ was administered endotracheally to rats and the leakage of Evans blue dye was used to measure the capsaicin-induced neurogenic albumin extravasation. These studies show that neurogenic albumin extravasation is significantly potentiated in the airways of rats after administration of Ad5CMVLacZ. This inflammatory response can be blocked by selective antagonists of the substance P receptor or by glucocorticoids. Therefore, (1) the acute airway inflammation observed in patients after exposure to adenoviral vectors may exhibit a neurogenic component, which can be blocked pharmacologically, and (2) preclinical adenoviral vector safety studies of other organs innervated by the tachykinin system, e.g., coronary arteries and gastrointestinal tract, should include assessment of neurogenic inflammation. PMID:9070609

  10. Neonatal helper-dependent adenoviral vector gene therapy mediates correction of hemophilia A and tolerance to human factor VIII

    PubMed Central

    Cela, Racel G.; Suzuki, Masataka; Lee, Brendan; Lipshutz, Gerald S.

    2011-01-01

    Neonatal gene therapy is a promising strategy for treating a number of congenital diseases diagnosed shortly after birth as expression of therapeutic proteins during postnatal life may limit the pathologic consequences and result in a potential “cure.” Hemophilia A is often complicated by the development of antibodies to recombinant protein resulting in treatment failure. Neonatal administration of vectors may avoid inhibitory antibody formation to factor VIII (FVIII) by taking advantage of immune immaturity. A helper-dependent adenoviral vector expressing human factor VIII was administered i.v. to neonatal hemophilia A knockout mice. Three days later, mice produced high levels of FVIII. Levels declined rapidly with animal growth to 5 wk of age with stable factor VIII expression thereafter to >1 y of age. Decline in factor VIII expression was not related to cell-mediated or humoral responses with lack of development of antibodies to capsid or human factor VIII proteins. Subsequent readministration and augmentation of expression was possible as operational tolerance was established to factor VIII without development of inhibitors; however, protective immunity to adenovirus remained. PMID:21245323

  11. Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors.

    PubMed

    Hanayama, Hiroyuki; Ohashi, Kazuo; Utoh, Rie; Shimizu, Hirofumi; Ise, Kazuya; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Tsuchiya, Hiroyuki; Okano, Teruo; Gotoh, Mitsukazu

    2015-12-17

    To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells. PMID:26858906

  12. Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors

    PubMed Central

    Hanayama, Hiroyuki; Ohashi, Kazuo; Utoh, Rie; Shimizu, Hirofumi; Ise, Kazuya; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Tsuchiya, Hiroyuki; Okano, Teruo; Gotoh, Mitsukazu

    2015-01-01

    To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells. PMID:26858906

  13. Sub-unit vaccine against S. aureus-mediated infections: set-up of nano-sized polymeric adjuvant.

    PubMed

    Colonna, C; Dorati, R; Conti, B; Caliceti, P; Genta, I

    2013-08-16

    The aim of this work was the design of a novel adjuvanted system for vaccination against S. aureus-mediated infections: in particular, poly-lactide-co-glycolide (PLGA) nanoparticles were developed in order to efficiently load and boost a sub-unit model vaccine, namely a purified recombinant collagen binding bacterial adhesin fragment (CNA19). At first, the assessment of the actual immunogenicity of free CNA19 via subcutaneous administration was evaluated, in order to consider it as subunit antigen model. Secondly, for the development of CNA19 loaded PLGA nanoparticles, a preliminary study was focused on the production of well-formed nanoparticles by w/o/w double emulsion method exploiting ultrasonication cycles under mild conditions, then the optimization of the freeze-drying conditions and different CNA19 loading methods were considered (encapsulation, adsorption of on blank or CNA19 encapsulated nanoparticles). The set-up preparation method (process yield of about 83%) permitted to obtain CNA19 loaded nanoparticles with spherical shape, narrow size distribution (187.41 ± 51.2 nm), a slightly negative zeta-potential (-2.91 ± 0.64 mV) and to elicit satisfactory protein encapsulation efficiency (75.91 ± 4.22%) and loading capacity (8.59 ± 0.33 μg CNA19/nanoparticles mg). Then, CNA19 loaded PLGA nanoparticles were characterized by (i) an in vitro release test performed at different temperatures, namely 4°C, 25°C and 37°C, testing the antigen integrity (SDS-PAGE) and activity (ELISA); (ii) an in vitro stability study in terms of dimension and surface charge performed in a 21 days period of time. At 37°C there was evidence of a sustained release of the antigen, in active form, for almost 240 h with a burst release of about 20% in the first 2h. At 4°C stability tests and activity assays allowed to identify storage conditions useful to maintain CNA19 activity and easily NP re-suspendability with intact physical characteristics. Furthermore the evaluation of CNA

  14. Prime/boost immunization with DNA and adenoviral vectors protects from hepatitis D virus (HDV) infection after simultaneous infection with HDV and woodchuck hepatitis virus.

    PubMed

    Fiedler, Melanie; Kosinska, Anna; Schumann, Alexandra; Brovko, Olena; Walker, Andreas; Lu, Mengji; Johrden, Lena; Mayer, Anja; Wildner, Oliver; Roggendorf, Michael

    2013-07-01

    Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8(+) and CD4(+) T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven. PMID:23637419

  15. Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

    PubMed Central

    Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

    2012-01-01

    Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

  16. Oncolytic adenoviral vectors which employ the survivin promoter induce glioma oncolysis via a process of beclin-dependent autophagy

    PubMed Central

    ULASOV, ILYA V.; TYLER, MATHEW A.; ZHU, ZENG B.; HAN, YU; HE, TONG-CHUAN; LESNIAK, MACIEJ S.

    2009-01-01

    Survivin has gained attention as a tumor-specific marker which is upregulated in a variety of neoplasms. Although the survivin protein is implicated in anti-apoptotic tumor pathways, little is known about the function of the survivin promoter. In this study, we constructed a conditionally replicative adenoviral vector (CRAd) that utilizes the survivin promoter and examined the mechanism of CRAd induced cell death in malignant glioma. Our results indicate that CRAd vectors which utilize the survivin promoter effectively replicate in glioma cells and exhibit a high oncolytic effect. The survivin-mediated CRAd appeared to induce apoptosis as measured by Annexin/7-AAD. Caspase-3 and BAX mRNAs were upregulated based on microarray data, however, Western blot analysis of infected cells showed no evidence of elevated caspase-3, BAX, or p53 protein expression. Of note, at each time point infected glioma cells showed no evidence of activated BAD or AKT. The inhibition of AKT signaling led us to examine autophagy in infected cells. Electron micrographs of virally infected glioma cells suggested autophagosomal-mediated cell death and selective blocking of beclin with siRNA prevented autophagy. These results indicate that the survivin promoter enhances viral replication and induces autophagy of infected glioma cells via a beclin-dependent mechanism. PMID:19212678

  17. Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

    PubMed

    Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

    2004-11-01

    Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury. PMID:15474356

  18. Role of the Polymerase ϵ sub-unit DPB2 in DNA replication, cell cycle regulation and DNA damage response in Arabidopsis

    PubMed Central

    Pedroza-Garcia, José Antonio; Domenichini, Séverine; Mazubert, Christelle; Bourge, Mickael; White, Charles; Hudik, Elodie; Bounon, Rémi; Tariq, Zakia; Delannoy, Etienne; del Olmo, Ivan; Piñeiro, Manuel; Jarillo, Jose Antonio; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2016-01-01

    Faithful DNA replication maintains genome stability in dividing cells and from one generation to the next. This is particularly important in plants because the whole plant body and reproductive cells originate from meristematic cells that retain their proliferative capacity throughout the life cycle of the organism. DNA replication involves large sets of proteins whose activity is strictly regulated, and is tightly linked to the DNA damage response to detect and respond to replication errors or defects. Central to this interconnection is the replicative polymerase DNA Polymerase ϵ (Pol ϵ) which participates in DNA replication per se, as well as replication stress response in animals and in yeast. Surprisingly, its function has to date been little explored in plants, and notably its relationship with DNA Damage Response (DDR) has not been investigated. Here, we have studied the role of the largest regulatory sub-unit of Arabidopsis DNA Pol ϵ: DPB2, using an over-expression strategy. We demonstrate that excess accumulation of the protein impairs DNA replication and causes endogenous DNA stress. Furthermore, we show that Pol ϵ dysfunction has contrasting outcomes in vegetative and reproductive cells and leads to the activation of distinct DDR pathways in the two cell types. PMID:27193996

  19. Synthesis and Comparative Study of Anti-Adenoviral Activity of 6-Azacytidine and Its Analogues.

    PubMed

    Alexeeva, Inna; Nosach, Lydia; Palchykovska, Larisa; Usenko, Lyubov; Povnitsa, Olga

    2015-01-01

    This paper presents the results of synthesis and study of cytotoxicity and the anti-adenoviral activity of new N4-derivatives of 6-azacytidine and its α-L-glycopyranosyl analogues obtained by the simplified one-pot version of the silyl condensation method. The resulting acylated 4-methylmercapto-1,2,4-triazin-3(2Н)-one glycosides then underwent the amination and/or ammonolysis to provide 6-azacytidine glycoside analogues (2-6, 12, 15, 17) and compounds with modifications at both base and sugar fragments (11, 15). The evaluation of cytotoxicity and antiviral activity of new compounds against AdV5 showed high selectivity indexes for N4-methyl-6-azacytidine (2) and N,O-tetraacetyl-6-azacytidine (8). High anti-adenoviral activity of N4-methyl-6-azacytidine as well as very low cytotoxicity may suggest its further investigation as potential compound for the therapy of AdV infection. PMID:26167665

  20. Adenoviral-mediated Gene Transfer into the Canine Brain In Vivo

    PubMed Central

    Candolfi, Marianela; Kroeger, Kurt M.; Pluhar, G. Elizabeth; Bergeron, Josee; Puntel, Mariana; Curtin, James F.; McNiel, Elizabeth A.; Freese, Andrew B.; Ohlfest, John R.; Moore, Peter; Lowenstein, Pedro R.; Castro, Maria G.

    2007-01-01

    OBJECTIVE: Glioblastoma multiforme (GBM) is a devastating brain tumor for which there is no cure. Adenoviral-mediated transfer of conditional cytotoxic (herpes simplex virus [HSV] 1-derived thymidine kinase [TK]) and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L]) transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. Dogs develop spontaneous GBM that closely resemble the human disease; therefore, they constitute an excellent large animal model. We assayed the transduction efficiency of adenoviral vectors (Ads) encoding β-galactosidase (βGal), TK, and Flt3L in J3T dog GBM cells in vitro and in the dog brain in vivo. METHODS: J3T cells were infected with Ads (30 plaque-forming units/cell; 72 h) encoding βGal (Ad-βGal), TK (Ad-TK), or Flt3L (Ad-Flt3L). We determined transgene expression by immunocytochemistry, βGal activity, Flt3L enzyme-linked immunosorbent assay, and TK-induced cell death. Ads were also injected intracranially into the parietal cortex of healthy dogs. We determined cell-type specific transgene expression and immune cell infiltration. RESULTS: Adenoviral-mediated gene transfer of HSV1-TK, Flt3L, and βGal was detected in dog glioma cells in vitro (45% transduction efficiency) and in the dog brain in vivo (10-mm2 area transduced surrounding each injection site). T cells and macrophages/activated microglia infiltrated the injection sites. Importantly, no adverse clinical or neuropathological side effects were observed. CONCLUSION: We demonstrate effective adenoviral-mediated gene transfer into the brain of dogs in vivo and support the use of these vectors to develop an efficacy trial for canine GBM as a prelude to human trials. PMID:17228266

  1. Treatment for retinopathy of prematurity in an infant with adenoviral conjunctivitis.

    PubMed

    Gunay, Murat; Celik, Gokhan; Con, Rahim

    2015-01-01

    Retinopathy of prematurity (ROP) has been a major problematic disorder during childhood. Laser photocoagulation (LPC) has been proven to be effective in most of the ROP cases. Adenoviral conjunctivitis (AVC) is responsible for epidemics among adult and pediatric population. It has also been reported to be a cause of outbreaks in neonatal intensive care units (NICU) several times. We herein demonstrate a case with AVC who underwent LPC for ROP. And we discuss the treatment methodology in such cases. PMID:25874149

  2. Adenoviral Delivery of the EMX2 Gene Suppresses Growth in Human Gastric Cancer

    PubMed Central

    Li, Jie; Mo, Minli; Chen, Zhao; Chen, Zhe; Sheng, Qing; Mu, Hang; Zhang, Fang; Zhang, Yi; Zhi, Xiu-Yi; Li, Hui; He, Biao; Zhou, Hai-Meng

    2012-01-01

    Background EMX2 is a human orthologue of the Drosophila empty spiracles homeobox gene that has been implicated in embryogenesis. Recent studies suggest possible involvement of EMX2 in human cancers; however, the role of EMX2 in carcinogenesis needs further exploration. Results In this study, we reported that down-regulation of EMX2 expression was significantly correlated with EMX2 promoter hypermethylation in gastric cancer. Restoring EMX2 expression using an adenovirus delivery system in gastric cancer cell lines lacking endogenous EMX2 expression led to inhibition of cell proliferation and Wnt signaling pathway both in vitro and in a gastric cancer xenograft model in vivo. In addition, we observed that animals treated with the adenoviral EMX2 expression vector had significantly better survival than those treated with empty adenoviral vector. Conclusion Our study suggests that EMX2 is a putative tumor suppressor in human gastric cancer. The adenoviral-EMX2 may have potential as a novel gene therapy for the treatment of patients with gastric cancer. PMID:23029345

  3. Advances and future challenges in recombinant adenoviral vectored H5N1 influenza vaccines.

    PubMed

    Zhang, Jianfeng

    2012-11-01

    The emergence of a highly pathogenic avian influenza virus H5N1 has increased the potential for a new pandemic to occur. This event highlights the necessity for developing a new generation of influenza vaccines to counteract influenza disease. These vaccines must be manufactured for mass immunization of humans in a timely manner. Poultry should be included in this policy, since persistent infected flocks are the major source of avian influenza for human infections. Recombinant adenoviral vectored H5N1 vaccines are an attractive alternative to the currently licensed influenza vaccines. This class of vaccines induces a broadly protective immunity against antigenically distinct H5N1, can be manufactured rapidly, and may allow mass immunization of human and poultry. Recombinant adenoviral vectors derived from both human and non-human adenoviruses are currently being investigated and appear promising both in nonclinical and clinical studies. This review will highlight the current status of various adenoviral vectored H5N1 vaccines and will outline novel approaches for the future. PMID:23202501

  4. An adenoviral vector regulated by hypoxia for the treatment of ischaemic disease and cancer.

    PubMed

    Binley, K; Iqball, S; Kingsman, A; Kingsman, S; Naylor, S

    1999-10-01

    Recombinant adenoviral vectors have a number of advantages for gene therapy, including transduction of a range of dividing and non-dividing cell types. However, this broad range may be a disadvantage if non-target cells are transduced and are adversely affected by expression of the transferred gene. Here we describe a novel adenoviral vector in which transcription of the transgene is restricted to the patho-physiological condition of low oxygen tension (hypoxia). Hypoxia activates the expression of a number of genes, principally via the stabilisation of members of the bHLH/PAS family of transcription factors that bind to a con- sensus DNA sequence, the hypoxia response element (HRE). We have configured an optimised HRE expression cassette into an adenoviral vector, AdOBHRE. A range of cell types, including primary human skeletal muscle, when transduced with AdOBHRE display a low basal level of transgene expression that is highly induced in hypoxia to levels equivalent to that obtained from the CMV promoter. The AdOBHRE vector could be exploited for transcriptionally targeted gene therapy for the treatment of diseases such as cancer, peripheral arterial disease, arthritis and anaemia where tissue hypoxia is a cardinal feature. PMID:10516721

  5. Advances and Future Challenges in Recombinant Adenoviral Vectored H5N1 Influenza Vaccines

    PubMed Central

    Zhang, Jianfeng

    2012-01-01

    The emergence of a highly pathogenic avian influenza virus H5N1 has increased the potential for a new pandemic to occur. This event highlights the necessity for developing a new generation of influenza vaccines to counteract influenza disease. These vaccines must be manufactured for mass immunization of humans in a timely manner. Poultry should be included in this policy, since persistent infected flocks are the major source of avian influenza for human infections. Recombinant adenoviral vectored H5N1 vaccines are an attractive alternative to the currently licensed influenza vaccines. This class of vaccines induces a broadly protective immunity against antigenically distinct H5N1, can be manufactured rapidly, and may allow mass immunization of human and poultry. Recombinant adenoviral vectors derived from both human and non-human adenoviruses are currently being investigated and appear promising both in nonclinical and clinical studies. This review will highlight the current status of various adenoviral vectored H5N1 vaccines and will outline novel approaches for the future. PMID:23202501

  6. Adenoviral vectors for prodrug activation-based gene therapy for cancer

    PubMed Central

    Doloff, Joshua C.; Waxman, David J.

    2013-01-01

    Cancer cell heterogeneity is a common feature - both between patients diagnosed with the same cancer and within an individual patient’s tumor - and leads to widely different response rates to cancer therapies and the potential for the emergence of drug resistance. Diverse therapeutic approaches have been developed to combat the complexity of cancer, including individual treatment modalities designed to target tumor heterogeneity. This review discusses adenoviral vectors and how they can be modified to replicate in a cancer-specific manner and deliver therapeutic genes under multi-tiered regulation to target tumor heterogeneity, including heterogeneity associated with cancer stem cell-like subpopulations. Strategies that allow for combination of prodrug-activation gene therapy with a novel replication-conditional, heterogeneous tumor-targeting adenovirus are discussed, as are the benefits of using adenoviral vectors as tumor-targeting oncolytic vectors. While the anticancer activity of many adenoviral vectors has been well established in preclinical studies, only limited successes have been achieved in the clinic, indicating a need for further improvements in activity, specificity, tumor cell delivery and avoidance of immunogenicity. PMID:23869779

  7. Adenoviral gene transfer of endothelial nitric-oxide synthase (eNOS) partially restores normal pulmonary arterial pressure in eNOS-deficient mice

    PubMed Central

    Champion, Hunter C.; Bivalacqua, Trinity J.; Greenberg, Stanley S.; Giles, Thomas D.; Hyman, Albert L.; Kadowitz, Philip J.

    2002-01-01

    It has been shown that mice deficient in the gene coding for endothelial nitric-oxide synthase (eNOS) have increased pulmonary arterial pressure and pulmonary vascular resistance. In the present study, the effect of transfer to the lung of an adenoviral vector encoding the eNOS gene (AdCMVeNOS) on pulmonary arterial pressure and pulmonary vascular resistance was investigated in eNOS-deficient mice. One day after intratracheal administration of AdCMVeNOS to eNOS−/− mice, there was an increase in eNOS protein, cGMP levels, and calcium-dependent conversion of l-arginine to l-citrulline in the lung. The increase in eNOS protein and activity in eNOS−/− mice was associated with a reduction in mean pulmonary arterial pressure and pulmonary vascular resistance when compared with values in eNOS-deficient mice treated with vehicle or a control adenoviral vector coding for β-galactosidase, AdCMVβgal. These data suggest that in vivo gene transfer of eNOS to the lung in eNOS−/− mice can increase eNOS staining, eNOS protein, calcium-dependent NOS activity, and cGMP levels and partially restore pulmonary arterial pressure and pulmonary vascular resistance to near levels measured in eNOS+/+ mice. Thus, the major finding in this study is that in vivo gene transfer of eNOS to the lung in large part corrects a genetic deficiency resulting from eNOS deletion and may be a useful therapeutic intervention for the treatment of pulmonary hypertensive disorders in which eNOS activity is reduced. PMID:12237402

  8. Adenoviral vector expressing murine β-defensin 2 enhances immunogenicity of an adenoviral vector based H5N1 influenza vaccine in aged mice.

    PubMed

    Vemula, Sai V; Pandey, Aseem; Singh, Neetu; Katz, Jacqueline M; Donis, Ruben; Sambhara, Suryaprakash; Mittal, Suresh K

    2013-10-01

    The ability to resist infections and respond to vaccinations is greatly reduced in the older adult population owing to a general decline in innate and adaptive immune functions with aging. Over the years several strategies such as increasing the vaccine dose, number of immunizations and using adjuvants have been evaluated to improve the immunogenicity and efficacy of vaccines in the older adult population. Murine β-defensin 2 (Mbd2) has been shown to function as a molecular adjuvant by recruiting and activating immature dendritic cells (DCs), professional antigen-presenting cells (APC), to the site of the immunization. In this study, we evaluated the potential utility of Mbd2 to enhance the efficacy of an adenoviral vector-based H5N1 influenza vaccine expressing hemagglutinin (HA) and nucleoprotein (NP) (HAd-HA-NP) in an aged mouse model. Our results indicated that immunostimulation with an adenoviral vector expressing Mbd2 (HAd-Mbd2) activated DCs and significantly enhanced the humoral and cellular immune responses induced by HAd-HA-NP. Furthermore, immunostimulation with HAd-Mbd2 followed by immunization with HAd-HA-NP resulted in significantly lower virus titers in the lungs following challenge with a H5N1 influenza virus compared to the group immunized with HAd-HA-NP without immunostimulation. Overall, our results highlight the potential utility of Mbd2 as a molecular adjuvant to enhance the immunogenicity and protective efficacy of vaccines for the elderly. PMID:23892144

  9. Adenoviral Vector Expressing Murine β-Defensin 2 Enhances Immunogenicity of an Adenoviral Vector based H5N1 Influenza Vaccine in Aged Mice

    PubMed Central

    Vemula, Sai V.; Pandey, Aseem; Singh, Neetu; Katz, Jacqueline M; Donis, Ruben; Sambhara, Suryaprakash; Mittal, Suresh K.

    2013-01-01

    The ability to resist infections and respond to vaccinations is greatly reduced in the older adult population owing to a general decline in innate and adaptive immune functions with aging. Over the years several strategies such as increasing the vaccine dose, number of immunizations and using adjuvants have been evaluated to improve the immunogenicity and efficacy of vaccines in the older adult population. Murine ß-defensin 2 (Mbd2) has been shown to function as a molecular adjuvant by recruiting and activating immature dendritic cells (DCs), professional antigen-presenting cells (APC), to the site of the immunization. In this study, we evaluated the potential utility of Mbd2 to enhance the efficacy of an adenoviral vector-based H5N1 influenza vaccine expressing hemagglutinin (HA) and nucleoprotein (NP) (HAd-HA-NP) in an aged mouse model. Our results indicated that immunostimulation with an adenoviral vector expressing Mbd2 (HAd-Mbd2) activated DCs and significantly enhanced the humoral and cellular immune responses induced by HAd-HA-NP. Furthermore, immunostimulation with HAd-Mbd2 followed by immunization with HAd-HA-NP resulted in significantly lower virus titers in the lungs following challenge with a H5N1 influenza virus compared to the group immunized with HAd-HA-NP without immunostimulation. Overall, our results highlight the potential utility of Mbd2 as a molecular adjuvant to enhance the immunogenicity and protective efficacy of vaccines for the elderly. PMID:23892144

  10. Live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy.

    PubMed

    Sakurai, Takashi; Lanahan, Anthony; Woolls, Melissa J; Li, Na; Tirziu, Daniela; Murakami, Masahiro

    2014-01-01

    Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope's autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators. PMID:24998400

  11. Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

    PubMed Central

    Sakurai, Takashi; Lanahan, Anthony; Woolls, Melissa J.; Li, Na; Tirziu, Daniela; Murakami, Masahiro

    2014-01-01

    Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope’s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators. PMID:24998400

  12. Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation.

    PubMed

    Knippertz, Ilka; Deinzer, Andrea; Dörrie, Jan; Schaft, Niels; Nettelbeck, Dirk M; Steinkasserer, Alexander

    2016-01-01

    To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B' core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8(+) T cells. Second, we generated the two-vector-based "modular promoter" system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B' core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo. PMID:27446966

  13. Alanine-glyoxylate aminotransferase-deficient mice, a model for primary hyperoxaluria that responds to adenoviral gene transfer.

    PubMed

    Salido, Eduardo C; Li, Xiao M; Lu, Yang; Wang, Xia; Santana, Alfredo; Roy-Chowdhury, Namita; Torres, Armando; Shapiro, Larry J; Roy-Chowdhury, Jayanta

    2006-11-28

    Mutations in the alanine-glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt(-/-) mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria. PMID:17110443

  14. The Effects of Adenoviral Transfection of the Keratinocyte Growth Factor Gene on Epidermal Stem Cells: an In Vitro Study

    PubMed Central

    Li, Xinping; Liang, Ling; Zhao, Pin; Uchida, Kenzo; Baba, Hisatoshi; Huang, Hong; Bai, Wenfang; Bai, Liming; Zhang, Mingsheng

    2013-01-01

    Epidermal stem cells (ESCs) are characterized as slow-cycling, multi-potent, and self-renewing cells that not only maintain somatic homeostasis but also participate in tissue regeneration and repair. To examine the feasibility of adenoviral vector-mediated keratinocyte growth factor (KGF) gene transfer into in vitro-expanded ESCs, ESCs were isolated from samples of human skin, cultured in vitro, and then transfected with recombinant adenovirus (Ad) carrying the human KGF gene (AdKGF) or green fluorescent protein gene (AdGFP). The effects of KGF gene transfer on cell proliferation, cell cycle arrest, cell surface antigen phenotype, and β-catenin expression were investigated. Compared to ESCs transfected with AdGFP, AdKGF-transfected ESCs grew well, maintained a high proliferative capacity in keratinocyte serum-free medium, and expressed high levels of β-catenin. AdKGF infection increased the number of ESCs in the G0/G1 phase and promoted ESCs entry into the G2/M phase, but had no effect on cell surface antigen phenotype (CD49f+/CD71−). The results suggest that KGF gene transfer can stimulate ESCs to grow and undergo cell division, which can be applied to enhance cutaneous wound healing. PMID:24170090

  15. Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation

    PubMed Central

    Deinzer, Andrea

    2016-01-01

    To specifically target dendritic cells (DCs) to simultaneously express different therapeutic transgenes for inducing immune responses against tumors, we used a combined promoter system of adenoviral vectors. We selected a 216 bp short Hsp70B′ core promoter induced by a mutated, constitutively active heat shock factor (mHSF) 1 to drive strong gene expression of therapeutic transgenes MelanA, BclxL, and IL-12p70 in HeLa cells, as well as in mature DCs (mDCs). As this involves overexpressing mHSF1, we first evaluated the resulting effects on DCs regarding upregulation of heat shock proteins and maturation markers, toxicity, cytokine profile, and capacity to induce antigen-specific CD8+ T cells. Second, we generated the two-vector-based “modular promoter” system, where one vector contains the mHSF1 under the control of the human CD83 promoter, which is specifically active only in DCs and after maturation. mHSF1, in turn, activates the Hsp70B′ core promotor-driven expression of transgenes MelanA and IL-12p70 in the DC-like cell line XS52 and in human mature and hence immunogenic DCs, but not in tolerogenic immature DCs. These in vitro experiments provide the basis for an in vivo targeting of mature DCs for the expression of multiple transgenes. Therefore, this modular promoter system represents a promising tool for future DC-based immunotherapies in vivo. PMID:27446966

  16. Evaluation of excipients for enhanced thermal stabilization of a human type 5 adenoviral vector through spray drying.

    PubMed

    LeClair, Daniel A; Cranston, Emily D; Xing, Zhou; Thompson, Michael R

    2016-06-15

    We have produced a thermally stable recombinant human type 5 adenoviral vector (AdHu5) through spray drying with three excipient formulations (l-leucine, lactose/trehalose and mannitol/dextran). Spray drying leads to immobilization of the viral vector which is believed to prevent viral protein unfolding, aggregation and inactivation. The spray dried powders were characterized by scanning electron microscopy, differential scanning calorimetry, Karl Fischer titrations, and X-ray diffraction to identify the effects of temperature and atmospheric moisture on the immobilizing matrix. Thermal stability of the viral vector was confirmed in vitro by infection of A549 lung epithelial cells. Mannitol/dextran powders showed the greatest improvement in thermal stability with almost no viral activity loss after storage at 20°C for 90days (0.7±0.3 log TCID50) which is a significant improvement over the current -80°C storage protocol. Furthermore, viral activity was retained over short term exposure (72h) to temperatures as high as 55°C. Conversely, all powders exhibited activity loss when subjected to moisture due to amplified molecular motion of the matrix. Overall, a straightforward method ideal for the production of thermally stable vaccines has been demonstrated through spray drying AdHu5 with a blend of mannitol and dextran and storing the powder under low humidity conditions. PMID:27130366

  17. HIV-1 Adenoviral Vector Vaccines Expressing Multi-Trimeric BAFF and 4-1BBL Enhance T Cell Mediated Anti-Viral Immunity

    PubMed Central

    Gupta, Sachin; Raffa, Francesca N.; Fuller, Katherine A.; Rivas, Yaelis; Philip, Sakhi; Kornbluth, Richard S.; Stone, Geoffrey W.

    2014-01-01

    Adenoviral vectored vaccines have shown considerable promise but could be improved by molecular adjuvants. Ligands in the TNF superfamily (TNFSF) are potential adjuvants for adenoviral vector (Ad5) vaccines based on their central role in adaptive immunity. Many TNFSF ligands require aggregation beyond the trimeric state (multi-trimerization) for optimal biological function. Here we describe Ad5 vaccines for HIV-1 Gag antigen (Ad5-Gag) adjuvanted with the TNFSF ligands 4-1BBL, BAFF, GITRL and CD27L constructed as soluble multi-trimeric proteins via fusion to Surfactant Protein D (SP-D) as a multimerization scaffold. Mice were vaccinated with Ad5-Gag combined with Ad5 expressing one of the SP-D-TNFSF constructs or single-chain IL-12p70 as adjuvant. To evaluate vaccine-induced protection, mice were challenged with vaccinia virus expressing Gag (vaccinia-Gag) which is known to target the female genital tract, a major route of sexually acquired HIV-1 infection. In this system, SP-D-4-1BBL or SP-D-BAFF led to significantly reduced vaccinia-Gag replication when compared to Ad5-Gag alone. In contrast, IL-12p70, SP-D-CD27L and SP-D-GITRL were not protective. Histological examination following vaccinia-Gag challenge showed a dramatic lymphocytic infiltration into the uterus and ovaries of SP-D-4-1BBL and SP-D-BAFF-treated animals. By day 5 post challenge, proinflammatory cytokines in the tissue were reduced, consistent with the enhanced control over viral replication. Splenocytes had no specific immune markers that correlated with protection induced by SP-D-4-1BBL and SP-D-BAFF versus other groups. IL-12p70, despite lack of anti-viral efficacy, increased the total numbers of splenic dextramer positive CD8+ T cells, effector memory T cells, and effector Gag-specific CD8+ T cells, suggesting that these markers are poor predictors of anti-viral immunity in this model. In conclusion, soluble multi-trimeric 4-1BBL and BAFF adjuvants led to strong protection from vaccinia

  18. Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia

    PubMed Central

    Koehler, David R.; Sajjan, Umadevi; Chow, Yu-Hua; Martin, Bernard; Kent, Geraldine; Tanswell, A. Keith; McKerlie, Colin; Forstner, Janet F.; Hu, Jim

    2003-01-01

    We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens. PMID:14673110

  19. Protection of Cftr knockout mice from acute lung infection by a helper-dependent adenoviral vector expressing Cftr in airway epithelia.

    PubMed

    Koehler, David R; Sajjan, Umadevi; Chow, Yu-Hua; Martin, Bernard; Kent, Geraldine; Tanswell, A Keith; McKerlie, Colin; Forstner, Janet F; Hu, Jim

    2003-12-23

    We developed a helper-dependent adenoviral vector for cystic fibrosis lung gene therapy. The vector expresses cystic fibrosis transmembrane conductance regulator (Cftr) using control elements from cytokeratin 18. The vector expressed properly localized CFTR in cultured cells and in the airway epithelia of mice. Cftr RNA and protein were present in whole lung and bronchioles, respectively, for 28 days after a vector dose. Acute inflammation was minimal to moderate. To test the therapeutic potential of the vector, we challenged mice with a clinical strain of Burkholderia cepacia complex (Bcc). Cftr knockout mice (but not Cftr+/+ littermates) challenged with Bcc developed severe lung histopathology and had high lung bacteria counts. Cftr knockout mice receiving gene therapy 7 days before Bcc challenge had less severe histopathology, and the number of lung bacteria was reduced to the level seen in Cftr+/+ littermates. These data suggest that gene therapy could benefit cystic fibrosis patients by reducing susceptibility to opportunistic pathogens. PMID:14673110

  20. Antitumor activity of adenoviral vector containing T42 and 4xT42 peptide gene through inducing apoptosis of tumor cells and suppressing angiogenesis.

    PubMed

    Zhang, Xiong; Qi, Dong-Dong; Zhang, Ting-Ting; Chen, Qing-Xin; Wang, Guang-Zhi; Sui, Guang-Yu; Hao, Xue-Wei; Sun, Shouli; Song, Xue; Chen, Ying-Li

    2015-03-01

    The T42 peptide, generated from two active fragments of tumstatin, has been shown to have anti‑tumor activity. The adenoviral vector is the most frequently used vector in research and clinical trials for gene therapy. In the present study, the anti‑tumor activity of the T42 peptide and quadruple T42 (4xT42) peptide adenoviral vectors were elucidated for the first time, to the best of our knowledge. Human embryonic kidney 293 cells were infected with plasmid adenovirus (pAd)‑enhanced green fluorescent protein (EGFP)‑T42 or pAd‑EGFP‑4xT42 and the expression of the T42 and 4xT42 genes was confirmed by the identification of GFP expression and reverse transcription polymerase chain reaction experiments. The anti‑cancer effects of pAd‑EGFP‑T42 and pAd‑EGFP‑4xT42 on breast cancer cells in vivo and in vitro were subsequently investigated. The results indicated that the packaging of the recombinant adenoviruses with the viral titer was successful, following purification at 5x109 plaque forming units/ml. The results also revealed that the recombinant adenoviruses promoted apoptosis in MCF‑7 breast cancer cells and inhibited cancer growth. Through the analysis of caspase‑3, B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein expression, it was demonstrated that the T42/4xT42 peptide may induce apoptosis via the mitochondrial pathway. In addition, mouse xenograft experiments confirmed that the T42 peptide inhibited tumor growth and reduced angiogenesis in vivo. In conclusion, the results of the present study indicated that the T42 and 4xT42 peptide genes, transfected by a recombinant adenovirus, may provide a potential novel strategy for the treatment of breast cancer. PMID:25384346

  1. Induction of Specific Humoral and Cellular Immune Responses in a Mouse Model following Gene Fusion of HSP70C and Hantaan Virus Gn and S0.7 in an Adenoviral Vector

    PubMed Central

    Li, Kai; Wang, Fang; Zhang, Liang; Ye, Wei; Li, Puyuan; Zhang, Fanglin; Xu, Zhikai

    2014-01-01

    Heat shock proteins (HSPs) display adjuvant functions when given as fusion proteins to enhance vaccination efficiency. To evaluate enhanced potency of Hantaan virus (HTNV) glycoprotein (GP) and nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), a recombinant adenovirus rAd-GnS0.7-pCAG-HSP70C expression vector was developed by genetically linking the HSP70 C-terminal gene (HSP70 359–610 aa, HSP70C) to the Gn and 0.7 kb fragment of the NP (aa1–274-S0.7). C57BL/6 mice were immunized with these recombinant adenoviral vectors. A series of immunological assays determined the immunogenicity of the recombinant adenoviral vectors. The results showed that rAd-GnS0.7-pCAG-HSP70C induced a stronger humoral and cellular immune response than other recombinant adenoviruses (rAd-GnS0.7-pCAG and rAd-GnS0.7) and the HFRS vaccine control. Animal protection experiments showed that rAd-GnS0.7-pCAG-HSP70C was effective at protecting C57BL/6 mice from HTNV infection. The results of the immunological experiments showed that HSP70C lead to enhanced vaccine potency, and suggested significant potential in the development of genetically engineered vaccines against HTNV. PMID:24505421

  2. A Novel and Simple Method for Rapid Generation of Recombinant Porcine Adenoviral Vectors for Transgene Expression

    PubMed Central

    Ma, Jing; Wang, Wenbin; Zhang, Lu; Tikoo, Suresh K.; Yang, Zengqi

    2015-01-01

    Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes. PMID:26011074

  3. Increased Mucosal CD4+ T Cell Activation in Rhesus Macaques following Vaccination with an Adenoviral Vector

    PubMed Central

    Bukh, Irene; Calcedo, Roberto; Roy, Soumitra; Carnathan, Diane G.; Grant, Rebecca; Qin, Qiuyue; Boyd, Surina; Ratcliffe, Sarah J.; Veeder, Christin L.; Bellamy, Scarlett L.; Betts, Michael R.

    2014-01-01

    ABSTRACT The possibility that vaccination with adenovirus (AdV) vectors increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of HIV acquisition within the Step trial. Modeling this within rhesus macaques is complicated because human adenoviruses, including human adenovirus type 5 (HAdV-5), are not endogenous to macaques. Here, we tested whether vaccination with a rhesus macaque-derived adenoviral vector (simian adenovirus 7 [SAdV-7]) enhances mucosal T cell activation within rhesus macaques. Following intramuscular SAdV-7 vaccination, we observed a pronounced increase in SAdV-7-specific CD4+ T cell responses in peripheral blood and, more dramatically, in rectal mucosa tissue. Vaccination also induced a significant increase in the frequency of activated memory CD4+ T cells in SAdV-7- and HAdV-5-vaccinated animals in the rectal mucosa but not in peripheral blood. These fluctuations within the rectal mucosa were also associated with a pronounced decrease in the relative frequency of naive resting CD4+ T cells. Together, these results indicate that peripheral vaccination with an AdV vector can increase the activation of mucosal CD4+ T cells, potentially providing an experimental model to further evaluate the role of host-vector interactions in increased HIV acquisition after AdV vector vaccination. IMPORTANCE The possibility that vaccination with a human adenovirus 5 vector increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of human immunodeficiency virus (HIV) acquisition within the Step trial. In this study, we tested whether vaccination with a rhesus macaque-derived adenoviral vector in rhesus macaques enhances mucosal CD4+ T cell activation, the main cell target of simian immunodeficiency virus (SIV)/HIV. The results showed that vaccination with an adenoviral vector indeed increases activation of mucosal CD4+ T cells and potentially increases susceptibility to SIV

  4. Vertical transmission and clinical signs in broiler breeders and broilers experiencing adenoviral gizzard erosion.

    PubMed

    Grafl, Beatrice; Aigner, Franz; Liebhart, Dieter; Marek, Ana; Prokofieva, Irina; Bachmeier, Josef; Hess, Michael

    2012-12-01

    The present report documents an outbreak of adenoviral gizzard erosion in 22 broiler flocks in Germany. The clinical picture was characterized by uneven growth of affected broilers that resulted in considerably lower than average weight at slaughtering. Fowl adenovirus serotype 1 (FAdV-1) was isolated from gizzard lesions and histological examinations demonstrated FAdV-1-positive intranuclear inclusion bodies in gizzard epithelial cells of affected broilers by in-situ hybridization. Birds from all affected flocks originated from one broiler breeder farm. During production of affected birds, broiler breeders were between 27 and 32 weeks old. Enzyme-linked immunosorbent assay and specific virus neutralization assay of sera from parent birds demonstrated an acute FAdV-1 infection within the first 5 weeks of the production cycle. Clinically, broiler breeders exhibited a moderate fall in the hatchability of their chicks, while egg production remained normal. No further clinical signs could be observed. Genetically identical FAdV-1 strains were isolated from gizzards of embryos at the lowest point of hatchability and from affected broiler flocks raised on independent farms. For the first time, direct detection of viable FAdV-1 from gizzards of embryos and progenies of one FAdV-1-seropositive broiler breeder farm in the course of an outbreak of adenoviral gizzard erosion could be demonstrated, highlighting the importance of vertical transmission of this disease. Additionally, growth retardation and subsequent reduced average weight at the time of slaughter of broiler chickens underline the economic impact of adenoviral gizzard erosion for poultry production. PMID:23237373

  5. A Human Vaccine Strategy Based On Chimpanzee Adenoviral and MVA Vectors That Primes, Boosts and Sustains Functional HCV Specific T-Cell Memory*

    PubMed Central

    Swadling, Leo; Capone, Stefania; Antrobus, Richard D.; Brown, Anthony; Richardson, Rachel; Newell, Evan W.; Halliday, John; Kelly, Christabel; Bowen, Dan; Fergusson, Joannah; Kurioka, Ayako; Ammendola, Virginia; Sorbo, Mariarosaria Del; Grazioli, Fabiana; Esposito, Maria Luisa; Siani, Loredana; Traboni, Cinzia; Hill, Adrian; Colloca, Stefano; Davis, Mark; Nicosia, Alfredo; Cortese, Riccardo; Folgori, Antonella; Klenerman, Paul; Barnes, Eleanor

    2015-01-01

    A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies and assessment of host immunity during acute infection highlight the critical role that effective T-cell immunity plays in viral control. In this first-in-man study we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A and NS5B proteins of HCV genotype-1b. Analysis employed single cell mass cytometry (CyTOF), and HLA class-I peptide tetramer technology in healthy human volunteers. We show that HCV specific T-cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8+ and CD4+ HCV specific T-cells targeting multiple HCV antigens. Sustained memory and effector T-cell populations are generated and T-cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) following heterologous MVA boost. We have developed a HCV vaccine strategy, with durable, broad, sustained and balanced T-cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine. PMID:25378645

  6. Intra-testicular injection of adenoviral constructs results in Sertoli cell-specific gene expression and disruption of the seminiferous epithelium

    PubMed Central

    Hooley, R P; Paterson, M; Brown, P; Kerr, K; Saunders, P T K

    2009-01-01

    Spermatogenesis is a complex process that cannot be modelled in vitro. The somatic Sertoli cells (SCs) within the seminiferous tubules perform a key role in supporting maturation of germ cells (GCs). Progress has been made in determining what aspects of SC function are critical to maintenance of fertility by developing rodent models based on the Cre/LoxP system; however, this is time-consuming and is only applicable to mice. The aim of the present study was to establish methods for direct injection of adenoviral vectors containing shRNA constructs into the testis as a way of inducing target-selective knock-down in vivo. This paper describes a series of experiments using adenovirus expressing a green fluorescent protein (GFP) transgene. Injection via the efferent ductules resulted in SC-specific expression of GFP; expression levels paralleled the amount of infective viral particles injected. At the highest doses of virus seminiferous tubule architecture were grossly disturbed and immune cell invasion noted. At lower concentrations, the expression of GFP was variable/negligible, the seminiferous tubule lumen was maintained but stage-dependent GC loss and development of numerous basal vacuoles was observed. These resembled intercellular dilations of SC junctional complexes previously described in rats and may be a consequence of disturbances in SC function due to interaction of the viral particles with the coxsackie/adenovirus receptor that is a component of the junctional complexes within the blood testis barrier. In conclusion, intra-testicular injection of adenoviral vectors disturbs SC function in vivo and future work will therefore focus on the use of lentiviral delivery systems. PMID:18955374

  7. A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustains functional HCV-specific T cell memory.

    PubMed

    Swadling, Leo; Capone, Stefania; Antrobus, Richard D; Brown, Anthony; Richardson, Rachel; Newell, Evan W; Halliday, John; Kelly, Christabel; Bowen, Dan; Fergusson, Joannah; Kurioka, Ayako; Ammendola, Virginia; Del Sorbo, Mariarosaria; Grazioli, Fabiana; Esposito, Maria Luisa; Siani, Loredana; Traboni, Cinzia; Hill, Adrian; Colloca, Stefano; Davis, Mark; Nicosia, Alfredo; Cortese, Riccardo; Folgori, Antonella; Klenerman, Paul; Barnes, Eleanor

    2014-11-01

    A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies, and assessment of host immunity during acute infection highlight the critical role that effective T cell immunity plays in viral control. In this first-in-man study, we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b. Analysis used single-cell mass cytometry and human leukocyte antigen class I peptide tetramer technology in healthy human volunteers. We show that HCV-specific T cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8(+) and CD4(+) HCV-specific T cells targeting multiple HCV antigens. Sustained memory and effector T cell populations are generated, and T cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) after heterologous MVA boost. We have developed an HCV vaccine strategy, with durable, broad, sustained, and balanced T cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine. PMID:25378645

  8. Immunogenicity and protective efficacy of a recombinant adenoviral based vaccine expressing heat-stable enterotoxin (STa) and K99 adhesion antigen of enterotoxigenic Escherichia coli in mice.

    PubMed

    Deng, Guangcun; Li, Wu; Wu, Xiaoling; Bao, Shaowen; Zeng, Jin; Zhao, Ning; Luo, Meihui; Liu, Xiaoming; Wang, Yujiong

    2015-12-01

    The diarrheal disease of domestic animals or in humans caused by enterotoxigenic Escherichia coli (ETEC) infections remains a major issue for public health in developing countries. Unfortunately, there is no effective vaccine available for preventing from an ETEC infection. Therefore, the development of a safe and effective vaccine against ETEC is urgently needed. In the present study, A recombinant adenoviral vector Ad5-STa-K99 that capable of expressing a fusion protein of heat-stable enterotoxin (STa) and K99 adhesion antigen of ETEC was generated and its immunogenicity was evaluated in a murine model. The intestinal mucosal secretory IgA(sIgA), serum anti-STa-K99 antibody responses, antigen-specific CD4(+) and CD8(+) T cells frequencies, as well as T-cell proliferation of mice immunized with the viral vector were determined as immunological indexes. The results demonstrated that Ad5-STa-K99 was able to enhance humoral responses with a dramatically augmented antigen-specific serum IgG antibody, and an elevated production of intestinal sIgA in immunized mice, suggesting the elicitation of both of humoral and mucosal immune responses. In addition, this adenoviral vector could significantly promote splenic T cell proliferation and increase the frequencies of CD4(+) and CD8(+) T cell populations in mice, indicative of a capacity to activate T cell responses. More importantly, vaccination of the Ad5-STa-K99 showed a potential to evoke a protective effect from ETEC challenge in mice. These data indicate that the Ad5-STa-K99 is a highly immunogenic vector able to induce a broad range of antigen-specific immune responses in vivo, and evoke a protective immune response against ETEC infections, implying that it may be a novel vaccine candidate warranted for further investigation. PMID:26589454

  9. Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

    PubMed Central

    Fishbein, Ilia; Forbes, Scott P.; Adamo, Richard F.; Chorny, Michael; Levy, Robert J.; Alferiev, Ivan S.

    2014-01-01

    In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of

  10. [Clinical syndrome of convulsive cough of adenoviral etiology in a children's collective].

    PubMed

    Grobnicu, M; Andreescu, V; Caffé, I; Măgureanu, E; Marion, M; Ivan, I; Botez, D; Cuteanu, I; Barbu, I

    1976-01-01

    Bacteriological, viral and serological investigators were carried out in a community with 100 prescholar children (Kindergarden), 34 of whom presented a clinical syndrome of whooping cough, in order to establish the bacteriologic or viral etiology of the syndrome. The etiologic role of organisms of the Bordetella, B. pertussis and B. parapertussis was invalidated by the bacteriologic and serological tests. Viral and serological tests, performed to demonstrate the participation of viral agents in the causation of this clinical syndrome, established an adenoviral diagnosis in 13 (43.3%) of the 30 children. Adenovirus type 6 was isolated and there was a significant increase in the titers of antibodies to adenoviruses. PMID:184515

  11. The catalase–hydrogen peroxide system. Role of sub-units in the thermal deactivation of bacterial catalase in the absence of substrate

    PubMed Central

    Jones, Peter; Suggett, A.

    1968-01-01

    1. Kinetic studies of the thermal deactivation of bacterial catalase in the absence of substrate suggest that the reaction involves a protonation-induced reversible dissociation of catalase into catalatically inactive sub-units, followed by an irreversible transformation of the sub-units into deactivated products. It is possible that the sub-units are mono-haem species. The rate of deactivation decreases with increasing pressure in accordance with the predictions of the proposed model. 2. The results also imply that the addition of hydrogen peroxide substrate induces the re-formation of active catalase. Under appropriate conditions the activity of catalase is found to increase with time in a manner that is quantitatively consistent with the results of deactivation studies. PMID:5673527

  12. Adenoviral Delivery of VEGF121 Early in Pregnancy Prevents Spontaneous Development of Preeclampsia in BPH/5 Mice

    PubMed Central

    Woods, Ashley K.; Hoffmann, Darren S.; Weydert, Christine J.; Butler, Scott D.; Zhou, Yi; Sharma, Ram V.; Davisson, Robin L.

    2011-01-01

    An imbalance in circulating pro-angiogenic and anti-angiogenic factors is postulated to play a causal role in pre-eclampsia (PE). We have described an inbred mouse strain, BPH/5, which spontaneously develops a PE-like syndrome including late-gestational hypertension, proteinuria, and poor feto-placental outcomes. Here we tested the hypothesis that an angiogenic imbalance during pregnancy in BPH/5 mice leads to the development of PE-like phenotypes in this model. Similar to clinical findings, plasma from pregnant BPH/5 showed reduced levels of free vascular endothelial growth factor (VEGF) and placental growth factor (PGF) compared to C57BL/6 controls. This was paralleled by a marked decrease in VEGF protein and Pgf mRNA in BPH/5 placentae. Surprisingly, antagonism by the soluble form of the FLT1 receptor (sFLT1) did not appear to be the cause of this reduction, as sFLT1 levels were unchanged or even reduced in BPH/5 compared to controls. Adenoviral-mediated delivery of VEGF121 (Ad-VEGF) via tail vein at e7.5 normalized both the plasma free VEGF levels in BPH/5 and restored the in vitro angiogenic capacity of serum from these mice. Ad-VEGF also reduced the incidence of fetal resorptions and prevented the late-gestational spike in blood pressure and proteinuria observed in BPH/5. These data underscore the importance of dysregulation of angiogenic factors in the pathogenesis of PE, and suggest the potential utility of early pro-angiogenic therapies in treating this disease. PMID:21079047

  13. Evaluation of the immune response to recombinant DNA vaccine and adenoviral vaccine co-expressing the M1 and HA genes of H5N1 influenza virus in mice.

    PubMed

    Guo, Jianqiang; Yao, Lihong; Chen, Aijun; Liu, Xiaoyu; Fu, Jinqi; Xu, Pengwei; Zhang, Zhiqing

    2011-06-01

    In order to evaluate the response to vector-expressed M1 and HA genes of influenza virus in mice, we prepared recombinant plasmid pStar-M1/HA and recombinant adenovirus Ad-M1/HA containing both the full-length matrix protein 1(M1) and hemagglutinin (HA) genes of human H5N1 influenza virus strain A/Anhui/1/2005. We then combined the DNA vaccine and adenoviral vaccine in immunization of BALB/c mice with a prime-boost regime. We immunized the mice with DNA vaccine at day 0 and 28 and with recombinant adenoviral vaccines at day 14 and 42. We took blood samples before each injection and 14 days after the final injection for detection of humoral immune responses. At day 56, we sacrificed the mice and collected splenocytes for detection of cellular immune responses. ELISA and hemagglutination inhibition (HI) assay showed that specific IgG Abs against H5N1 influenza virus was induced in serum of the immunized mice. ELISPOT results confirmed that the specific cellular immune responses were successfully induced against the M1 and HA proteins of H5N1 influenza virus. This study provides new strategy for development of novel influenza vaccines. PMID:22034816

  14. Development of an adenoviral vector with robust expression driven by p53

    SciTech Connect

    Bajgelman, Marcio C.; Strauss, Bryan E.

    2008-02-05

    Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx{beta}, containing a p53-responsive element, a minimal promoter and the first intron of the rabbit {beta}-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG served as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities.

  15. An adenoviral vector for probing promoter activity in primary immune cells

    PubMed Central

    Tripathi, Pulak; Madan, Rajat; Chougnet, Claire; Divanovic, Senad; Ma, Xiaojing; Wahl, Larry M.; Gajewski, Thomas; Karp, Christopher L.; Hildeman, David A.

    2010-01-01

    Functional analysis of the DNA regulatory regions that control gene expression has largely been performed through transient transfection of promoter–reporter constructs into transformed cells. However, transformed cells are often poor models of primary cells. To directly analyze DNA regulatory regions in primary cells, we generated a novel adenoviral luciferase reporter vector, pShuttle-luciferase-GFP (pSLUG) that contains a promoterless luciferase cassette (with an upstream cloning site) for probing promoter activity, and a GFP expression cassette that allows for the identification of transduced cells. Recombinant adenoviruses generated from this vector can transduce a wide range of primary immune cells with high efficiency, including human macrophages, dendritic cells and T cells; and mouse T cells transgenic for the coxsackie and adenoviral receptor (CAR). In primary T cells, we show inducible nuclear factor of activated T cells (NF-AT) activity using a recombinant pSLUG adenovirus containing a consensus NF-AT promoter. We further show inducible IL-12/23 p40 promoter activity in primary macrophages and dendritic cells using a recombinant pSLUG adenovirus containing the proximal human IL-12/23 p40 promoter. The pSLUG system promises to be a powerful tool for the analysis of DNA regulatory regions in diverse types of primary immune cells. PMID:16563424

  16. Disseminated adenoviral infection masquerading as lower urinary tract voiding dysfunction in a kidney transplant recipient.

    PubMed

    Aboumohamed, Ahmed; Flechner, Stuart M; Chiesa-Vottero, Andres; Srinivas, Titte R; Mossad, Sherif B

    2014-11-01

    Viral infections continue to cause significant morbidity in immunosuppressed kidney transplant patients. Although cytomegalovirus, Epstein-Barr virus and polyoma "BK" virus are more frequently encountered, the Adenovirus can cause multi-organ system infections, and may be difficult to diagnose because it is not often considered in the initial work up in kidney transplant recipients. We present an unusual case of a kidney recipient 1 year post-transplant with disseminated adenoviral infection, who had an initial presentation of lower urinary tract voiding dysfunction with hematuria and sterile pyuria. This progressed to a severe tubulointerstitial nephritis and acute kidney injury that improved with reduction of immunosuppression. Serial blood viral loads are useful for monitoring the course of infection. Urinary adenoviral infection should be considered in the differential diagnosis whenever a kidney transplant recipient presents with unexplained lower tract voiding dysfunction, hematuria, and sterile pyuria. The allograft kidney and bladder can be targets of viral proliferation. Early diagnosis with reduction of immunosuppressive therapy is essential to clear the virus and maintain allograft function. PMID:23816478

  17. Adenoviral infection or deferoxamine? Two approaches to overexpress VEGF in beta-cell lines.

    PubMed

    Langlois, Allan; Bietiger, William; Sencier, Marie-Christine; Maillard, Elisa; Pinget, Michel; Kessler, Laurence; Sigrist, Severine

    2009-07-01

    Rapid and adequate revascularization of transplanted islets is important for their survival and function during transplantation. Vascular endothelial growth factor (VEGF) could play a critical role with respect to islet revascularization. The aim of this study was to compare two strategies that are used to overexpress VEGF in beta-cells: (1) gene therapy through adenoviral infection and (2) a pharmacological approach using deferoxamine (DFO). beta-Cell lines from rat insulinoma (RINm5F) were either infected using an adenovirus encoding the gene of human VEGF 165 or incubated with DFO. One day after treatment, the viability of RINm5F cells was preserved with 10 micromol/L of DFO (103.95 +/- 5.66% toward control; n = 4). In addition, adenoviral infection maintained the viability of cells for all the concentrations used. In both treatments, overexpression of VEGF was in a comparable level. Finally, the ratio of Bax/Bcl-2 indicated that the apoptosis increased in infected beta-cells whereas treatment with DFO seems to be antiapoptotic. Our results suggest that the use of DFO could be a realistic approach to improve the vascularization of islets during transplantation. PMID:19527112

  18. ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser298 Augments Hepatic Gluconeogenesis

    PubMed Central

    Parsa, Kishore V. L.; Kain, Vasundhara; Behera, Soma; Suraj, Sashidhara Kaimal; Babu, Phanithi Prakash; Kar, Anand; Panda, Sunanda; Zhu, Yi-jun; Jia, Yuzhi; Thimmapaya, Bayar; Reddy, Janardan K.; Misra, Parimal

    2013-01-01

    PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser298 and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMTS298D) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMTS298D but not PIMTS298A augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser298 phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser298 is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. PMID:24358311

  19. ERK2-mediated phosphorylation of transcriptional coactivator binding protein PIMT/NCoA6IP at Ser298 augments hepatic gluconeogenesis.

    PubMed

    Kapadia, Bandish; Viswakarma, Navin; Parsa, Kishore V L; Kain, Vasundhara; Behera, Soma; Suraj, Sashidhara Kaimal; Babu, Phanithi Prakash; Kar, Anand; Panda, Sunanda; Zhu, Yi-jun; Jia, Yuzhi; Thimmapaya, Bayar; Reddy, Janardan K; Misra, Parimal

    2013-01-01

    PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser(298) and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMT(S298D)) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMT(S298D) but not PIMT(S298A) augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser(298) phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser(298) is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. PMID:24358311

  20. Chemokine CXCL1/KC and its Receptor CXCR2 Are Responsible for Neutrophil Chemotaxis in Adenoviral Keratitis

    PubMed Central

    Chintakuntlawar, Ashish V.

    2009-01-01

    Epidemic keratoconjunctivitis (EKC), caused by human adenovirus (HAdV), is one of the most common ocular infections and results in corneal inflammation and subepithelial infiltrates. Adenoviral keratitis causes significant morbidity to the patients, and is characterized by infiltration of leukocytes in the corneal stroma, and expression of chemokines. The exact role of these chemokines in adenoviral infection has not been studied due to lack of animal models. Here, we have characterized the role of chemokine CXCL1/KC and receptor CXCR2 in adenoviral keratitis using a novel mouse model. Analysis of chemokine expression, leukocyte infiltration, and development of keratitis was performed by ELISA, flow cytometry, and histopathology, respectively. Deficiency of CXCL1 and CXCR2 resulted in delayed infiltration of neutrophils, but not inflammatory monocytes in HAdV-37 corneal infection. CXCL1−/− mice showed decreased expression of CXCL2/MIP-2, but not CCL2/MCP-1. CXCR2−/− mice showed increased expression of CXCL1 and CXCL2, but not CCL2. Both CXCL1−/− and CXCR2−/− mice demonstrated keratitis similar to wild-type mice. In conclusion, both CXCL1 and CXCR2 play an important role in chemokine expression and neutrophil infiltration following adenoviral corneal infection, but have a redundant role in the development of keratitis. PMID:19642907

  1. A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration.

    PubMed

    Boehme, Philip; Zhang, Wenli; Solanki, Manish; Ehrke-Schulz, Eric; Ehrhardt, Anja

    2016-01-01

    For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose. PMID:27434682

  2. Adenoviral-based foot-and-mouth disease virus vaccine: effect of duration of antigen expression on immunogenicity in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An adenoviral (Ad5)-based foot-and-mouth disease virus (FMDV) subunit vaccine, Ad5-A24, is an effective alternative to the current inactivated whole virus vaccine and has a number of advantages including no requirement for infectious FMDV and DIVA (Differentiation of Infected from Vaccinated Animals...

  3. Osteogenic gene regulation and relative acceleration of healing by adenoviral-mediated transfer of human BMP-2 or -6 in equine osteotomy and ostectomy models.

    PubMed

    Ishihara, Akikazu; Shields, Kathleen M; Litsky, Alan S; Mattoon, John S; Weisbrode, Steven E; Bartlett, Jeffrey S; Bertone, Alicia L

    2008-06-01

    This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)-2, Ad-BMP-6, or beta-galactosidase protein vector control (Ad-LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad-BMP-2 or Ad-BMP-6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad-BMP-2 than Ad-BMP-6, and both Ad-BMP-2- and Ad-BMP-6-treated osteotomies were greater than Ad-LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta-1, cathepsin H, and gelsolin-like capping protein were greater in Ad-BMP-2- and Ad-BMP-6-treated callus compared to Ad-LacZ-treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad-BMP-2 over Ad-BMP-6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites. PMID:18241059

  4. Helper-Dependent Adenoviral Vectors and Their Use for Neuroscience Applications.

    PubMed

    Montesinos, Mónica S; Satterfield, Rachel; Young, Samuel M

    2016-01-01

    Neuroscience research has been revolutionized by the use of recombinant viral vector technology from the basic, preclinical and clinical levels. Currently, multiple recombinant viral vector types are employed with each having its strengths and weaknesses depending on the proposed application. Helper-dependent adenoviral vectors (HdAd) are emerging as ideal viral vectors that solve a major need in the neuroscience field: (1) expression of transgenes that are too large to be packaged by other viral vectors and (2) rapid onset of transgene expression in the absence of cytotoxicity. Here, we describe the methods for large-scale production of HdAd viral vectors for in vivo use with neurospecific transgene expression. PMID:27515075

  5. Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

    PubMed Central

    Campos, Samuel K.; Barry, Michael A.

    2008-01-01

    Gene delivery vectors based on Adenoviral (Ad) vectors have enormous potential for the treatment of both hereditary and acquired disease. Detailed structural analysis of the Ad virion, combined with functional studies has broadened our knowledge of the structure/function relationships between Ad vectors and host cells/tissues and substantial achievement has been made towards a thorough understanding of the biology of Ad vectors. The widespread use of Ad vectors for clinical gene therapy is compromised by their inherent immunogenicity. The generation of safer and more effective Ad vectors, targeted to the site of disease, has therefore become a great ambition in the field of Ad vector development. This review provides a synopsis of the structure/function relationships between Ad vectors and host systems and summarizes the many innovative approaches towards achieving Ad vector targeting. PMID:17584037

  6. Hepatic Delivery of Artificial Micro RNAs Using Helper-Dependent Adenoviral Vectors.

    PubMed

    Crowther, Carol; Mowa, Betty; Arbuthnot, Patrick

    2016-01-01

    The potential of RNA interference (RNAi)-based gene therapy has been demonstrated in many studies. However, clinical application of this technology has been hampered by a paucity of efficient and safe methods of delivering the RNAi activators. Prolonged transgene expression and improved safety of helper-dependent adenoviral vectors (HD AdVs) makes them well suited to delivery of engineered artificial intermediates of the RNAi pathway. Also, AdVs' natural hepatotropism makes them potentially useful for liver-targeted gene delivery. HD AdVs may be used for efficient delivery of cassettes encoding short hairpin RNAs and artificial primary microRNAs to the mouse liver. Methods for the characterization of HD AdV-mediated delivery of hepatitis B virus-targeting RNAi activators are described here. PMID:26472456

  7. Magnetically Responsive Biodegradable Nanoparticles Enhance Adenoviral Gene Transfer in Cultured Smooth Muscle and Endothelial Cells

    PubMed Central

    Chorny, Michael; Fishbein, Ilia; Alferiev, Ivan; Levy, Robert J.

    2012-01-01

    Replication-defective adenoviral (Ad) vectors have shown promise as a tool for gene delivery-based therapeutic applications. Their clinical use is however limited by therapeutically suboptimal transduction levels in cell types expressing low levels of Coxsackie-Ad receptor (CAR), the primary receptor responsible for the cell entry of the virus, and by systemic adverse reactions. Targeted delivery achievable with Ad complexed with biodegradable magnetically responsive nanoparticles (MNP) may therefore be instrumental for improving both the safety and efficiency of these vectors. Our hypothesis was that magnetically driven delivery of Ad affinity-bound to biodegradable MNP can substantially increase transgene expression in CAR deficient vascular cells in culture. Fluorescently labeled MNP were formulated from polylactide with inclusion of iron oxide and surface-modified with the D1 domain of CAR as an affinity linker. MNP cellular uptake and GFP reporter transgene expression were assayed fluorimetrically in cultured endothelial and smooth muscle cells using λex/λem of 540 nm/575 nm and 485 nm/535 nm, respectively. Stable vector-specific association of Ad with MNP resulted in formation of MNP–Ad complexes displaying rapid cell binding kinetics following a brief exposure to a high gradient magnetic field with resultant gene transfer levels significantly increased compared to free vector or nonmagnetic control treatment. Multiple regression analysis suggested a mechanism of MNP–Ad mediated transduction distinct from that of free Ad, and confirmed the major contribution of the complexes to the gene transfer under magnetic conditions. The magnetically enhanced transduction was achieved without compromising the cell viability or growth kinetics. The enhancement of adenoviral gene delivery by affinity complexation with biodegradable MNP represents a promising approach with a potential to extend the applicability of the viral gene therapeutic strategies. PMID:19496618

  8. Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference

    PubMed Central

    CAO, Huibi; WANG, Anan; MARTIN, Bernard; KOEHLER, David R.; ZEITLIN, Pamela L.; TANAWELL, A. Keith; HU, Jim

    2015-01-01

    Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr−/−; C38, Cftr-corrected) stimulated with TNF-α, IL-1β or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels of IκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases. PMID:15740640

  9. Adenoviral vectors elicit humoral immunity against variable loop 2 of clade C HIV-1 gp120 via "Antigen Capsid-Incorporation" strategy.

    PubMed

    Gu, Linlin; Krendelchtchikova, Valentina; Krendelchtchikov, Alexandre; Farrow, Anitra L; Derdeyn, Cynthia A; Matthews, Qiana L

    2016-01-01

    Adenoviral (Ad) vectors in combination with the "Antigen Capsid-Incorporation" strategy have been applied in developing HIV-1 vaccines, due to the vectors׳ abilities in incorporating and inducing immunity of capsid-incorporated antigens. Variable loop 2 (V2)-specific antibodies were suggested in the RV144 trial to correlate with reduced HIV-1 acquisition, which highlights the importance of developing novel HIV-1 vaccines by targeting the V2 loop. Therefore, the V2 loop of HIV-1 has been incorporated into the Ad capsid protein. We generated adenovirus serotype 5 (Ad5) vectors displaying variable loop 2 (V2) of HIV-1 gp120, with the "Antigen Capsid-Incorporation" strategy. To assess the incorporation capabilities on hexon hypervariable region1 (HVR1) and protein IX (pIX), 20aa or full length (43aa) of V2 and V1V2 (67aa) were incorporated, respectively. Immunizations with the recombinant vectors significantly generated antibodies against both linear and discontinuous V2 epitopes. The immunizations generated durable humoral immunity against V2. This study will lead to more stringent development of various serotypes of adenovirus-vectored V2 vaccine candidates, based on breakthroughs regarding the immunogenicity of V2. PMID:26499044

  10. Adenoviral vectors elicit humoral immunity against variable loop 2 of clade C HIV-1 gp120 via “Antigen Capsid-Incorporation” strategy

    PubMed Central

    Gu, Linlin; Krendelchtchikova, Valentina; Krendelchtchikov, Alexandre; Farrow, Anitra L.; Derdeyn, Cynthia A.; Matthews, Qiana L.

    2016-01-01

    Adenoviral (Ad) vectors in combination with the “Antigen Capsid-Incorporation” strategy have been applied in developing HIV-1 vaccines, due to the vectors’ abilities in incorporating and inducing immunity of capsid-incorporated antigens. Variable loop 2 (V2)-specific antibodies were suggested in the RV144 trial to correlate with reduced HIV-1 acquisition, which highlights the importance of developing novel HIV-1 vaccines by targeting the V2 loop. Therefore, the V2 loop of HIV-1 has been incorporated into the Ad capsid protein. We generated adenovirus serotype 5 (Ad5) vectors displaying variable loop 2 (V2) of HIV-1 gp120, with the “Antigen Capsid-Incorporation” strategy. To assess the incorporation capabilities on hexon hypervariable region1 (HVR1) and protein IX (pIX), 20aa or full length (43aa) of V2 and V1V2 (67aa) were incorporated, respectively. Immunizations with the recombinant vectors significantly generated antibodies against both linear and discontinuous V2 epitopes. The immunizations generated durable humoral immunity against V2. This study will lead to more stringent development of various serotypes of adenovirus-vectored V2 vaccine candidates, based on breakthroughs regarding the immunogenicity of V2. PMID:26499044

  11. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine

    PubMed Central

    Nielsen, Morten A.; Resende, Mafalda; de Jongh, Willem A.; Ditlev, Sisse B.; Mordmüller, Benjamin; Houard, Sophie; Ndam, Nicaise Tuikue; Agerbæk, Mette Ø.; Hamborg, Mette; Massougbodji, Achille; Issifou, Saddou; Strøbæk, Anette; Poulsen, Lars; Leroy, Odile; Kremsner, Peter G.; Chippaux, Jean-Philippe; Luty, Adrian J. F.; Deloron, Philippe; Theander, Thor G.; Dyring, Charlotte; Salanti, Ali

    2015-01-01

    The disease caused by Plasmodium falciparum (Pf) involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM) is one such manifestation in which Pf infected erythrocytes (IE) bind to chondroitin sulphate A (CSA) through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a

  12. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine.

    PubMed

    Nielsen, Morten A; Resende, Mafalda; de Jongh, Willem A; Ditlev, Sisse B; Mordmüller, Benjamin; Houard, Sophie; Ndam, Nicaise Tuikue; Agerbæk, Mette Ø; Hamborg, Mette; Massougbodji, Achille; Issifou, Saddou; Strøbæk, Anette; Poulsen, Lars; Leroy, Odile; Kremsner, Peter G; Chippaux, Jean-Philippe; Luty, Adrian J F; Deloron, Philippe; Theander, Thor G; Dyring, Charlotte; Salanti, Ali

    2015-01-01

    The disease caused by Plasmodium falciparum (Pf) involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM) is one such manifestation in which Pf infected erythrocytes (IE) bind to chondroitin sulphate A (CSA) through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a

  13. Surmounting limited gene delivery into primary immune cell populations: Efficient cell type-specific adenoviral transduction by CAR.

    PubMed

    Clausen, Björn E; Brand, Anna; Karram, Khalad

    2015-06-01

    Ectopic gene expression studies in primary immune cells have been notoriously difficult to perform due to the limitations in conventional transfection and viral transduction methods. Although replication-defective adenoviruses provide an attractive alternative for gene delivery, their use has been hampered by the limited susceptibility of murine leukocytes to adenoviral infection, due to insufficient expression of the human coxsackie/adenovirus receptor (CAR). In this issue of the European Journal of Immunology, Heger et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] report the generation of transgenic mice that enable conditional Cre/loxP-mediated expression of human CAR. The authors demonstrate that this R26/CAG-CAR∆1(StopF) mouse strain facilitates the faithful monitoring of Cre activity in situ as well as the specific and efficient adenoviral transduction of primary immune cell populations in vitro. Further tweaking of the system towards more efficient gene transfer in vivo remains a future challenge. PMID:25903647

  14. A novel Cre recombinase reporter mouse strain facilitates selective and efficient infection of primary immune cells with adenoviral vectors.

    PubMed

    Heger, Klaus; Kober, Maike; Rieß, David; Drees, Christoph; de Vries, Ingrid; Bertossi, Arianna; Roers, Axel; Sixt, Michael; Schmidt-Supprian, Marc

    2015-06-01

    Replication-deficient recombinant adenoviruses are potent vectors for the efficient transient expression of exogenous genes in resting immune cells. However, most leukocytes are refractory to efficient adenoviral transduction as they lack expression of the coxsackie/adenovirus receptor (CAR). To circumvent this obstacle, we generated the R26/CAG-CARΔ1(StopF) (where R26 is ROSA26 and CAG is CMV early enhancer/chicken β actin promoter) knock-in mouse line. This strain allows monitoring of in situ Cre recombinase activity through expression of CARΔ1. Simultaneously, CARΔ1 expression permits selective and highly efficient adenoviral transduction of immune cell populations, such as mast cells or T cells, directly ex vivo in bulk cultures without prior cell purification or activation. Furthermore, we show that CARΔ1 expression dramatically improves adenoviral infection of in vitro differentiated conventional and plasmacytoid dendritic cells (DCs), basophils, mast cells, as well as Hoxb8-immortalized hematopoietic progenitor cells. This novel dual function mouse strain will hence be a valuable tool to rapidly dissect the function of specific genes in leukocyte physiology. PMID:25787118

  15. SR-A and SREC-I Are Kupffer and Endothelial Cell Receptors for Helper-dependent Adenoviral Vectors

    PubMed Central

    Piccolo, Pasquale; Vetrini, Francesco; Mithbaokar, Pratibha; Grove, Nathan C; Bertin, Terry; Palmer, Donna; Ng, Philip; Brunetti-Pierri, Nicola

    2013-01-01

    Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes with no chronic toxicity. However, a toxic acute response with potentially lethal consequences has hindered their clinical applications. Liver sinusoidal endothelial cells (LSECs) and Kupffer cells are major barriers to efficient hepatocyte transduction. Understanding the mechanisms of adenoviral vector uptake by non-parenchymal cells may allow the development of strategies aimed at overcoming these important barriers and to achieve preferential hepatocyte gene transfer with reduced toxicity. Scavenger receptors on Kupffer cells bind adenoviral particles and remove them from the circulation, thus preventing hepatocyte transduction. In the present study, we show that HDAd particles interact in vitro and in vivo with scavenger receptor-A (SR-A) and with scavenger receptor expressed on endothelial cells-I (SREC-I) and we exploited this knowledge to increase the efficiency of hepatocyte transduction by HDAd vectors in vivo through blocking of SR-A and SREC-I with specific fragments antigen-binding (Fabs). PMID:23358188

  16. Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis

    PubMed Central

    Dolzhikova, Inna V.; Shcherbinin, Dmitry N.; Zubkova, Olga V.; Ivanova, Tatiana I.; Tukhvatulin, Amir I.; Shmarov, Maxim M.; Logunov, Denis Y.; Naroditsky, Boris S.; Gintsburg, Aleksandr L.

    2016-01-01

    Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection. PMID:26962869

  17. Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis.

    PubMed

    Burmistrova, Daria A; Tillib, Sergey V; Shcheblyakov, Dmitry V; Dolzhikova, Inna V; Shcherbinin, Dmitry N; Zubkova, Olga V; Ivanova, Tatiana I; Tukhvatulin, Amir I; Shmarov, Maxim M; Logunov, Denis Y; Naroditsky, Boris S; Gintsburg, Aleksandr L

    2016-01-01

    Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection. PMID:26962869

  18. Differential Type I Interferon-dependent Transgene Silencing of Helper-dependent Adenoviral vs. Adeno-associated Viral Vectors In Vivo

    PubMed Central

    Suzuki, Masataka; Bertin, Terry K; Rogers, Geoffrey L; Cela, Racel G; Zolotukhin, Irene; Palmer, Donna J; Ng, Philip; Herzog, Roland W; Lee, Brendan

    2013-01-01

    We previously dissected the components of the innate immune response to Helper-dependent adenoviral vectors (HDAds) using genetic models, and demonstrated that multiple pattern recognition receptor signaling pathways contribute to this host response to HDAds in vivo. Based on analysis of cytokine expression profiles, type I interferon (IFN) mRNA is induced in host mouse livers at 1 hour post-injection. This type I IFN signaling amplifies cytokine expression in liver independent of the nature of vector DNA sequences after 3 hours post-injection. This type I IFN signaling in response to HDAds administration contributes to transcriptional silencing of both HDAd prokaryotic and eukaryotic DNA in liver. This silencing occurs early and is mediated by epigenetic modification as shown by in vivo chromatin immunoprecipitation (ChIP) with anti-histone deacetylase (HDAC) and promyelocytic leukemia protein (PML). In contrast, self-complementary adeno-associated viral vectors (scAAVs) showed significantly lower induction of type I IFN mRNA in liver compared to HDAds at both early and late time points. These results show that the type I IFN signaling dependent transgene silencing differs between AAV and HDAd vectors after liver-directed gene transfer. PMID:23319058

  19. The route of immunization with adenoviral vaccine influences the recruitment of cytotoxic T lymphocytes in the lung that provide potent protection from influenza A virus.

    PubMed

    Suda, Tatsuya; Kawano, Masaaki; Nogi, Yasuhisa; Ohno, Naohito; Akatsuka, Toshitaka; Matsui, Masanori

    2011-09-01

    Virus-specific cytotoxic T lymphocytes (CTLs) in the lung are considered to confer protection from respiratory viruses. Several groups demonstrated that the route of priming was likely to have an implication for the trafficking of antigen-specific CTLs. Therefore, we investigated whether the route of immunization with adenoviral vaccine influenced the recruitment of virus-specific CTLs in the lung that should provide potent protection from influenza A virus. Mice were immunized with recombinant adenovirus expressing the matrix (M1) protein of influenza A virus via various immunization routes involving intraperitoneal, intranasal, intramuscular, or intravenous administration as well as subcutaneous administration in the hind hock. We found that the immunization route dramatically impacted the recruitment of M1-specific IFN-γ(+) CD8(+) T cells both in the lung and the spleen. Surprisingly, hock immunization was most effective for the accumulation in the lung of IFN-γ-producing CD8(+) T cells that possessed M1-specific cytolytic activity. Further, antigen-driven IFN-γ(+) CD8(+) T cells in the lung, but not in the spleen, were likely to be correlated with the resistance to challenge with influenza A virus. These results may improve our ability to design vaccines that target virus-specific CTL responses to respiratory viruses such as influenza A virus. PMID:21722671

  20. Intranasal immunization with a replication-deficient adenoviral vector expressing the fusion glycoprotein of respiratory syncytial virus elicits protective immunity in BALB/c mice

    SciTech Connect

    Fu, Yuanhui; He, Jinsheng; Zheng, Xianxian; Wu, Qiang; Zhang, Mei; Wang, Xiaobo; Wang, Yan; Xie, Can; Tang, Qian; Wei, Wei; Wang, Min; Song, Jingdong; Qu, Jianguo; Zhang, Ying; Wang, Xin; Hong, Tao

    2009-04-17

    Human respiratory syncytial virus (RSV) is a serious pediatric pathogen of the lower respiratory tract worldwide. There is currently no clinically approved vaccine against RSV infection. Recently, it has been shown that a replication-deficient first generation adenoviral vector (FGAd), which encodes modified RSV attachment glycoprotein (G), elicits long-term protective immunity against RSV infection in mice. The major problem in developing such a vaccine is that G protein lacks MHC-I-restricted epitopes. However, RSV fusion glycoprotein (F) is a major cytotoxic T-lymphocyte epitope in humans and mice, therefore, an FGAd-encoding F (FGAd-F) was constructed and evaluated for its potential as an RSV vaccine in a murine model. Intranasal (i.n.) immunization with FGAd-F generated serum IgG, bronchoalveolar lavage secretory IgA, and RSV-specific CD8+ T-cell responses in BALB/c mice, with characteristic balanced or mixed Th1/Th2 CD4+ T-cell responses. Serum IgG was significantly elevated after boosting with i.n. FGAd-F. Upon challenge, i.n. immunization with FGAd-F displayed an effective protective role against RSV infection. These results demonstrate FGAd-F is able to induce effective protective immunity and is a promising vaccine regimen against RSV infection.

  1. Highly efficient adenoviral transduction of pancreatic islets using a microfluidic device.

    PubMed

    Silva, Pamuditha N; Atto, Zaid; Regeenes, Romario; Tufa, Uilki; Chen, Yih Yang; Chan, Warren C W; Volchuk, Allen; Kilkenny, Dawn M; Rocheleau, Jonathan V

    2016-08-01

    Tissues are challenging to genetically manipulate due to limited penetration of viral particles resulting in low transduction efficiency. We are particularly interested in expressing genetically-encoded sensors in ex vivo pancreatic islets to measure glucose-stimulated metabolism, however poor viral penetration biases these measurements to only a subset of cells at the periphery. To increase mass transfer of viral particles, we designed a microfluidic device that holds islets in parallel hydrodynamic traps connected by an expanding by-pass channel. We modeled viral particle flow into the tissue using fluorescently-labelled gold nanoparticles of varying sizes and showed a penetration threshold of only ∼5 nm. To increase this threshold, we used EDTA to transiently reduce cell-cell adhesion and expand intercellular space. Ultimately, a combination of media flow and ETDA treatment significantly increased adenoviral transduction to the core of the islet. As proof-of-principle, we used this protocol to transduce an ER-targeted redox sensitive sensor (eroGFP), and revealed significantly greater ER redox capacity at core islet cells. Overall, these data demonstrate a robust method to enhance transduction efficiency of islets, and potentially other tissues, by using a combination of microfluidic flow and transient tissue expansion. PMID:27378588

  2. PCR diagnostics and monitoring of adenoviral infections in hematopoietic stem cell transplantation recipients.

    PubMed

    Bil-Lula, Iwona; Ussowicz, Marek; Rybka, Blanka; Wendycz-Domalewska, Danuta; Ryczan, Renata; Gorczyńska, Ewa; Kałwak, Krzysztof; Woźniak, Mieczysław

    2010-12-01

    After stem cell transplantation, human patients are prone to life-threatening opportunistic infections with a plethora of microorganisms. We report a retrospective study on 116 patients (98 children, 18 adults) who were transplanted in a pediatric bone marrow transplantation unit. Blood, urine and stool samples were collected and monitored for adenovirus (AdV) DNA using polymerase chain reaction (PCR) and real-time PCR (RT-PCR) on a regular basis. AdV DNA was detected in 52 (44.8%) patients, with mortality reaching 19% in this subgroup. Variables associated with adenovirus infection were transplantations from matched unrelated donors and older age of the recipient. An increased seasonal occurrence of adenoviral infections was observed in autumn and winter. Analysis of immune reconstitution showed a higher incidence of AdV infections during periods of low T-lymphocyte count. This study also showed a strong interaction between co-infections of AdV and BK polyomavirus in patients undergoing hematopoietic stem cell transplantations. PMID:20848295

  3. Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1.

    PubMed

    Castello, R; Borzone, R; D'Aria, S; Annunziata, P; Piccolo, P; Brunetti-Pierri, N

    2016-02-01

    Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT), which catalyzes conversion of glyoxylate into glycine. AGT deficiency results in overproduction of oxalate that ultimately leads to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only available therapy to prevent disease progression. Gene therapy is an attractive option to provide an alternative treatment for PH1. Toward this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. Compared with saline controls, AGT-deficient mice injected with an HDAd encoding the AGT under the control of a liver-specific promoter showed a significant reduction of hyperoxaluria and less increase of urinary oxalate following challenge with ethylene glycol, a precursor of glyoxylate. These studies may thus pave the way to clinical application of HDAd for PH1 gene therapy. PMID:26609667

  4. Factors involved in the maturation of murine dendritic cells transduced with adenoviral vector variants

    SciTech Connect

    Kanagawa, Naoko; Koretomo, Ryosuke; Murakami, Sayaka |; Sakurai, Fuminori; Mizuguchi, Hiroyuki |; Nakagawa, Shinsaku; Fujita, Takuya |; Yamamoto, Akira; Okada, Naoki |

    2008-05-10

    Adenoviral vector (Ad)-mediated gene transfer is an attractive method for manipulating the immunostimulatory properties of dendritic cells (DCs) for cancer immunotherapy. DCs treated with Ad have phenotype alterations (maturation) that facilitate T cell sensitization. We investigated the mechanisms of DC maturation with Ad transduction. Expression levels of a maturation marker (CD40) on DCs treated with conventional Ad, fiber-modified Ads (AdRGD, AdF35, AdF35{delta}RGD), or a different serotype Ad (Ad35) were correlated with their transduction efficacy. The {alpha}{sub v}-integrin directional Ad, AdRGD, exhibited the most potent ability to enhance both foreign gene expression and CD40 expression, and induced secretion of interleukin-12, tumor necrosis factor-{alpha}, and interferon-{alpha} in DCs. The presence of a foreign gene expression cassette in AdRGD was not necessary for DC maturation. Maturation of DCs treated with AdRGD was suppressed by destruction of the Ad genome, inhibition of endocytosis, or endosome acidification, whereas proteasome inhibition increased CD40 expression levels on DCs. Moreover, inhibition of {alpha}{sub v}-integrin signal transduction and blockade of cytokine secretion affected the maturation of DCs treated with AdRGD only slightly or not at all, respectively. Thus, our data provide evidence that Ad-induced DC maturation is due to Ad invasion of the DCs, followed by nuclear transport of the Ad genome, and not to the expression of foreign genes.

  5. PCR diagnostics and monitoring of adenoviral infections in hematopoietic stem cell transplantation recipients

    PubMed Central

    Ussowicz, Marek; Rybka, Blanka; Wendycz-Domalewska, Danuta; Ryczan, Renata; Gorczyńska, Ewa; Kałwak, Krzysztof; Woźniak, Mieczysław

    2010-01-01

    After stem cell transplantation, human patients are prone to life-threatening opportunistic infections with a plethora of microorganisms. We report a retrospective study on 116 patients (98 children, 18 adults) who were transplanted in a pediatric bone marrow transplantation unit. Blood, urine and stool samples were collected and monitored for adenovirus (AdV) DNA using polymerase chain reaction (PCR) and real-time PCR (RT-PCR) on a regular basis. AdV DNA was detected in 52 (44.8%) patients, with mortality reaching 19% in this subgroup. Variables associated with adenovirus infection were transplantations from matched unrelated donors and older age of the recipient. An increased seasonal occurrence of adenoviral infections was observed in autumn and winter. Analysis of immune reconstitution showed a higher incidence of AdV infections during periods of low T-lymphocyte count. This study also showed a strong interaction between co-infections of AdV and BK polyomavirus in patients undergoing hematopoietic stem cell transplantations. PMID:20848295

  6. Evaluation of CD46 re-targeted adenoviral vectors for clinical ovarian cancer intraperitoneal therapy.

    PubMed

    Hulin-Curtis, S L; Uusi-Kerttula, H; Jones, R; Hanna, L; Chester, J D; Parker, A L

    2016-07-01

    Ovarian cancer accounts for >140 000 deaths globally each year. Typically, disease is asymptomatic until an advanced, incurable stage. Although response to cytotoxic chemotherapy is frequently observed, resistance to conventional platinum-based therapies develop rapidly. Improved treatments are therefore urgently required. Virotherapy offers great potential for ovarian cancer, where the application of local, intraperitoneal delivery circumvents some of the limitations of intravenous strategies. To develop effective, adenovirus (Ad)-based platforms for ovarian cancer, we profiled the fluid and cellular components of patient ascites for factors known to influence adenoviral transduction. Levels of factor X (FX) and neutralizing antibodies (nAbs) in ascitic fluid were quantified and tumor cells were assessed for the expression of coxsackie virus and adenovirus receptor (CAR) and CD46. We show that clinical ascites contains significant levels of FX but consistently high CD46 expression. We therefore evaluated in vitro the relative transduction of epithelial ovarian cancers (EOCs) by Ad5 (via CAR) and Ad5 pseudotyped with the fiber of Ad35 (Ad5T*F35++) via CD46. Ad5T*F35++ achieved significantly increased transduction in comparison to Ad5 (P<0.001), independent of FX and nAb levels. We therefore propose selective transduction of CD46 over-expressing EOCs using re-targeted, Ad35-pseudotyped Ad vectors may represent a promising virotherapy for ovarian cancer. PMID:27229159

  7. Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1

    PubMed Central

    Castello, Raffaele; Borzone, Roberta; D’Aria, Stefania; Annunziata, Patrizia; Piccolo, Pasquale; Brunetti-Pierri, Nicola

    2015-01-01

    Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT) which catalyzes conversion of glyoxylate into glycine. AGT deficiency results in overproduction of oxalate which ultimately leads to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only available therapy to prevent disease progression. Gene therapy is an attractive option to provide an alternative treatment for PH1. Towards this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. Compared to saline controls, AGT-deficient mice injected with an HDAd encoding the AGT under the control of a liver-specific promoter showed a significant reduction of hyperoxaluria and less increase of urinary oxalate following challenge with Ethylene Glycol (EG), a precursor of glyoxylate. These studies may thus pave the way to clinical application of HDAd for PH1 gene therapy. PMID:26609667

  8. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

    PubMed

    Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R

    2003-10-01

    High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle. PMID:12960972

  9. Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir.

    PubMed

    Pozzuto, Tanja; Röger, Carsten; Kurreck, Jens; Fechner, Henry

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs. PMID:26026665

  10. Production of first generation adenoviral vectors for preclinical protocols: amplification, purification and functional titration.

    PubMed

    Armendáriz-Borunda, Juan; Bastidas-Ramírez, Blanca Estela; Sandoval-Rodríguez, Ana; González-Cuevas, Jaime; Gómez-Meda, Belinda; García-Bañuelos, Jesús

    2011-11-01

    Gene therapy represents a promising approach in the treatment of several diseases. Currently, the ideal vector has yet to be designed; though, adenoviral vectors (Ad-v) have provided the most utilized tool for gene transfer due principally to their simple production, among other specific characteristics. Ad-v viability represents a critical variable that may be affected by storage or shipping conditions and therefore it is advisable to be assessed previously to protocol performance. The present work is unique in this matter, as the complete detailed process to obtain Ad-v of preclinical grade is explained. Amplification in permissive HEK-293 cells, purification in CsCl gradients in a period of 10 h, spectrophotometric titration of viral particles (VP) and titration of infectious units (IU), yielding batches of AdβGal, AdGFP, AdHuPA and AdMMP8, of approximately 10¹³-10¹⁴ VP and 10¹²-10¹³ IU were carried out. In vivo functionality of therapeutic AdHuPA and AdMMP8 was evidenced in rats presenting CCl₄-induced fibrosis, as more than 60% of fibrosis was eliminated in livers after systemic delivery through iliac vein in comparison with irrelevant AdβGal. Time required to accomplish the whole Ad-v production steps, including IU titration was 20 to 30 days. We conclude that production of Ad-v following standard operating procedures assuring vector functionality and the possibility to effectively evaluate experimental gene therapy results, leaving aside the use of high-cost commercial kits or sophisticated instrumentation, can be performed in a conventional laboratory of cell culture. PMID:21856222

  11. Retrograde optogenetic characterization of the pontospinal module of the locus coeruleus with a canine adenoviral vector.

    PubMed

    Li, Yong; Hickey, Louise; Perrins, Ray; Werlen, Emilie; Patel, Amisha A; Hirschberg, Stefan; Jones, Matt W; Salinas, Sara; Kremer, Eric J; Pickering, Anthony E

    2016-06-15

    Noradrenergic neurons of the brainstem extend projections throughout the neuraxis to modulate a wide range of processes including attention, arousal, autonomic control and sensory processing. A spinal projection from the locus coeruleus (LC) is thought to regulate nociceptive processing. To characterize and selectively manipulate the pontospinal noradrenergic neurons in rats, we implemented a retrograde targeting strategy using a canine adenoviral vector to express channelrhodopsin2 (CAV2-PRS-ChR2-mCherry). LC microinjection of CAV2-PRS-ChR2-mCherry produced selective, stable, transduction of noradrenergic neurons allowing reliable opto-activation in vitro. The ChR2-transduced LC neurons were opto-identifiable in vivo and functional control was demonstrated for >6 months by evoked sleep-wake transitions. Spinal injection of CAV2-PRS-ChR2-mCherry retrogradely transduced pontine noradrenergic neurons, predominantly in the LC but also in A5 and A7. A pontospinal LC (ps:LC) module was identifiable, with somata located more ventrally within the nucleus and with a discrete subset of projection targets. These ps:LC neurons had distinct electrophysiological properties with shorter action potentials and smaller afterhyperpolarizations compared to neurons located in the core of the LC. In vivo recordings of ps:LC neurons showed a lower spontaneous firing frequency than those in the core and they were all excited by noxious stimuli. Using this CAV2-based approach we have demonstrated the ability to retrogradely target, characterise and optogenetically manipulate a central noradrenergic circuit and show that the ps:LC module forms a discrete unit. This article is part of a Special Issue entitled SI: Noradrenergic System. PMID:26903420

  12. Comparison of Efficacy of Two Different Topical 0.05% Cyclosporine A Formulations in the Treatment of Adenoviral Keratoconjunctivitis-Related Subepithelial Infiltrates

    PubMed Central

    Bayraktutar, Betül N.; Uçakhan, Ömur Ö.

    2016-01-01

    Subepithelial infiltrates secondary to adenoviral keratoconjunctivitis may persist for years and cause blurred vision, halos, glare, and photophobia. These infiltrates arise from immune reaction against the virus, and few studies have reported topical cyclosporine A to be effective in the treatment of subepithelial infiltrates. Herein, we describe a patient with adenoviral keratoconjunctivitis-related subepithelial infiltrates who did not respond to treatment with a new topical cyclosporine A emulsion prepared with castor oil (Depores 0.05%; Deva İlaç, Kocaeli, Turkey), while the FDA-approved nanoemulsion formulation provided improvement in symptoms and reduced the inflammatory reaction (Restasis 0.05%; Allergan, Irvine, Calif., USA). PMID:27065851

  13. Comparison of Efficacy of Two Different Topical 0.05% Cyclosporine A Formulations in the Treatment of Adenoviral Keratoconjunctivitis-Related Subepithelial Infiltrates.

    PubMed

    Bayraktutar, Betül N; Uçakhan, Ömur Ö

    2016-01-01

    Subepithelial infiltrates secondary to adenoviral keratoconjunctivitis may persist for years and cause blurred vision, halos, glare, and photophobia. These infiltrates arise from immune reaction against the virus, and few studies have reported topical cyclosporine A to be effective in the treatment of subepithelial infiltrates. Herein, we describe a patient with adenoviral keratoconjunctivitis-related subepithelial infiltrates who did not respond to treatment with a new topical cyclosporine A emulsion prepared with castor oil (Depores 0.05%; Deva İlaç, Kocaeli, Turkey), while the FDA-approved nanoemulsion formulation provided improvement in symptoms and reduced the inflammatory reaction (Restasis 0.05%; Allergan, Irvine, Calif., USA). PMID:27065851

  14. Amelioration of carbon tetrachloride-induced cirrhosis and portal hypertension in rat using adenoviral gene transfer of Akt

    PubMed Central

    Deng, Gang; Huang, Xiang-Jun; Luo, Hong-Wu; Huang, Fei-Zhou; Liu, Xun-Yang; Wang, Yong-Heng

    2013-01-01

    AIM: To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride (CCl4). METHODS: Using cre-loxp technique, we created an Ad-myr-HA-Akt virus, in which Akt is labeled by a HA tag and its expression is driven by myr promoter. Further, through measuring enzyme levels and histological structure, we determined the efficacy of this Ad-myr-HA-Akt virus in inhibiting the development of cirrhosis induced by CCl4 in rats. Lastly, using western blotting, we examined the expression levels and/or phosphorylation status of Akt, apoptotic mediators, endothelial nitric oxide synthase (eNOS), and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus. RESULTS: The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment, which was consistent with the sequence reported in the GenBank. The concentrations of Ad-myr-HA-Akt and adenoviral enhanced green fluorescent protein (Ad-EGFP) virus used in the current study were 5.5 × 1011 vp/mL. The portal vein diameter, peak velocity of blood flow, portal blood flow and congestion index were significantly increased in untreated, saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection. In contrast, these parameters in the Akt cirrhosis group were comparable to normal control group. Compared to the normal control, the liver function (Alanine aminotransferase, Aspartate aminotransferase and Albumin) was significantly impaired in the untreated, saline and Ad-EGFP cirrhosis groups. The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated, saline and Ad-EGFP cirrhosis groups. The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups

  15. Standard Free Droplet Digital Polymerase Chain Reaction as a New Tool for the Quality Control of High-Capacity Adenoviral Vectors in Small-Scale Preparations

    PubMed Central

    Boehme, Philip; Stellberger, Thorsten; Solanki, Manish; Zhang, Wenli; Schulz, Eric; Bergmann, Thorsten; Liu, Jing; Doerner, Johannes; Baiker, Armin E.

    2015-01-01

    Abstract High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy. PMID:25640117

  16. Standard free droplet digital polymerase chain reaction as a new tool for the quality control of high-capacity adenoviral vectors in small-scale preparations.

    PubMed

    Boehme, Philip; Stellberger, Thorsten; Solanki, Manish; Zhang, Wenli; Schulz, Eric; Bergmann, Thorsten; Liu, Jing; Doerner, Johannes; Baiker, Armin E; Ehrhardt, Anja

    2015-02-01

    High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy. PMID:25640117

  17. A reproducible and quantifiable model of choroidal neovascularization induced by VEGF A165 after subretinal adenoviral gene transfer in the rabbit

    PubMed Central

    Kreppel, Florian; Beck, Susanne; Heiduschka, Peter; Brito, Veronica; Schnichels, Sven; Kochanek, Stefan; Schraermeyer, Ulrich

    2008-01-01

    Purpose To determine the effects of the vascular endothelial growth factor (VEGF)-A165 delivered using a high capacity adenoviral vector (HC Ad.VEGF-A) on vascular growth and pathological changes in the rabbit eye. To combine different detection methods of VEGF-A165 overexpression-induced neovascularization in the rabbit. Methods HC Ad.VEGF-A165 was constructed and injected at 5x106 infectious units (iu) into the subretinal space of rabbit eyes. Two and four weeks postinjection, the development of neovascularization and the expression of HC Ad-transduced VEGF-A165 protein were followed up in vivo by scanning laser ophthalmoscopy, fluorescein and indocyanine green angiographies and ex vivo by electron microscopy and immunohistochemistry Results We observed a choroidal neovascularization (CNV) with leakage in 83% of the rabbit eyes. Our findings present clear indications that there is a significant effect on the endothelial cells of the choriocapillaris after subretinal transduction of the retinal pigment epithelium (RPE) with VEGF-A165 vector. The choroidal endothelial cells were activated, adherent junctions opened, and the fenestration was minimized, while the extracellular matrix localized between the RPE and the endothelium of the choriocapillaris was enlarged toward the lumen of the vessels, inducing a deep invagination of the endothelial cells into the vessel lumen. They also proliferated and formed pathological vessels in the subretinal space. Moreover,there was an increased expression of basic fibroblast growth factor and VEGF-A accompanied by macrophage stimulation, retinal edema, and photoreceptor loss. Conclusions This is the first model of VEGF-induced CNV in the rabbit in which the pathological events following overexpression of VEGF by RPE cells have been described in detail. Many of the features of our experimental CNV resemble those observed clinically in patients having wet age-related macular degeneration. PMID:18682809

  18. Helper virus-mediated downregulation of transgene expression permits production of recalcitrant helper-dependent adenoviral vector

    PubMed Central

    Palmer, Donna J; Grove, Nathan C; Ng, Philip

    2016-01-01

    Helper-dependent adenoviral vectors (HDAd) that express certain transgene products are impossible to produce because the transgene product is toxic to the producer cells, especially when made in large amounts during vector production. Downregulating transgene expression from the HDAd during vector production is a way to solve this problem. In this report, we show that this can be accomplished by inserting the target sequence for the adenoviral VA RNAI into the 3’ untranslated region of the expression cassette in the HDAd. Thus during vector production, when the producer cells are coinfected with both the helper virus (HV) and the HDAd, the VA RNAI produced by the HV will target the transgene mRNA from the HDAd via the endogenous cellular RNAi pathway. Once the HDAd is produced and purified, transduction of the target cells results in unimpeded transgene expression because of the absence of HV. This simple and universal strategy permits for the robust production of otherwise recalcitrant HDAds. PMID:27331077

  19. STANDARDIZATION AND VALIDATION OF ADENOVIRAL TRANSDUCTION OF AN ANDROGEN RECEPTOR POSITIVE CELL LINE WITH AN MMTV-LUC REPORTER FOR ENDOCRINE SCREENING

    EPA Science Inventory

    Standardization and Validation of Adenoviral Transduction of an Androgen Receptor Positive Cell Line with an MMTV-Luc Reporter for Endocrine Screening P. Hartig, K . Bobseine,
    M. Cardon, C. Lambright and L. E. Gray, Jr. USEPA, Reproductive Toxicology Division, NHEERL, RTP, NC...

  20. AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED CU,ZN-SOD AND MN-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED Cu,Zn-SOD AND Mn-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO. JB Smith1, PC Hartig3, MR Blanton3, KK Sulik1,2, and ES Hunter3. 1Department of Cell and Developmental Biology and 2Bowles Cente...

  1. Intradermal vaccination with un-adjuvanted sub-unit vaccines triggers skin innate immunity and confers protective respiratory immunity in domestic swine.

    PubMed

    Le Luduec, Jean-Benoît; Debeer, Sabine; Piras, Fabienne; Andréoni, Christine; Boudet, Florence; Laurent, Philippe; Kaiserlian, Dominique; Dubois, Bertrand

    2016-02-10

    Intradermal (ID) vaccination constitutes a promising approach to induce anti-infectious immunity. This route of immunization has mostly been studied with influenza split-virion vaccines. However, the efficacy of ID vaccination for sub-unit vaccines in relation to underlying skin innate immunity remains to be explored for wider application in humans. Relevant animal models that more closely mimic human skin immunity than the widely used mouse models are therefore necessary. Here, we show in domestic swine, which shares striking anatomic and functional properties with human skin, that a single ID delivery of pseudorabies virus (PRV) glycoproteins without added adjuvant is sufficient to trigger adaptive cellular and humoral immune responses, and to confer protection from a lethal respiratory infection with PRV. Analysis of early events at the skin injection site revealed up-regulation of pro-inflammatory cytokine and chemokine genes, recruitment of neutrophils and monocytes and accumulation of inflammatory DC. We further show that the sustained induction of pro-inflammatory cytokine genes results from the combined effects of skin puncture, liquid injection in the dermis and viral antigens. These data highlight that immune protection against respiratory infection can be induced by ID vaccination with a subunit vaccine and reveal that adjuvant requirements are circumvented by the mechanical and antigenic stress caused by ID injection, which triggers innate immunity and mobilization of inflammatory DC at the immunization site. ID vaccination with sub-unit vaccines may thus represent a safe and efficient solution for protection against respiratory infections in swine and possibly also in humans, given the similarity of skin structure and function in both species. PMID:26768129

  2. INGN 201: Ad-p53, Ad5CMV-p53, Adenoviral p53, INGN 101, p53 gene therapy--Introgen, RPR/INGN 201.

    PubMed

    2003-01-01

    undergoing phase I trials for the potential treatment of lung, breast, ovarian, bladder, liver and brain cancers. Introgen and Aventis Pharma had signed a Cooperative Research and Development Agreement (CRADA) with the National Cancer Institute (NCI). NCI will sponsor clinical trials to evaluate and develop RPR/INGN 201 as a potential anticancer agent for these cancer indications. The trials conducted under a NCI-sponsored IND will evaluate RPR/INGN 201 alone and in combination with other anticancer agents. This agreement was originally signed by Rhône-Poulenc Rorer's Gencell. Introgen has completed three phase I clinical trials with INGN 201 in patients with bronchioalveolar cell lung carcinoma, ovarian cancer and recurrent glioblastomas, respectively. Intratumoural injection of RPR/INGN 201 in patients with recurrent glioblastomas was well tolerated and resulted in expression of the p53 protein. Direct administration of RPR/INGN 201 to the lower airways of patients with bronchioalveolar cell lung carcinoma resulted in symptomatic improvement and improved lung function in some patients. In February 2003, Introgen announced that the US Patent and Trademark Office has issued to The Board of Regents of The University of Texas System, patent No. 6,511,847 entitled "Recombinant p53 Adenovirus Methods and Compositions". Introgen Therapeutics is the exclusive licensee of this patent. The patent covers any adenoviral DNA molecules that encode the p53 gene positioned under the control of a promoter. Such a DNA molecule forms the genetic core of Introgen's ADVEXIN cancer therapy. Introgen's ADVEXIN therapy is now covered by up to ten separate US patents relevant to the product including compositions, therapeutic methods of administering the product in virtually any form, alone and in conjunction with the most widely used chemotherapeutic and radiation treatments, as well as its production. Introgen has a number of US patents that relate to the clinical use of ADVEXIN in cancer as

  3. Immunogenicity without Efficacy of an Adenoviral Tuberculosis Vaccine in a Stringent Mouse Model for Immunotherapy during Treatment.

    PubMed

    Alyahya, S Anisah; Nolan, Scott T; Smith, Cara M R; Bishai, William R; Sadoff, Jerald; Lamichhane, Gyanu

    2015-01-01

    To investigate if bacterial persistence during TB drug treatment could be overcome by modulation of host immunity, we adapted a clinically-relevant model developed for the evaluation of new drugs and examined if immunotherapy with two adenoviral vaccines, Ad35-TBS (AERAS-402) and Ad26-TBS, could shorten therapy in mice. Even though immunotherapy resulted in strong splenic IFN-γ responses, no effect on bacterial replication in the lungs was seen. Multiplex assay analysis of lung samples revealed the absence of cytokine augmentation such as IFN-γ, TNF-α and IL-2, suggesting that immunization failed to induce immunity in the lungs. In this model, we show that IFN-γ levels were not associated with protection against disease relapse. The results obtained from our study raise questions regarding the traits of protective TB immunity that are relevant for the development of future immunotherapeutic and post-exposure vaccination strategies. PMID:25996375

  4. Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy

    PubMed Central

    Weymann, Alexander; Arif, Rawa; Weber, Antje; Zaradzki, Marcin; Richter, Karsten; Ensminger, Stephan; Robinson, Peter Nicholas; Wagner, Andreas H.; Karck, Matthias; Kallenbach, Klaus

    2016-01-01

    Objectives Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis. Methods We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM). Results IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1

  5. Anti-Inflammatory Effects of Modified Adenoviral Vectors for Gene Therapy: A View through Animal Models Tested.

    PubMed

    Castañeda-Lopez, M E; Garza-Veloz, I; Lopez-Hernandez, Y; Barbosa-Cisneros, O Y; Martinez-Fierro, M L

    2016-07-01

    The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases. PMID:27245510

  6. Construction and evaluation of replication-defective recombinant optimized triosephosphate isomerase adenoviral vaccination in Schistosoma japonicum challenged mice.

    PubMed

    Dai, Yang; Wang, Xiaoting; Zhao, Song; Tang, Jianxia; Zhang, Lu; Dai, Jianrong; Zeng, Mingtao; Lu, Shan; Zhu, Yinchang; Su, Chuan

    2014-02-01

    Schistosomiasis is an endemic, zoonotic parasitic disease that remains a public health concern in China. Development of transmission blocking veterinary vaccines against Schistosoma japonicum infection is urgently needed. Replication-defective adenoviral vector is an efficient vaccine delivery system that has been widely used. Its use is associated with high levels of gene insertion and expression. It is easy to construct and prepare, and is safe. It is not known whether this delivery system can improve the protective effect of schistosome vaccination. Triosephosphate isomerase from S. japonicum (SjTPI) is a promising vaccine candidate. Thus far it has induced only partial protection in animal models and needs to be further enhanced to be effective. We constructed a replication-defective adenoviral vector-based vaccine with optimized SjTPI (rAdV-SjTPI.opt). The specific immune responses and protective efficiency in mice were evaluated. Results showed that intramuscular rAdV-SjTPI.opt induced Th1 biased immune responses in the host, while subcutaneous rAdV-SjTPI.opt induced Th2 predominant immune responses. Oral rAdV-SjTPI.opt induced low levels of immune responses and no significant protection. Intramuscular rAdV-SjTPI.opt provided a consistent and repeatable higher protective effect in mice (more than 50%). These findings may be due to the associated higher levels of specific Th1, antibody responses and partially lower level of IL-17A. This report provides a foundation for developing transmission-blocking veterinary vaccines in larger animals. PMID:24397904

  7. Clinical and Antiviral Efficacy of an Ophthalmic Formulation of Dexamethasone Povidone-Iodine in a Rabbit Model of Adenoviral Keratoconjunctivitis

    PubMed Central

    Clement, Christian; Capriotti, Joseph A.; Kumar, Manish; Hobden, Jeffery A.; Foster, Timothy P.; Bhattacharjee, Partha S.; Thompson, Hilary W.; Mahmud, Rashed; Liang, Bo

    2011-01-01

    Purpose. To determine the efficacy of a new formulation of topical dexamethasone 0.1%/povidone-iodine 0.4% (FST-100) in reducing clinical symptoms and infectious viral titers in a rabbit model of adenoviral keratoconjunctivitis. Methods. Rabbit corneas were inoculated bilaterally with 2 × 106 plaque-forming-units (PFU) of adenovirus type 5 (Ad5) after corneal scarification. Animals were randomized 1:1:1:1 (five rabbits per group) to FST-100, 0.5% cidofovir, tobramycin/dexamethasone (Tobradex; Alcon Laboratories, Fort Worth, TX) ophthalmic suspension, and balanced salt solution (BSS; Alcon Laboratories). Treatment began 12 hours after viral inoculation and continued for 7 consecutive days. The eyes were clinically scored daily for scleral inflammation (injection), ocular neovascularization, eyelid inflammation (redness), friability of vasculature, inflammatory discharge (pus), and epiphora (excessive tearing). Eye swabs were collected daily before treatment for the duration of the study. Virus was eluted from the swabs and PFU determined by titration on human A549 cells, according to standard procedures. Results. The FST-100 treatment resulted in significantly lower clinical scores (P < 0.05) than did the other treatments. The 0.5% cidofovir exhibited the most ocular toxicity compared with FST-100, tobramycin/dexamethasone, and balanced salt solution treatments. FST-100 and 0.5% cidofovir significantly (P < 0.05) reduced viral titers compared with tobramycin/dexamethasone or balanced salt solution. Conclusions. FST-100 was the most efficacious in minimizing the clinical symptoms of adenovirus infection in rabbit eyes. FST-100 and 0.5% cidofovir were both equally effective in reducing viral titers and decreasing the duration of viral shedding. By providing symptomatic relief in addition to reducing infectious virus titers, FST-100 should be a valuable addition to treatment of epidemic adenoviral keratoconjunctivitis. PMID:20702820

  8. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    SciTech Connect

    Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  9. Adenoviral-Mediated Glial Cell Line–Derived Neurotrophic Factor Gene Transfer Has a Protective Effect on Sciatic Nerve Following Constriction-Induced Spinal Cord Injury

    PubMed Central

    Chou, An-Kuo; Yang, Ming-Chang; Tsai, Hung-Pei; Chai, Chee-Yin; Tai, Ming-Hong; Kwan, Aij-Li; Hong, Yi-Ren

    2014-01-01

    Neuropathic pain due to peripheral nerve injury may be associated with abnormal central nerve activity. Glial cell-line-derived neurotrophic factor (GDNF) can help attenuate neuropathic pain in different animal models of nerve injury. However, whether GDNF can ameliorate neuropathic pain in the spinal cord dorsal horn (SCDH) in constriction-induced peripheral nerve injury remains unknown. We investigated the therapeutic effects of adenoviral-mediated GDNF on neuropathic pain behaviors, microglial activation, pro-inflammatory cytokine expression and programmed cell death in a chronic constriction injury (CCI) nerve injury animal model. In this study, neuropathic pain was produced by CCI on the ipsilateral SCDH. Mechanical allodynia was examined with von Frey filaments and thermal sensitivity was tested using a plantar test apparatus post-operatively. Target proteins GDNF-1, GDNFRa-1, MMP2, MMP9, p38, phospho-p38, ED1, IL6, IL1β, AIF, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, cleaved PARP, SPECTRIN, cleaved SPECTRIN, Beclin-1, PKCσ, PKCγ, iNOS, eNOS and nNOS were detected. Microglial activity was measured by observing changes in immunoreactivity with OX-42. NeuN and TUNEL staining were used to reveal whether apoptosis was attenuated by GDNF. Results showed that administrating GDNF began to attenuate both allodynia and thermal hyperalgesia at day 7. CCI-rats were found to have lower GDNF and GDNFRa-1 expression compared to controls, and GDNF re-activated their expression. Also, GDNF significantly down-regulated CCI-induced protein expression except for MMP2, eNOS and nNOS, indicating that the protective action of GDNF might be associated with anti-inflammation and prohibition of microglia activation. Immunocytochemistry staining showed that GDNF reduced CCI-induced neuronal apoptosis. In sum, GDNF enhanced the neurotrophic effect by inhibiting microglia activation and cytokine production via p38 and PKC signaling. GDNF could be a good

  10. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  11. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  12. Induction of a Protective Heterosubtypic Immune Response Against the Influenza Virus by using Recombinant Adenoviral Vectors Expressing Hemagglutinin of the Influenza H5 Virus.

    PubMed

    Shmarov, M M; Sedova, E S; Verkhovskaya, L V; Rudneva, I A; Bogacheva, E A; Barykova, Yu A; Shcherbinin, D N; Lysenko, A A; Tutykhina, I L; Logunov, D Y; Smirnov, Yu A; Naroditsky, B S; Gintsburg, A L

    2010-04-01

    Influenza viruses are characterized by a high degree of antigenic variability, which causes the annual emergence of flu epidemics and irregularly timed pandemics caused by viruses with new antigenic and biological traits. Novel approaches to vaccination can help circumvent this problem. One of these new methods incorporates genetic vaccines based on adenoviral vectors. Recombinant adenoviral vectors which contain hemagglutinin-encoding genes from avian H5N1 and H5N2 (Ad-HA5-1 and Ad-HA5-2) influenza viruses were obtained using the AdEasy Adenoviral Vector System (Stratagene). Laboratory mice received a double intranasal vaccination with Ad-HA5-1 and Ad-HA5-2. This study demonstrates that immunization with recombinant adenoviruses bearing the Н 5 influenza virus hemagglutinin gene induces a immune response which protects immunized mice from a lethal dose of the H5 influenza virus. Moreover, it also protects the host from a lethal dose of the H1 virus, which belongs to the same clade as H5, but does not confer protection from the subtype H3 influenza virus, which belongs to a different clade. PMID:22649637

  13. A Genetically Modified Adenoviral Vector with a Phage Display-Derived Peptide Incorporated into Fiber Fibritin Chimera Prolongs Survival in Experimental Glioma.

    PubMed

    Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Morshed, Ramin A; Miska, Jason; Ahmed, Atique U; Balyasnikova, Irina V; Han, Yu; Zhang, Lingjiao; Curiel, David T; Lesniak, Maciej S

    2015-09-01

    The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as "GliomaFF." We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy. PMID:26058317

  14. Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5.

    PubMed

    Ehrke-Schulz, Eric; Zhang, Wenli; Schiwon, Maren; Bergmann, Thorsten; Solanki, Manish; Liu, Jing; Boehme, Philip; Leitner, Theo; Ehrhardt, Anja

    2016-01-01

    High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks and shortcuts developed over the past years working with this HCAdV vector system are presented. PMID:26863087

  15. Emphysematous lung destruction by cigarette smoke. The effects of latent adenoviral infection on the lung inflammatory response.

    PubMed

    Meshi, Bernard; Vitalis, Timothy Z; Ionescu, Diana; Elliott, W Mark; Liu, Chun; Wang, Xiang-Dong; Hayashi, Shizu; Hogg, James C

    2002-01-01

    This study was designed to test the hypothesis that cigarette smoke-induced inflammation and emphysema are amplified by the presence of latent adenoviral (Ad) infection, and to determine whether this emphysematous process can be reversed by all-trans-retinoic acid (RA) treatment. The results confirm that in guinea pigs, chronic cigarette-smoke exposure caused lesions similar to human centrilobular emphysema. They also show that latent Ad infection combined with cigarette-smoke exposure caused an excess increase in lung volume (P < 0.001), air-space volume (P < 0.001), and lung weight (P < 0.01), and further decrease in surface-to-volume ratio (P < 0.001) compared with smoke exposure alone. RA treatment failed to reverse these emphysematous changes. Analysis of inflammatory response in parenchymal and airway tissue showed that smoking caused an increase of polymorphonuclear leukocytes (PMNs) (P < 0.0002), macrophages (P < 0.001), and CD4 cells (P < 0.0009), and that latent Ad infection independently increased PMNs (P < 0.001), macrophages (P = 0.003), and CD8 cells (P < 0.001). We conclude that latent Ad infection amplifies the emphysematous lung destruction and increases the inflammatory response produced by cigarette-smoke exposure. In this study, the increase in CD4 was associated with cigarette smoke and the increase in CD8 cells with latent Ad infection. PMID:11751203

  16. Neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer.

    PubMed

    Li, Rongying; Oka, Kazuhiro; Yechoor, Vijay

    2012-01-01

    Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes. Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression(1). They lack chronic toxicity because they lack viral coding sequences(2-5) and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes. In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice. PMID:23093064

  17. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients.

    PubMed

    Dasari, Vijayendra; Schuessler, Andrea; Smith, Corey; Wong, Yide; Miles, John J; Smyth, Mark J; Ambalathingal, George; Francis, Ross; Campbell, Scott; Chambers, Daniel; Khanna, Rajiv

    2016-01-01

    Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as "off-the-shelf" therapeutics as well as autologous T-cell therapies for transplant patients. PMID:27606351

  18. Prolonged mechanical support in children with severe adenoviral infections: a case series and review of the literature.

    PubMed

    Gupta, Punkaj; Tobias, Joseph D; Goyal, Sunali; Hervie, Peter; Harris, Jason B; Sadot, Efraim; Noviski, Natan

    2011-01-01

    Adenovirus infections occur primarily in infants and children less than 5 years of age, accounting for 2% to 5% of respiratory illnesses in the pediatric population and 4% to 10% of childhood pneumonias. Although the majority of children with adenovirus disease develop mild upper respiratory tract disease, more severe disease may occur with involvement of the lower respiratory tract characterized by pneumonitis and/or small airways disease. The authors present a case series of 3 high-risk children with severe lower respiratory tract adenoviral infections. These cases demonstrate the potential for the development of severe respiratory involvement from adenovirus in infants and children with comorbid conditions and illustrate that there may be a rapid progression of the disease as well as the need, in selected circumstances, for prolonged mechanical support. We review the role of adenovirus in lower respiratory tract infections in infants and children, its potential to result in life-threatening complications in pediatric patients with comorbid conditions, and the potential life-saving role of mechanical ventilation and extracorporeal life support (ECLS) in these children. PMID:21320864

  19. Hexon-modified recombinant E1-deleted adenoviral vectors as bivalent vaccine carriers for Coxsackievirus A16 and Enterovirus 71.

    PubMed

    Zhang, Chao; Yang, Yong; Chi, Yudan; Yin, Jieyun; Yan, Lijun; Ku, Zhiqiang; Liu, Qingwei; Huang, Zhong; Zhou, Dongming

    2015-09-22

    Hand, foot and mouth disease (HFMD) is a major public health concern in Asia; more efficient vaccines against HFMD are urgently required. Adenoviral (Ad) capsids have been used widely for the presentation of foreign antigens to induce specific immune responses in the host. Here, we describe a novel bivalent vaccine for HFMD based on the hexon-modified, E1-deleted chimpanzee adenovirus serotype 68 (AdC68). The novel vaccine candidate was generated by incorporating the neutralising epitope of Coxsackievirus A16 (CA16), PEP71, into hypervariable region 1 (HVR1), and a shortened neutralising epitope of Enterovirus 71 (EV71), sSP70, into HVR2 of the AdC68 hexon. In order to enhance the immunogenicity of EV71, VP1 of EV71 was cloned into the E1-region of the AdC68 vectors. The results demonstrated that these two epitopes were well presented on the virion surface and had high affinity towards specific antibodies, and VP1 of EV71 was also significantly expressed. In pre-clinical mouse models, the hexon-modified AdC68 elicited neutralising antibodies against both CA16 and EV71, which conferred protection to suckling mice against a lethal challenge of CA16 and EV71. In summary, this study demonstrates that the hexon-modified AdC68 may represent a promising bivalent vaccine carrier against EV71 and CA16 and an epitope-display platform for other pathogens. PMID:26296491

  20. Adenoviral Vector Vaccination Induces a Conserved Program of CD8+ T Cell Memory Differentiation in Mouse and Man

    PubMed Central

    Bolinger, Beatrice; Sims, Stuart; Swadling, Leo; O’Hara, Geraldine; de Lara, Catherine; Baban, Dilair; Saghal, Natasha; Lee, Lian Ni; Marchi, Emanuele; Davis, Mark; Newell, Evan; Capone, Stefania; Folgori, Antonella; Barnes, Ellie; Klenerman, Paul

    2015-01-01

    Summary Following exposure to vaccines, antigen-specific CD8+ T cell responses develop as long-term memory pools. Vaccine strategies based on adenoviral vectors, e.g., those developed for HCV, are able to induce and sustain substantial CD8+ T cell populations. How such populations evolve following vaccination remains to be defined at a transcriptional level. We addressed the transcriptional regulation of divergent CD8+ T cell memory pools induced by an adenovector encoding a model antigen (beta-galactosidase). We observe transcriptional profiles that mimic those following infection with persistent pathogens, murine and human cytomegalovirus (CMV). Key transcriptional hallmarks include upregulation of homing receptors and anti-apoptotic pathways, driven by conserved networks of transcription factors, including T-bet. In humans, an adenovirus vaccine induced similar CMV-like phenotypes and transcription factor regulation. These data clarify the core features of CD8+ T cell memory following vaccination with adenovectors and indicate a conserved pathway for memory development shared with persistent herpesviruses. PMID:26586434

  1. Balloon Catheter Delivery of Helper-dependent Adenoviral Vector Results in Sustained, Therapeutic hFIX Expression in Rhesus Macaques

    PubMed Central

    Brunetti-Pierri, Nicola; Liou, Aimee; Patel, Priti; Palmer, Donna; Grove, Nathan; Finegold, Milton; Piccolo, Pasquale; Donnachie, Elizabeth; Rice, Karen; Beaudet, Arthur; Mullins, Charles; Ng, Philip

    2012-01-01

    Hemophilia B is an excellent candidate for gene therapy because low levels of factor IX (FIX) (≥1%) result in clinically significant improvement of the bleeding diathesis. Helper-dependent adenoviral (HDAd) vectors can mediate long-term transgene expression without chronic toxicity. To determine the potential for HDAd-mediated liver-directed hemophilia B gene therapy, we administered an HDAd expressing hFIX into rhesus macaques through a novel and minimally invasive balloon occlusion catheter-based method that permits preferential, high-efficiency hepatocyte transduction with low, subtoxic vector doses. Animals given 1 × 1012 and 1 × 1011 virus particle (vp)/kg achieved therapeutic hFIX levels for the entire observation period (up to 1,029 days). At 3 × 1010 and 1 × 1010 vp/kg, only subtherapeutic hFIX levels were achieved which were not sustained long-term. Balloon occlusion administration of HDAd was well tolerated with negligible toxicity. Five of six animals developed inhibitors to hFIX. These results provide important information in assessing the clinical utility of HDAd for hemophilia B gene therapy. PMID:22828499

  2. Adenoviral p53 gene transfer and gemcitabine in three patients with liver metastases due to advanced pancreatic carcinoma

    PubMed Central

    Thiede, Christian; Fischer, Rainer; Ehninger, Gerhard; Haag, Cornelie

    2007-01-01

    Background. Current therapies for adenocarcinoma of the pancreas do not improve the life expectancy of patients. Methods. In a non-randomized pilot trail we tested whether a local therapy based upon an adenoviral gene transfer of wild type p53 in combination with gemcitabine administration would be safe in patients with liver metastases due to pancreatic carcinoma. We report on the clinical course of three patients with respect to safety, tolerability and tumor response. Results. Transient grade III toxicities occurred with fever, leucopenia, elevation of AP, ALT, AST, GGT, while grade IV toxicity occurred for bilirubin only. Laboratory tests suggested disseminated intravascular coagulation in all three patients, but fine needle biopsies of liver did not show any histological evidence of thrombus or clot formation. Progression of liver metastases was documented in one and stable disease in another patient two months after treatment. However, a major improvement with regression of the indexed lesion by 80% occurred in a third patient after a single administration of 7.5×1012 viral particles, and time to progression was extended to six months. Conclusion. The combination therapy of viral gene transfer and chemotherapy temporarily controls and diminishes tumor burden. Improvement of the toxicity profile is necessary. Further trials are warranted to improve treatment and life expectancy of patients suffering from fatal diseases such as pancreatic carcinoma. PMID:18333108

  3. Adenoviral delivery of the beta2-adrenoceptor gene in sepsis: a subcutaneous approach in rat for kidney protection.

    PubMed

    Nakamura, Akio; Imaizumi, Akira; Niimi, Ryo; Yanagawa, Yukishige; Kohsaka, Takao; Johns, Edward J

    2005-12-01

    Successful gene therapy requires gene delivery that is efficient, has an optimal route of administration and has biosafety. The aims of the present study were to evaluate the safety and applicability of the subcutaneous delivery route for adenoviral transgenes containing the human beta(2)-adrenoceptor (adeno-beta(2)-AR) and to investigate whether this approach prevented renal dysfunction in a rat model of endotoxaemic shock induced by LPS (lipopolysaccharide). Subcutaneous administration of adeno-beta(2)-AR (a total of 10(10) viral particles) significantly increased beta-AR density in the kidney, lung and liver, but was without effect on physiological and plasma biochemical parameters. Moreover, this dose of virus did not cause any of the potential toxic responses of viral administration, such as inflammation and tissue TNF (tumour necrosis factor)-alpha expression. Although the LPS challenge caused a decrease in glomerular filtration rate, fractional excretion of sodium and renal beta-AR density in all groups, the reduction in renal function was significantly less in the rats given adeno-beta(2)-AR compared with non-treated rats. Thus, although further evaluation will be required, this initial study demonstrated that the subcutaneous injection of adeno-beta(2)-AR was efficient, comparatively non-pathogenic and potentially therapeutic to deal with acute renal failure associated with sepsis. PMID:16076286

  4. Prophylactic and therapeutic adenoviral vector-based multivirus-specific T-cell immunotherapy for transplant patients

    PubMed Central

    Dasari, Vijayendra; Schuessler, Andrea; Smith, Corey; Wong, Yide; Miles, John J; Smyth, Mark J; Ambalathingal, George; Francis, Ross; Campbell, Scott; Chambers, Daniel; Khanna, Rajiv

    2016-01-01

    Viral infections including cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus are a common and predictable problem in transplant recipients. While cellular immune therapies have been successfully used to tackle infectious complications in transplant recipients, manufacturing immunotherapies to address the multitude of possible pathogens can be technically challenging and labor-intensive. Here we describe a novel adenoviral antigen presentation platform (Ad-MvP) as a tool for rapid generation of multivirus-specific T-cells in a single step. Ad-MvP encodes 32 CD8+ T-cell epitopes from cytomegalovirus, Epstein-Barr virus, adenovirus, and BK virus as a contiguous polyepitope. We demonstrate that Ad-MvP vector can be successfully used for rapid in vitro expansion of multivirus-specific T-cells from transplant recipients and in vivo priming of antiviral T-cell immunity. Most importantly, using an in vivo murine model of Epstein-Barr virus-induced lymphoma, we also show that adoptive immunotherapy with Ad-MvP expanded autologous and allogeneic multivirus-specific T-cells is highly effective in controlling Epstein-Barr virus tumor outgrowth and improving overall survival. We propose that Ad-MvP has wide ranging therapeutic applications in greatly facilitating in vivo priming of antiviral T-cells, the generation of third-party T-cell banks as “off-the-shelf” therapeutics as well as autologous T-cell therapies for transplant patients. PMID:27606351

  5. Short-term Correction of Arginase Deficiency in a Neonatal Murine Model With a Helper-dependent Adenoviral Vector

    PubMed Central

    Gau, Chia-Ling; Rosenblatt, Robin A; Cerullo, Vincenzo; Lay, Fides D; Dow, Adrienne C; Livesay, Justin; Brunetti-Pierri, Nicola; Lee, Brendan; Cederbaum, Stephen D; Grody, Wayne W; Lipshutz, Gerald S

    2009-01-01

    Neonatal gene therapy has the potential to ameliorate abnormalities before disease onset. Our gene knockout of arginase I (AI) deficiency is characterized by increasing hyperammonemia, neurological deterioration, and early death. We constructed a helper-dependent adenoviral vector (HDV) carrying AI and examined for correction of this defect. Neonates were administered 5 × 109 viral particles/g and analyzed for survival, arginase activity, and ammonia and amino acids levels. The life expectancy of arg−/− mice increased to 27 days while controls died at 14 days with hyperammonemia and in extremis. Death correlated with a decrease in viral DNA/RNA per cell as liver mass increased. Arginase assays demonstrated that vector-injected hepatocytes had ~20% activity of heterozygotes at 2 weeks of age. Hepatic arginine and ornithine in treated mice were similar to those of saline-injected heterozygotes at 2 weeks, whereas ammonia was normal. By 26 days, arginase activity in the treated arg−/− livers declined to <10%, and arginine and ornithine increased. Ammonia levels began increasing by day 25, suggesting the cause of death to be similar to that of uninjected arg−/− mice, albeit at a later time. These studies demonstrate that the AI deficient newborn mouse can be temporarily corrected and rescued using a HDV. PMID:19367256

  6. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors

    PubMed Central

    Suzuki, Keiichiro; Mitsui, Kaoru; Aizawa, Emi; Hasegawa, Kouichi; Kawase, Eihachiro; Yamagishi, Toshiyuki; Shimizu, Yoshihiko; Suemori, Hirofumi; Nakatsuji, Norio; Mitani, Kohnosuke

    2008-01-01

    Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey (Macaca fasicularis) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was ≈100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only ≈1% with other gene delivery methods. Furthermore, in combination with negative selection, ≈45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells. PMID:18768795

  7. Genetic incorporation of HSV-1 thymidine kinase into the adenovirus protein IX for functional display on the virion

    SciTech Connect

    Li Jing; Le, Long; Sibley, Don A.; Mathis, J. Michael; Curiel, David T. . E-mail: david.curiel@ccc.uab.edu

    2005-08-01

    Adenoviral vectors have been exploited for a wide range of gene therapy applications. Direct genetic modification of the adenovirus capsid proteins has been employed to achieve alteration of vector tropism. We have defined the carboxy-terminus of the minor capsid protein pIX as a locus capable of presenting incorporated ligands on the virus capsid surface. Thus, we sought to exploit the possibility of incorporating functional proteins at pIX. In our current study, we incorporated the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) within pIX to determine if a larger protein of this type could retain functionality in this context. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional HSV-1 TK as a component of their capsid surface. DNA packaging and cytopathic effect were not affected by this genetic modification to the virus, while CAR-dependent binding was only marginally affected. Using an in vitro [{sup 3}H]-thymidine phosphorylation assay, we demonstrated that the kinase activity of the protein IX-TK fusion protein incorporated into adenoviral virions is functional. Analysis of cell killing after adenovirus infection showed that the protein IX-TK fusion protein could also serve as a therapeutic gene by rendering transduced cells sensitive to gancyclovir. Using 9-[4-[{sup 18}F]-fluoro-3-(hydroxymethyl)butyl]guanine ([{sup 18}F]-FHBG; a positron-emitting TK substrate), we demonstrated that we could detect specific cell binding and uptake of adenoviral virions containing the protein IX-TK fusion protein at 1 h post-infection. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional HSV-1 TK as a component of their capsid surface. The alternative display of HSV-1 TK on the capsid may offer advantages with respect to direct functional applications of this gene product. In addition, the determination of an expanded upper limit of incorporable

  8. The HDAC Inhibitor FK228 Enhances Adenoviral Transgene Expression by a Transduction-Independent Mechanism but Does Not Increase Adenovirus Replication

    PubMed Central

    Danielsson, Angelika; Dzojic, Helena; Rashkova, Victoria; Cheng, Wing-Shing; Essand, Magnus

    2011-01-01

    The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected. PMID:21379379

  9. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    SciTech Connect

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M.; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

  10. Oncolytic Adenoviral Mutants with E1B19K Gene Deletions Enhance Gemcitabine-induced Apoptosis in Pancreatic Carcinoma Cells and Anti-Tumor Efficacy In vivo

    PubMed Central

    Leitner, Stephan; Sweeney, Katrina; Öberg, Daniel; Davies, Derek; Miranda, Enrique; Lemoine, Nick R.; Halldén, Gunnel

    2010-01-01

    Purpose Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal.We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. Experimental Design Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdΔE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. Results The ΔE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdΔE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. Conclusions Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways.These findings imply that less toxic doses than currently practicedin the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants. PMID:19223497

  11. Proteins encoded near the adenovirus late messenger RNA leader segments

    SciTech Connect

    Lewis, J.B.; Anderson, C.W.

    1983-01-01

    Small fragments of adenovirus 2 DNA cloned into the single-strand phage M13 were used to select adenoviral messenger RNAs transcribed from the R-strand between map positions 16 and 30. Cell-free translation of these mRNAs produced proteins of 13.5K, 13.6K, and 11.5K, respectively encoded between the first and second segments of the tripartite major late leader, within the ''i''-leader segment, and immediately preceding the third leader segment. Partial sequence analysis of the 13.6K protein is consistent with the hypothesis that it is encoded within the i-leader segment.

  12. Large-Scale Production of High-Quality Helper-Dependent Adenoviral Vectors Using Adherent Cells in Cell Factories

    PubMed Central

    Suzuki, Masataka; Cela, Racel; Clarke, Christian; Bertin, Terry K.; Mouriño, Susana

    2010-01-01

    Abstract The most efficient and widely used system for generating helper-dependent adenoviral vectors (HDAds) is the Cre/loxP system developed by Graham and co-workers (Parks, R.J., Chen, L., Anton, M., Sankar, U., Rudnicki, M.A., and Graham, F.L. [1996]. Proc. Natl. Acad. Sci. U. S. A. 93, 13565–13570). Alternative systems have been developed for HDAd production, but all are limited by the technical complexity of a three-component vector production system for reproducibly generating large quantities of adenovirus with high infectivity and low helper virus (HV) contamination. Recently, these problems were addressed by Ng and co-workers (Palmer, D., and Ng, P. [2003]. Mol Ther. 8, 846–852), who developed an improved system that combines the use of a suspension-adapted producer cell line expressing high levels of Cre recombinase, a HV resistant to mutation, and a refined purification protocol. With this system, >1 × 1013 highly infectious vector particles are easily produced without vector genome rearrangements and having very low HV contamination levels. However, the Ng system incorporates a spinner flask culture system that involves considerable time, effort, and tissue culture medium to produce HDAds. We have an alternative system to obtain comparable quantities with equivalent quality to the spinner flask approach but requiring reduced labor and lower volumes of medium. This method utilizes a 10-chamber cell factory with adherent cells to produce high infectivity of HDAds with minimal HV contamination while improving yield and reducing technical complexity, effort, and medium requirements. This system is easily translatable to the production of clinical-grade HDAds for human trials. PMID:19719388

  13. Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

    PubMed Central

    2013-01-01

    Introduction Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Methods Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Results Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Conclusion Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the

  14. Comparison of the effect of adenoviral delivery of three superoxide dismutase genes against hepatic ischemia-reperfusion injury.

    PubMed

    Wheeler, M D; Katuna, M; Smutney, O M; Froh, M; Dikalova, A; Mason, R P; Samulski, R J; Thurman, R G

    2001-12-10

    The purpose of this study was to investigate the effectiveness of superoxide dismutase (SOD) overexpression in an acute model of hepatic oxidative stress. Oxidative stress was established using a warm ischemia-reperfusion model, where nearly 70% of the liver was made hypoxic by clamping the hepatic artery and a branch of the portal vein for 1 hr followed by restoration of blood flow. Animals were infected i.v. with 1 x 10(9) plaque-forming units (PFU) of adenovirus containing the transgene for cytosolic Cu/Zn-SOD (Ad.SOD1), mitochondrial Mn-SOD (Ad.SOD2), extracellular Cu/Zn-SOD (Ad.SOD3), or the bacterial reporter gene for beta-galactosidase (Ad.lacZ) 3 days prior to experiments. Ad.SOD1 and Ad.SOD2 caused a three-fold increase in SOD expression and activity in liver compared to Ad.lacZ-treated control animals. Intravenous administration of Ad.SOD3 increased SOD activity slightly in serum but not in liver. Increases in serum transaminases and pathology due to ischemia-reperfusion were blunted by Ad.SOD1 and Ad.SOD2; however, extracellular SOD had no significant effect. Moreover, lipid-derived free radical adducts (a(N) = 15.65 G and a(H)(beta) = 2.78 G) were increased by ischemia-reperfusion. This effect was blunted by about 60% in Ad.SOD1- and Ad.SOD2-infected animals, but was unaffected by Ad.SOD3. However, when high doses of Ad.SOD3 (3 x 10(10) PFU) were administered. serum SOD activity was elevated three-fold and was protective against hepatic ischemia-reperfusion injury under these conditions. These data demonstrate that adenoviral delivery of superoxide dismutase can effectively reduce hepatic oxidative stress. PMID:11779401

  15. Bone formation in vivo induced by Cbfa1-carrying adenoviral vectors released from a biodegradable porous β-tricalcium phosphate (β-TCP) material

    NASA Astrophysics Data System (ADS)

    Uemura, Toshimasa; Kojima, Hiroko

    2011-06-01

    Overexpression of Cbfa1 (a transcription factor indispensable for osteoblastic differentiation) is expected to induce the formation of bone directly and indirectly in vivo by accelerating osteoblastic differentiation. Adenoviral vectors carrying the cDNA of Cbfa1/til-1(Adv-Cbf1) were allowed to be adsorbed onto porous blocks of β-tricalcium phosphate (β-TCP), a biodegradable ceramic, which were then implanted subcutaneously and orthotopically into bone defects. The adenoviral vectors were released sustainingly by biodegradation, providing long-term expression of the genes. Results of the subcutaneous implantation of Adv-Cbfa1-adsorbed β-TCP/osteoprogenitor cells suggest that a larger amount of bone formed in the pores of the implant than in the control material. Regarding orthotopic implantation into bone defects, the released Adv-Cbfa1 accelerated regeneration in the cortical bone, whereas it induced bone resorption in the marrow cavity. A safer gene transfer using a smaller amount of the vector was achieved using biodegradable porous β-TCP as a carrier.

  16. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

    PubMed

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. PMID:24995995

  17. Inhibition of rabies virus multiplication by siRNA delivered through adenoviral vector in vitro in BHK-21 cells and in vivo in mice.

    PubMed

    Sonwane, Arvind A; Dahiya, Shyam S; Saini, Mohini; Chaturvedi, V K; Singh, R P; Gupta, Praveen K

    2012-08-01

    To evaluate antiviral potential of adenoviral vector-delivered small interfering RNA (siRNA) against rabies, recombinant, replication-defective adenoviral vectors (rAdV) encoding siRNAs targeting rabies virus (RV) polymerase (L) and nucleoprotein (N) genes were developed. The siRNAs were delivered as small hairpin RNAs (shRNAs) through these vectors. Treatment of BHK-21 cells with rAdV expressing siRNA targeting L gene (rAdV-L) and N gene (rAdV-N) (100 MOI) and their subsequent infection with RV (0.001 MOI, RV PV-11), reduced RV fluorescent foci by 48.2% (mean±SEM; 48.17±0.6540, N=6) and 41.8% (mean±SEM; 41.83±0.3073, N=6), respectively, with respect to that of BHK-21 cells treated with rAdV expressing negative control siRNA (rAdV-Neg) indicating inhibition of multiplication of RV in BHK-21 cells in response to adenoviral vector mediated siRNA delivery. Also, the similar treatment of BHK-21 cells with rAdV-L and rAdV-N and similar subsequent infection of them with RV resulted in reduction in RV mRNA transcript levels for their respective targets (RV L gene for rAdV-L and N gene for rAdV-N). mRNA transcript level for RV L gene was reduced by 17.88-fold (mean±SEM; 17.88±0.06638, N=6) in cells treated with rAdV-L and that for RV N gene was reduced by 5.7-fold (mean±SEM; 5.7±0.04472, N=6), in cells treated with rAdV-N, in comparison with that in cells treated with rAdV-Neg, as analyzed by using real-time PCR. These in vitro studies showed that between these two, adenoviral vector mediated delivery of siRNA targeting RV L gene was comparatively more effective in inhibiting RV multiplication in BHK-21 cells than that of siRNA targeting RV N gene (p<0.0001). Localized treatment (intramuscular injection in masseter muscle) of mice with 10(7) plaque forming units of either rAdV-L or rAdV-N and subsequent lethal RV infection (15-20LD(50) of CVS-11) at the same site, through the same route, although resulted in 50% protection (3 out of 6 mice survived) against lethal

  18. Peri- and Postnatal Effects of Prenatal Adenoviral VEGF Gene Therapy in Growth-Restricted Sheep.

    PubMed

    Carr, David J; Wallace, Jacqueline M; Aitken, Raymond P; Milne, John S; Martin, John F; Zachary, Ian C; Peebles, Donald M; David, Anna L

    2016-06-01

    Uterine artery (UtA) adenovirus (Ad) vector-mediated overexpression of vascular endothelial growth factor (VEGF) enhances uterine blood flow in normal sheep pregnancy and increases fetal growth in the overnourished adolescent sheep model of fetal growth restriction (FGR). Herein, we examined its impact on gestation length, neonatal survival, early postnatal growth and metabolism. Singleton-bearing ewes were evenly allocated to receive Ad.VEGF-A165 (5 × 10(10) particles/ml, 10 ml, n = 17) or saline (10 ml, n = 16) injected into each UtA at laparotomy (0.6 gestation). Fetal growth was serially monitored (blind) by ultrasound until delivery. Lambs were weighed and blood was sampled weekly and a glucose tolerance test performed (68-day postnatal age). Hepatic DNA/RNA was extracted at necropsy (83-day postnatal age) to examine methylation status of eight somatotropic axis genes. IGF1 mRNA and protein expression were measured by RT-PCR and radioimmunoassay, respectively. All pregnancies remained viable following Ad.VEGF-A165 treatment. Fetal abdominal circumference and renal volume were greater in the Ad.VEGF-A165 group compared with the saline group at 21/28 days (P ≤ 0.04) postinjection. At delivery, gestation length (P = 0.07), lamb birthweight (P = 0.08), umbilical girth (P = 0.06), and plasma glucose (P = 0.09) tended to be greater in Ad.VEGF-A165-treated lambs. Levels of neonatal intervention required to ensure survival was equivalent between groups. Absolute postnatal growth rate (P = 0.02), insulin area under the curve (P = 0.04) and carcass weight at necropsy (P = 0.04) were increased by Ad.VEGF-A165 treatment. There was no impact on markers of insulin sensitivity or methylation/expression of key genes involved in somatic growth. Ad.VEGF-A165 gene therapy increased fetal growth in a sheep FGR model, and lambs continued to thrive during the neonatal and early postnatal period. PMID:27103444

  19. Adenoviral-E2F-1 radiosensitizes p53{sup wild-type} and p53{sup null} human prostate cancer cells

    SciTech Connect

    Nguyen, Khanh H.; Hachem, Paul; Khor, L.-Y.; Salem, Naji; Hunt, Kelly K.; Calkins, Peter R.; Pollack, Alan . E-mail: Alan.Pollack@fccc.edu

    2005-09-01

    Purpose: E2F-1 is a transcription factor that enhances the radiosensitivity of various cell lines by inducing apoptosis. However, there are conflicting data concerning whether this enhancement is mediated via p53 dependent pathways. Additionally, the role of E2F-1 in the response of human prostate cancer to radiation has not been well characterized. In this study, we investigated the effect of Adenoviral-E2F-1 (Ad-E2F-1) on the radiosensitivity of p53{sup wild-type} (LNCaP) and p53{sup null} (PC3) prostate cancer cell lines. Methods and Materials: LNCaP and PC3 cells were transduced with Ad-E2F-1, Adenoviral-Luciferase (Ad-Luc) control vector, or Adenoviral-p53 (Ad-p53). Expression of E2F-1 and p53 was examined by Western blot analysis. Annexin V and caspase 3 + 7 assays were performed to estimate the levels of apoptosis. Clonogenic survival assays were used to determine overall cell death. Statistical significance was determined by analysis of variance, using the Bonferroni method to correct for multiple comparisons. Results: Western blot analysis confirmed the efficacy of transductions with Ad-E2F-1 and Ad-p53. Ad-E2F-1 transduction significantly enhanced apoptosis and decreased clonogenic survival in both cell lines. These effects were compounded by the addition of RT. Although E2F-1-mediated radiosensitization was independent of p53 status, this effect was more pronounced in p53{sup wild-type} LNCaP cells. When PC3 cells were treated with Ad-p53 in combination with RT and Ad-E2F-1, there was at least an additive reduction in clonogenic survival. Conclusions: Our results suggest that Ad-E2F-1 significantly enhances the response of p53{sup wild-type} and p53{sup null} prostate cancer cells to radiation therapy, although radiosensitization is more pronounced in the presence of p53. Ad-E2F-1 may be a useful adjunct to radiation therapy in the treatment of prostate cancer.

  20. Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells

    PubMed Central

    Thompson, David B.; Cronican, James J.; Liu, David R.

    2012-01-01

    Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high net positive or negative theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat, and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells. PMID:22230574

  1. New pre-pandemic influenza vaccines: an egg- and adjuvant-independent human adenoviral vector strategy induces long-lasting protective immune responses in mice.

    PubMed

    Hoelscher, M A; Jayashankar, L; Garg, S; Veguilla, V; Lu, X; Singh, N; Katz, J M; Mittal, S K; Sambhara, S

    2007-12-01

    Highly pathogenic avian H5N1 influenza viruses that are currently circulating in southeast Asia may acquire the potential to cause the next influenza pandemic. A number of alternate approaches are being pursued to generate cross-protective, dose-sparing, safe, and effective vaccines, as traditional vaccine approaches, i.e., embryonated egg-grown, are not immunogenic. We developed a replication-incompetent adenoviral vector-based, adjuvant- and egg-independent pandemic influenza vaccine strategy as a potential alternative to conventional egg-derived vaccines. In this paper, we address suboptimal dose and longevity of vaccine-induced protective immunity and demonstrate that a vaccine dose as little as 1 x 10(6) plaque-forming unit (PFU) is sufficient to induce protective immune responses against a highly pathogenic H5N1 virus. Furthermore, the vaccine-induced humoral and cellular immune responses and protective immunity persisted at least for a year. PMID:17957181

  2. Micro-computed tomography of pulmonary fibrosis in mice induced by adenoviral gene transfer of biologically active transforming growth factor-β1

    PubMed Central

    2010-01-01

    Background Micro-computed tomography (micro-CT) is a novel tool for monitoring acute and chronic disease states in small laboratory animals. Its value for assessing progressive lung fibrosis in mice has not been reported so far. Here we examined the importance of in vivo micro-CT as non-invasive tool to assess progression of pulmonary fibrosis in mice over time. Methods Pulmonary fibrosis was induced in mice by intratracheal delivery of an adenoviral gene vector encoding biologically active TGF-ß1 (AdTGF-ß1). Respiratory gated and ungated micro-CT scans were performed at 1, 2, 3, and 4 weeks post pulmonary adenoviral gene or control vector delivery, and were then correlated with respective histopathology-based Ashcroft scoring of pulmonary fibrosis in mice. Visual assessment of image quality and consolidation was performed by 3 observers and a semi-automated quantification algorithm was applied to quantify aerated pulmonary volume as an inverse surrogate marker for pulmonary fibrosis. Results We found a significant correlation between classical Ashcroft scoring and micro-CT assessment using both visual assessment and the semi-automated quantification algorithm. Pulmonary fibrosis could be clearly detected in micro-CT, image quality values were higher for respiratory gated exams, although differences were not significant. For assessment of fibrosis no significant difference between respiratory gated and ungated exams was observed. Conclusions Together, we show that micro-CT is a powerful tool to assess pulmonary fibrosis in mice, using both visual assessment and semi-automated quantification algorithms. These data may be important in view of pre-clinical pharmacologic interventions for the treatment of lung fibrosis in small laboratory animals. PMID:21176193

  3. Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

    PubMed

    Moayeri, Mahtab; Tremblay, Jacqueline M; Debatis, Michelle; Dmitriev, Igor P; Kashentseva, Elena A; Yeh, Anthony J; Cheung, Gordon Y C; Curiel, David T; Leppla, Stephen; Shoemaker, Charles B

    2016-03-01

    Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. PMID:26740390

  4. Adenoviral gene delivery of elafin and secretory leukocyte protease inhibitor attenuates NF-kappa B-dependent inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli.

    PubMed

    Henriksen, Peter A; Hitt, Mary; Xing, Zhou; Wang, Jun; Haslett, Chris; Riemersma, Rudolph A; Webb, David J; Kotelevtsev, Yuri V; Sallenave, Jean-Michel

    2004-04-01

    Atherosclerosis is a chronic inflammatory disease affecting arterial vessels. Strategies to reduce the inflammatory responses of endothelial cells and macrophages may slow lesion development and prevent complications such as plaque rupture. The human protease human neutrophil elastase (HNE), oxidized low density lipoprotein, LPS, and TNF-alpha were chosen as model stimuli of arterial wall inflammation and led to production of the chemokine IL-8 in endothelial cells. To counteract the activity of HNE, we have examined the effects of adenoviral gene delivery of the anti-elastases elafin, previously demonstrated within human atheroma, and murine secretory leukocyte protease inhibitor (SLPI), a related molecule, on the inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli. We developed a technique of precomplexing adenovirus with cationic lipid to augment adenoviral infection efficiency in endothelial cells and to facilitate infection in macrophages. Elafin overexpression protected endothelial cells from HNE-induced IL-8 production and cytotoxicity. Elafin and murine SLPI also reduced endothelial IL-8 release in response to oxidized low density lipoprotein, LPS, and TNF-alpha and macrophage TNF-alpha production in response to LPS. This effect was associated with reduced activation of the inflammatory transcription factor NF-kappaB, through up-regulation of IkappaBalpha, in both cell types. Our work suggests a novel and extended anti-inflammatory role for these HNE inhibitors working as effectors of innate immunity to protect tissues against maladaptive inflammatory responses. Our findings indicate that elafin and SLPI may be gene therapy targets for the treatment of atheroma. PMID:15034071

  5. Comparison of adenovirus fiber, protein IX, and hexon capsomeres as scaffolds for vector purification and cell targeting

    SciTech Connect

    Campos, Samuel K.; Barry, Michael A. . E-mail: mab@bcm.edu

    2006-06-05

    The direct genetic modification of adenoviral capsid proteins with new ligands is an attractive means to confer targeted tropism to adenoviral vectors. Although several capsid proteins have been reported to tolerate the genetic fusion of foreign peptides and proteins, direct comparison of cell targeting efficiencies through the different capsomeres has been lacking. Likewise, direct comparison of with one or multiple ligands has not been performed due to a lack of capsid-compatible ligands available for retargeting. Here we utilize a panel of metabolically biotinylated Ad vectors to directly compare targeted transduction through the fiber, protein IX, and hexon capsomeres using a variety of biotinylated ligands including antibodies, transferrin, EGF, and cholera toxin B. These results clearly demonstrate that cell targeting with a variety of high affinity receptor-binding ligands is only effective when transduction is redirected through the fiber protein. In contrast, protein IX and hexon-mediated targeting by the same set of ligands failed to mediate robust vector targeting, perhaps due to aberrant trafficking at the cell surface or inside targeted cells. These data suggest that vector targeting by genetic incorporation of high affinity ligands will likely be most efficient through modification of the adenovirus fiber rather than the protein IX and hexon capsomeres. In contrast, single-step monomeric avidin affinity purification of Ad vectors using the metabolic biotinylation system is most effective through capsomeres like protein IX and hexon.

  6. Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model

    PubMed Central

    Ahmed, Atique U; Rolle, Cleo E; Tyler, Matthew A; Han, Yu; Sengupta, Sadhak; Wainwright, Derek A; Balyasnikova, Irina V; Ulasov, Ilya V; Lesniak, Maciej S

    2010-01-01

    Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow–derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response. PMID:20588259

  7. Bone marrow mesenchymal stem cells loaded with an oncolytic adenovirus suppress the anti-adenoviral immune response in the cotton rat model.

    PubMed

    Ahmed, Atique U; Rolle, Cleo E; Tyler, Matthew A; Han, Yu; Sengupta, Sadhak; Wainwright, Derek A; Balyasnikova, Irina V; Ulasov, Ilya V; Lesniak, Maciej S

    2010-10-01

    Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow-derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response. PMID:20588259

  8. Permissive environment in postnatal wounds induced by adenoviral-mediated overexpression of the anti-inflammatory cytokine interleukin-10 prevents scar formation.

    PubMed

    Gordon, Ashley; Kozin, Elliott D; Keswani, Sundeep G; Vaikunth, Sachin S; Katz, Anna B; Zoltick, Philip W; Favata, Michele; Radu, Antoneta P; Soslowsky, Louis J; Herlyn, Meenhard; Crombleholme, Timothy M

    2008-01-01

    Wound healing in the mid-gestation fetus is scarless with minimal inflammation and a unique extracellular matrix. We have previously documented the relative lack of inflammatory cytokines in this environment. We demonstrate that interleukin (IL)-10 is highly expressed in mid-gestation human fetal skin but is absent in postnatal human skin. We hypothesize that overexpression of IL-10 in postnatal skin may replicate a permissive environment for scarless healing. To study the mechanism underlying this process we performed immunohistochemistry for IL-10 in human mid-gestation fetal and postnatal skin. We also determined if adenoviral-mediated overexpression of IL-10 could allow for scarless wound healing in a murine incisional wound model. Wounds were analyzed at 1-90 days postwounding for effects on scar formation, inflammatory response, and biomechanical properties. Ad-IL-10 reconstitutes a permissive environment for scarless healing as shown by reconstitution of a normal dermal reticular collagen pattern and distribution of dermal elements. Compared with controls, Ad-IL-10 treated wounds showed reduced inflammatory response and no difference in biomechanical parameters. Therefore, overexpression of IL-10 in postnatal wounds results in a permissive environment for scarless wound repair, possibly by replicating a fetal wound environment. PMID:18086289

  9. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations

    PubMed Central

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M.; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A.F.V.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  10. Developing Adenoviral Vectors Encoding Therapeutic Genes Toxic to Host Cells: Comparing Binary and Single Inducible Vectors Expressing Truncated E2F-1

    PubMed Central

    Gomez-Gutierrez, Jorge G.; Rao, Xiao-Mei; Garcia-Garcia, Aracely; Hao, Hongying; McMasters, Kelly M.; Zhou, H. Sam

    2010-01-01

    Adenoviral vectors are highly efficient at transferring genes into cells and are broadly used in cancer gene therapy. However, many therapeutic genes are toxic to vector host cells and thus inhibit vector production. The truncated form of E2F-1 (E2Ftr), which lacks the transactivation domain, can significantly induce cancer cell apoptosis, but is also toxic to HEK-293 cells and inhibits adenovirus replication. To overcome this, we have developed binary- and single-vector systems with a modified tetracycline-off inducible promoter to control E2Ftr expression. We compared several vectors and found that the structure of expression cassettes in vectors significantly affects E2Ftr expression. One construct expresses high levels of inducible E2Ftr and efficiently causes apoptotic cancer cell death by activation of caspase-3. The approach developed in this study may be applied in other viral vectors for encoding therapeutic genes that are toxic to their host cells and/or inhibit vector propagation. PMID:20003994

  11. Configurations of a two-tiered amplified gene expression system in adenoviral vectors designed to improve the specificity of in vivo prostate cancer imaging

    PubMed Central

    Sato, M; Figueiredo, ML; Burton, JB; Johnson, M; Chen, M; Powell, R; Gambhir, SS; Carey, M; Wu, L

    2009-01-01

    Effective treatment for recurrent, disseminated prostate cancer is notably limited. We have developed adenoviral vectors with a prostate-specific two-step transcriptional amplification (TSTA) system that would express therapeutic genes at a robust level to target metastatic disease. The TSTA system employs the prostate-specific antigen (PSA) promoter/enhancer to drive a potent synthetic activator, which in turn activates the expression of the therapeutic gene. In this study, we explored different configurations of this bipartite system and discovered that physical separation of the two TSTA components into E1 and E3 regions of adenovirus was able to enhance androgen regulation and cell-discriminatory expression. The TSTA vectors that express imaging reporter genes were assessed by noninvasive imaging technologies in animal models. The improved selectivity of the E1E3 configured vector was reflected in silenced ectopic expression in the lung. Significantly, the enhanced specificity of the E1E3 vector enabled the detection of lung metastasis of prostate cancer. An E1E3 TSTA vector that expresses the herpes simplex virus thymidine kinase gene can effectively direct positron emission tomography (PET) imaging of the tumor. The prostate-targeted gene delivery vectors with robust and cell-specific expression capability will advance the development of safe and effective imaging guided therapy for recurrent metastatic stages of prostate cancer. PMID:18305574

  12. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.

    PubMed

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A F V

    2016-02-18

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  13. Adenoviral targeting of malignant melanoma for fluorescence-guided surgery prevents recurrence in orthotopic nude-mouse models

    PubMed Central

    Yano, Shuya; Takehara, Kiyoto; Kishimoto, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Bouvet, Michael; Fujiwara, Toshiyoshi; Hoffman, Robert M.

    2016-01-01

    Malignant melanoma requires precise resection in order to avoid metastatic recurrence. We report here that the telomerase-dependent, green fluorescent protein (GFP)-containing adenovirus OBP-401 could label malignant melanoma with GFP in situ in orthotopic mouse models. OBP-401-based fluorescence-guided surgery (FGS) resulted in the complete resection of malignant melanoma in the orthotopic models, where conventional bright-light surgery (BLS) could not. High-dose administration of OBP-401 enabled FGS without residual cancer cells or recurrence, due to its dual effect of cancer-cell labeling with GFP and killing. PMID:26701857

  14. Intratracheal Instillation of High Dose Adenoviral Vectors Is Sufficient to Induce Lung Injury and Fibrosis in Mice

    PubMed Central

    Zhou, Qiyuan; Chen, Tianji; Bozkanat, Melike; Ibe, Joyce Christina F.; Christman, John W.; Raj, J. Usha; Zhou, Guofei

    2014-01-01

    Rationale Replication deficient adenoviruses (Ad) vectors are common tools in gene therapy. Since Ad vectors are known to activate innate and adaptive immunity, we investigated whether intratracheal administration of Ad vectors alone is sufficient to induce lung injury and pulmonary fibrosis. Methods We instilled Ad viruses ranging from 107 to 1.625×109 ifu/mouse as well as the same volume of PBS and bleomycin. 14 and 21 days after administration, we collected bronchoalveolar lavage fluid (BALF) and mouse lung tissues. We measured the protein concentration, total and differential cell counts, and TGF-β1 production, performed Trichrome staining and Sircol assay, determined gene and protein levels of profibrotic cytokines, MMPs, and Wnt signaling proteins, and conducted TUNEL staining and co-immunofluorescence for GFP and α-SMA staining. Results Instillation of high dose Ad vectors (1.625×109 ifu/mouse) into mouse lungs induced high levels of protein content, inflammatory cells, and TGF-β1 in BALF, comparable to those in bleomycin-instilled lungs. The collagen content and mRNA levels of Col1a1, Col1a2, PCNA, and α-SMA were also increased in the lungs. Instillation of both bleomycin and Ad vectors increased expression levels of TNFα and IL-1β but not IL-10. Instillation of bleomycin but not Ad increased the expression of IL-1α, IL-13 and IL-16. Treatment with bleomycin or Ad vectors increased expression levels of integrin α1, α5, and αv, MMP9, whereas treatment with bleomycin but not Ad vectors induced MMP2 expression levels. Both bleomycin and Ad vectors induced mRNA levels of Wnt2, 2b, 5b, and Lrp6. Intratracheal instillation of Ad viruses also induced DNA damages and Ad viral infection-mediated fibrosis is not limited to the infection sites. Conclusions Our results suggest that administration of Ad vectors induces an inflammatory response, lung injury, and pulmonary fibrosis in a dose dependent manner. PMID:25551570

  15. Competitive Inhibition of Lysine Acetyltransferase 2B by a Small Motif of the Adenoviral Oncoprotein E1A.

    PubMed

    Shi, Shasha; Liu, Ke; Chen, Yanheng; Zhang, Shijun; Lin, Juanyu; Gong, Chenfang; Jin, Quanwen; Yang, Xiang-Jiao; Chen, Ruichuan; Ji, Zhiliang; Han, Aidong

    2016-07-01

    The adenovirus early region 1A (E1A) oncoprotein hijacks host cells via direct interactions with many key cellular proteins, such as KAT2B, also known as PCAF (p300/CBP associated factor). E1A binds the histone acetyltransferase (HAT) domain of KAT2B to repress its transcriptional activation. However, the molecular mechanism by which E1A inhibits the HAT activity is not known. Here we demonstrate that a short and relatively conserved N-terminal motif (cNM) in the intrinsically disordered E1A protein is crucial for KAT2B interaction, and inhibits its HAT activity through a direct competition with acetyl-CoA, but not its substrate histone H3. Molecular modeling together with a series of mutagenesis experiments suggests that the major helix of E1A cNM binds to a surface of the acetyl-CoA pocket of the KAT2B HAT domain. Moreover, transient expression of the cNM peptide is sufficient to inhibit KAT2B-specific H3 acetylation H3K14ac in vivo Together, our data define an essential motif cNM in N-terminal E1A as an acetyl-CoA entry blocker that directly associates with the entrance of acetyl-CoA binding pocket to block the HAT domain access to its cofactor. PMID:27143356

  16. Adenoviral modification of mouse brain derived endothelial cells, bEnd3, to induce apoptosis by vascular endothelial growth factor.

    PubMed

    Mitsuuchi, Y; Powell, D R; Gallo, J M

    2006-02-01

    A second generation genetically-engineered cell-based drug delivery system, referred to as apoptotic-induced drug delivery (AIDD), was developed using endothelial cells (ECs) that undergo apoptosis upon binding of vascular endothelial growth factor (VEGF) to a Flk-1:Fas fusion protein (FF). This new AIDD was redesigned using mouse brain derived ECs, bEnd3 cells, and an adenovirus vector in order to enhance and control the expression of FF. The FF was tagged with a HA epitope (FFHA) and designed to be coexpressed with green fluorescence protein (GFP) by the regulation of cytomegalovirus promoters in the adenovirus vector. bEnd3 cells showed favorable coexpression of FFHA and GFP consistent with the multiplicity of infection of the adenovirus. Immunofluorescence analysis demonstrated that FFHA was localized at the plasma membrane, whereas GFP was predominantly located in the cytoplasm of ECs. Cell death was induced by VEGF, but not by platelet derived growth factor or fibroblast growth factor in a dose-dependent manner (range 2-20 ng/ml), and revealed caspase-dependent apoptotic profiles. The FFHA expressing bEnd3 cells underwent apoptosis when cocultured with a glioma cell (SF188V+) line able to overexpress VEGF. The combined data indicated that the FFHA adenovirus system can induce apoptotic signaling in ECs in response to VEGF, and thus, is an instrumental modification to the development of AIDD. PMID:16247462

  17. Long-Term Reduction of Cocaine Self-Administration in Rats Treated with Adenoviral Vector-Delivered Cocaine Hydrolase: Evidence for Enzymatic Activity

    PubMed Central

    Zlebnik, Natalie E; Brimijoin, Stephen; Gao, Yang; Saykao, Amy T; Parks, Robin J; Carroll, Marilyn E

    2014-01-01

    A new pharmacokinetic approach treating cocaine addiction involves rapidly metabolizing cocaine before it reaches brain reward centers using mutated human butyrylcholinesterase (BChE) or cocaine hydrolase (CocH). Recent work has shown that helper-dependent adenoviral (hdAD) vector-mediated plasma CocH reduced the locomotor-activating effects of cocaine and prevented reinstatement of cocaine-seeking behavior up to 6 months in rats. The present study investigated whether hdAD-CocH could decrease ongoing intravenous cocaine (0.4 mg/kg) self-administration. The hdAD-CocH vector was injected into self-administering rats, and after accumulation of plasma CocH, there was a dramatic reduction in cocaine infusions earned under a fixed ratio 1 schedule of reinforcement that lasted for the length of the study (>2 months). Pretreatment with the selective BChE and CocH inhibitor iso-OMPA (1.5 mg/kg) restored cocaine intake; therefore, the decline in self-administration was likely due to rapid CocH-mediated cocaine metabolism. Direct measurements of cocaine levels in plasma and brain samples taken after the conclusion of behavioral studies provided strong support for this conclusion. Further, rats injected with hdAD-CocH did not experience a deficit in operant responding for drug reinforcement and self-administered methamphetamine (0.05 mg/kg) at control levels. Overall, these outcomes suggest that viral gene transfer can yield plasma CocH levels that effectively diminish long-term cocaine intake and may have potential treatment implications for cocaine-dependent individuals seeking to become and remain abstinent. PMID:24407266

  18. Electric cell-substrate impedance sensing (ECIS) based real-time measurement of titer dependent cytotoxicity induced by adenoviral vectors in an IPI-2I cell culture model.

    PubMed

    Müller, Jakob; Thirion, Christian; Pfaffl, Michael W

    2011-01-15

    Recombinant viral vectors are widespread tools for transfer of genetic material in various modern biotechnological applications like for example RNA interference (RNAi). However, an accurate and reproducible titer assignment represents the basic step for most downstream applications regarding a precise multiplicity of infection (MOI) adjustment. As necessary scaffold for the studies described in this work we introduce a quantitative real-time PCR (qPCR) based approach for viral particle measurement. Still an implicated problem concerning physiological effects is that the appliance of viral vectors is often attended by toxic effects on the individual target. To determine the critical viral dose leading to cell death we developed an electric cell-substrate impedance sensing (ECIS) based assay. With ECIS technology the impedance change of a current flow through the cell culture medium in an array plate is measured in a non-invasive manner, visualizing effects like cell attachment, cell-cell contacts or proliferation. Here we describe the potential of this online measurement technique in an in vitro model using the porcine ileal epithelial cell line IPI-2I in combination with an adenoviral transfection vector (Ad5-derivate). This approach shows a clear dose-depending toxic effect, as the amount of applied virus highly correlates (p<0.001) with the level of cell death. Thus this assay offers the possibility to discriminate the minimal non-toxic dose of the individual transfection method. In addition this work suggests that the ECIS-device bears the feasibility to transfer this assay to multiple other cytotoxicological questions. PMID:20875729

  19. Adenovirus Specific Pre-Immunity Induced by Natural Route of Infection Does Not Impair Transduction by Adenoviral Vaccine Vectors in Mice

    PubMed Central

    de Andrade Pereira, Bruna; E. Maduro Bouillet, Leoneide; Dorigo, Natalia A.; Fraefel, Cornel; Bruna-Romero, Oscar

    2015-01-01

    Recombinant human adenovirus serotype 5 (HAd5V) vectors are gold standards of T-cell immunogenicity as they efficiently induce also humoral responses to exogenous antigens, in particular when used in prime-boost protocols. Some investigators have shown that pre-existing immunity to adenoviruses interferes with transduction by adenoviral vectors, but the actual extent of this interference is not known since it has been mostly studied in mice using unnatural routes of infection and virus doses. Here we studied the effects of HAd5V-specific immune responses induced by intranasal infection on the transduction efficiency of recombinant adenovirus vectors. Of interest, when HAd5V immunity was induced in mice by the natural respiratory route, the pre-existing immunity against HAd5V did not significantly interfere with the B and T-cell immune responses against the transgene products induced after a prime/boost inoculation protocol with a recombinant HAd5V-vector, as measured by ELISA and in vivo cytotoxic T-cell assays, respectively. We also correlated the levels of HAd5V-specific neutralizing antibodies (Ad5NAbs) induced in mice with the levels of Ad5NAb titers found in humans. The data indicate that approximately 60% of the human serum samples tested displayed Ad5NAb levels that could be overcome with a prime-boost vaccination protocol. These results suggest that recombinant HAd5V vectors are potentially useful for prime-boost vaccination strategies, at least when pre-existing immunity against HAd5V is at low or medium levels. PMID:26679149

  20. Preclinical Efficacy and Safety Profile of Allometrically Scaled Doses of Doxycycline Used to Turn "On" Therapeutic Transgene Expression from High-Capacity Adenoviral Vectors in a Glioma Model.

    PubMed

    VanderVeen, Nathan; Raja, Nicholas; Yi, Elizabeth; Appelman, Henry; Ng, Philip; Palmer, Donna; Zamler, Daniel; Dzaman, Marta; Lowenstein, Pedro R; Castro, Maria G

    2016-06-01

    Glioblastoma multiforme (GBM) is the most commonly occurring primary brain cancer in adults, in whom its highly infiltrative cells prevent total surgical resection, often leading to tumor recurrence and patient death. Our group has discovered a gene therapy approach for GBM that utilizes high-capacity "gutless" adenoviral vectors encoding regulatable therapeutic transgenes. The herpes simplex type 1-thymidine kinase (TK) actively kills dividing tumor cells in the brain when in the presence of the prodrug, ganciclovir (GCV), whereas the FMS-like tyrosine kinase 3 ligand (Flt3L) is an immune-stimulatory molecule under tight regulation by a tetracycline-inducible "Tet-On" activation system that induces anti-GBM immunity. As a prelude to a phase I clinical trial, we evaluated the safety and efficacy of Food and Drug Administration (FDA)-approved doses of the tetracycline doxycycline (DOX) allometrically scaled for rats. DOX initiates the expression of Flt3L, which has been shown to recruit dendritic cells to the brain tumor microenvironment-an integral first step in the development of antitumor immunity. The data revealed a highly safe profile surrounding these human-equivalent doses of DOX under an identical therapeutic window as proposed in the clinical trial. This was confirmed through a neuropathological analysis, liver and kidney histopathology, detection of neutralizing antibodies, and systemic toxicities in the blood. Interestingly, we observed a significant survival advantage in rats with GBM receiving the 300 mg/day equivalent dosage of DOX versus the 200 mg/day equivalent. Additionally, rats rejected "recurrent" brain tumor threats implanted 90 days after their primary brain tumors. We also show that DOX detection within the plasma can be an indicator of optimal dosing of DOX to attain therapeutic levels. This work has significant clinical relevance for an ongoing phase I clinical trial in humans with primary GBM and for other therapeutic approaches using

  1. Phosphodiesterase 5a Inhibition with Adenoviral Short Hairpin RNA Benefits Infarcted Heart Partially through Activation of Akt Signaling Pathway and Reduction of Inflammatory Cytokines

    PubMed Central

    Jin, Zhe; Zhang, Jian; Paul, Christian; Wang, Yigang

    2015-01-01

    Introduction Treatment with short hairpin RNA (shRNA) interference therapy targeting phosphodiesterase 5a after myocardial infarction (MI) has been shown to mitigate post-MI heart failure. We investigated the mechanisms that underpin the beneficial effects of PDE5a inhibition through shRNA on post-MI heart failure. Methods An adenoviral vector with an shRNA sequence inserted was adopted for the inhibition of phosphodiesterase 5a (Ad-shPDE5a) in vivo and in vitro. Myocardial infarction (MI) was induced in male C57BL/6J mice by left coronary artery ligation, and immediately after that, the Ad-shPDE5a was injected intramyocardially around the MI region and border areas. Results Four weeks post-MI, the Ad-shPDE5a-treated mice showed significant mitigation of the left ventricular (LV) dilatation and dysfunction compared to control mice. Infarction size and fibrosis were also significantly reduced in Ad-shPDE5a-treated mice. Additionally, Ad-shPDE5a treatment decreased the MI-induced inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and transforming growth factor-β1, which was confirmed in vitro in Ad-shPDE5a transfected myofibroblasts cultured under oxygen glucose deprivation. Finally, Ad-shPDE5a treatment was found to activate the myocardial Akt signaling pathway in both in vivo and in vitro experiments. Conclusion These findings indicate that PDE5a inhibition by Ad-shPDE5a via the Akt signal pathway could be of significant value in the design of future therapeutics for post-MI heart failure. PMID:26709517

  2. A Multi-Antigenic Adenoviral-Vectored Vaccine Improves BCG-Induced Protection of Goats against Pulmonary Tuberculosis Infection and Prevents Disease Progression

    PubMed Central

    Pérez de Val, Bernat; Vidal, Enric; Villarreal-Ramos, Bernardo; Gilbert, Sarah C.; Andaluz, Anna; Moll, Xavier; Martín, Maite; Nofrarías, Miquel; McShane, Helen; Vordermeier, H. Martin; Domingo, Mariano

    2013-01-01

    The “One world, one health” initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG. PMID:24278420

  3. Adenoviral gene transfer into the normal and injured spinal cord: enhanced transgene stability by combined administration of temperature-sensitive virus and transient immune blockade.

    PubMed

    Romero, M I; Smith, G M

    1998-12-01

    This study characterized gene transfer into both normal and injured adult rat dorsal spinal cord using first (E1-/E3-) or second (E1-/E2A125/E3-, temperature-sensitive; ts) generation of replication-defective adenoviral (Ad) vectors. A novel immunosuppressive regimen aimed at blocking CD4/CD45 lymphocytic receptors was tested for improving transgene persistence. In addition, the effect of gene transfer on nociception was also evaluated. Seven days after treatment, numerous LacZ-positive cells were observed after transfection with either viral vector. By 21 days after transfection, beta-galactosidase staining was reduced and suggestive of ongoing cytopathology in both Ad-treated groups, despite the fact that the immunogenicity of LacZ/Adts appeared less when compared with that elicited by the LacZ/Ad vector. In contrast, immunosuppressed animals showed a significant (P < or = 0.05) increase in the number of LacZ-positive cells not displaying cytopathology. In these animals, a concomitant reduction in numbers of macrophages/microglia and CD4 and CD8 lymphocytes was observed. Only animals that received LacZ/Adts and immunosuppression showed transgene expression after 60 days. Similar results were observed in animals in which the L4-L5 dorsal roots were lesioned before transfection. Gene transfer into the dorsal spinal cord did not affect nociception, independent of the adenovirus vector. These results indicate that immune blockade of the CD4/CD45 lymphocytic receptors enhanced transgene stability in adult animals with normal or injured spinal cords and that persistent transgene expression in the spinal cord does not interfere with normal neural function. PMID:10023440

  4. Long-Term Blockade of Cocaine Self-Administration and Locomotor Activation in Rats by an Adenoviral Vector-Delivered Cocaine Hydrolase.

    PubMed

    Smethells, John R; Swalve, Natashia; Brimijoin, Stephen; Gao, Yang; Parks, Robin J; Greer, Adam; Carroll, Marilyn E

    2016-05-01

    A promising approach in treating cocaine abuse is to metabolize cocaine in the blood using a mutated butyrylcholinesterase (BChE) that functions as a cocaine hydrolase (CocH). In rats, a helper-dependent adenoviral (hdAD) vector-mediated delivery of CocH abolished ongoing cocaine use and cocaine-primed reinstatement of drug-seeking for several months. This enzyme also metabolizes ghrelin, an effect that may be beneficial in maintaining healthy weights. The effect of a single hdAD-CocH vector injection was examined in rats on measures of anxiety, body weight, cocaine self-administration, and cocaine-induced locomotor activity. To examine anxiety, periadolescent rats were tested in an elevated-plus maze. Weight gain was then examined under four rodent diets. Ten months after CocH-injection, adult rats were trained to self-administer cocaine intravenously and, subsequently, cocaine-induced locomotion was tested. Viral gene transfer produced sustained plasma levels of CocH for over 13 months of testing. CocH-treated rats did not differ from controls in measures of anxiety, and only showed a transient reduction in weight gain during the first 3 weeks postinjection. However, CocH-treated rats were insensitive to cocaine. At 10 months postinjection, none of the CocH-treated rats initiated cocaine self-administration, unlike 90% of the control rats. At 13 months postinjection, CocH-treated rats showed no cocaine-induced locomotion, whereas control rats showed a dose-dependent enhancement of locomotion. CocH vector produced a long-term blockade of the rewarding and behavioral effects of cocaine in rats, emphasizing its role as a promising therapeutic intervention in cocaine abuse. PMID:26968195

  5. Recombinant adenoviral expression of IL-10 protects beta cell from impairment induced by pro-inflammatory cytokine.

    PubMed

    Xu, Ai-Jing; Zhu, Wei; Tian, Fei; Yan, Li-Hua; Li, Tang

    2010-11-01

    Interleukin-10 (IL-10) is a pleiotropic immunosuppressive and immunostimulatory cytokine. In autoimmune diabetes of the nonobese diabetic (NOD) mouse, IL-10 has exhibited paradoxical effects. Systemic IL-10 expression prevented or delayed diabetes onset in NOD mice while local expression of IL-10 did not. As antigen-presenting cells (APCs) play a central role in the generation of primary T cell responses, the direct role of this gene in pancreatic beta (β) cell is not clear. The effects of IL-10 on the protection of β cells in vitro were examined. In the present study, we examined the effects of adenovirus vector-mediated murine IL-10 (mIL-10) gene transfer to islet cell line RINm5F cells in vitro and to explore if IL-10 overexpression may prevent cytokine-mediated cytotoxicity. We had established the recombinant adenovirus vector containing mIL-10 genes (Ad-mIL-10) successfully. After infection of Ad-mIL-10, both mRNA and protein were expressed in RINm5F cells. Moreover, RINm5F cells secreted IL-10 protein into culture medium. Ad-mIL-10 prevented IL-1β-mediated nitric oxide production from β cells in vitro as well as the suppression of β cells function as determined by glucose-stimulated insulin production. Furthermore, Ad-mIL-10 gene transfer led to a profound reduction of Fas-expressing β cells and caspase-3 activity which were induced by IL-1β and the apoptotic rates of Ad-mIL-10 group were decreased. These findings show that IL-10 gene transfer to β cells may be beneficial in maintaining cells function, protecting islet cells from apoptosis-mediated by factors, which showed the potential therapy for type 1 diabetes mellitus. PMID:20658311

  6. Analysis of Fluorescent Proteins with a Nanoparticle Probe

    PubMed Central

    Fernandez-Lima, Francisco A.; Eller, Michael J.; DeBord, J. Daniel; Levy, Michaella J.; Verkhoturov, Stanislav V.; Della-Negra, Serge; Schweikert, Emile A.

    2012-01-01

    This letter presents the first application of high energy, single nanoparticle probes (e.g., 520 keV Au400 2nm NP) in the characterization of surfaces containing fluorescent proteins (e.g., GFP variants) by their co-emitted photon, electron and secondary ion signals. NP induced protein luminescence increases with the NP incident energy, is originated by the NP impact and is transferred to the protein fluorophor via electronic energy transfer. Multi-electron emission is observed per single NP impacts and their distributions are specific to the target morphology and composition. Fragment ions of protein sub-units consisting of 2–7 amino acid peptides are observed under individual NP impacts that can be correlated to the random protein orientation relative to the impact site (e.g., outer layer or “skin” of the protein). PMID:22308203

  7. Adenoviral gene transfer corrects the ion transport defect in the sinus epithelia of a porcine CF model.

    PubMed

    Potash, Andrea E; Wallen, Tanner J; Karp, Philip H; Ernst, Sarah; Moninger, Thomas O; Gansemer, Nicholas D; Stoltz, David A; Zabner, Joseph; Chang, Eugene H

    2013-05-01

    Cystic fibrosis (CF) pigs spontaneously develop sinus and lung disease resembling human CF. The CF pig presents a unique opportunity to use gene transfer to test hypotheses to further understand the pathogenesis of CF sinus disease. In this study, we investigated the ion transport defect in the CF sinus and found that CF porcine sinus epithelia lack cyclic AMP (cAMP)-stimulated anion transport. We asked whether we could restore CF transmembrane conductance regulator gene (CFTR) current in the porcine CF sinus epithelia by gene transfer. We quantified CFTR transduction using an adenovirus expressing CFTR and green fluorescent protein (GFP). We found that as little as 7% of transduced cells restored 6% of CFTR current with 17-28% of transduced cells increasing CFTR current to 50% of non-CF levels. We also found that we could overcorrect cAMP-mediated current in non-CF epithelia. Our findings indicate that CF porcine sinus epithelia lack anion transport, and a relatively small number of cells expressing CFTR are required to rescue the ion transport phenotype. These studies support the use of the CF pig as a preclinical model for future gene therapy trials in CF sinusitis. PMID:23511247

  8. Peptide-Based Technologies to Alter Adenoviral Vector Tropism: Ways and Means for Systemic Treatment of Cancer

    PubMed Central

    Reetz, Julia; Herchenröder, Ottmar; Pützer, Brigitte M.

    2014-01-01

    Due to the fundamental progress in elucidating the molecular mechanisms of human diseases and the arrival of the post-genomic era, increasing numbers of therapeutic genes and cellular targets are available for gene therapy. Meanwhile, the most important challenge is to develop gene delivery vectors with high efficiency through target cell selectivity, in particular under in situ conditions. The most widely used vector system to transduce cells is based on adenovirus (Ad). Recent endeavors in the development of selective Ad vectors that target cells or tissues of interest and spare the alteration of all others have focused on the modification of the virus broad natural tropism. A popular way of Ad targeting is achieved by directing the vector towards distinct cellular receptors. Redirecting can be accomplished by linking custom-made peptides with specific affinity to cellular surface proteins via genetic integration, chemical coupling or bridging with dual-specific adapter molecules. Ideally, targeted vectors are incapable of entering cells via their native receptors. Such altered vectors offer new opportunities to delineate functional genomics in a natural environment and may enable efficient systemic therapeutic approaches. This review provides a summary of current state-of-the-art techniques to specifically target adenovirus-based gene delivery vectors. PMID:24699364

  9. A case of autism with an interstitial deletion on 4q leading to hemizygosity for genes encoding for glutamine and glycine neurotransmitter receptor sub-units (AMPA 2, GLRA3, GLRB) and neuropeptide receptors NPY1R, NPY5R

    PubMed Central

    Ramanathan, Subhadra; Woodroffe, Abigail; Flodman, Pamela L; Mays, Lee Z; Hanouni, Mona; Modahl, Charlotte B; Steinberg-Epstein, Robin; Bocian, Maureen E; Spence, M Anne; Smith, Moyra

    2004-01-01

    Background Autism is a pervasive developmental disorder characterized by a triad of deficits: qualitative impairments in social interactions, communication deficits, and repetitive and stereotyped patterns of behavior. Although autism is etiologically heterogeneous, family and twin studies have established a definite genetic basis. The inheritance of idiopathic autism is presumed to be complex, with many genes involved; environmental factors are also possibly contributory. The analysis of chromosome abnormalities associated with autism contributes greatly to the identification of autism candidate genes. Case presentation We describe a child with autistic disorder and an interstitial deletion on chromosome 4q. This child first presented at 12 months of age with developmental delay and minor dysmorphic features. At 4 years of age a diagnosis of Pervasive Developmental Disorder was made. At 11 years of age he met diagnostic criteria for autism. Cytogenetic studies revealed a chromosome 4q deletion. The karyotype was 46, XY del 4 (q31.3-q33). Here we report the clinical phenotype of the child and the molecular characterization of the deletion using molecular cytogenetic techniques and analysis of polymorphic markers. These studies revealed a 19 megabase deletion spanning 4q32 to 4q34. Analysis of existing polymorphic markers and new markers developed in this study revealed that the deletion arose on a paternally derived chromosome. To date 33 genes of known or inferred function are deleted as a consequence of the deletion. Among these are the AMPA 2 gene that encodes the glutamate receptor GluR2 sub-unit, GLRA3 and GLRB genes that encode glycine receptor subunits and neuropeptide Y receptor genes NPY1R and NPY5R. Conclusions The deletion in this autistic subject serves to highlight specific autism candidate genes. He is hemizygous for AMPA 2, GLRA3, GLRB, NPY1R and NPY5R. GluR2 is the major determinant of AMPA receptor structure. Glutamate receptors maintain structural

  10. Immunization with Hexon Modified Adenoviral Vectors Integrated with gp83 Epitope Provides Protection against Trypanosoma cruzi Infection

    PubMed Central

    Gu, Linlin; Krendelchtchikova, Valentina; Nde, Pius N.; Pratap, Siddharth; Lima, Maria F.; Villalta, Fernando; Matthews, Qiana L.

    2014-01-01

    Background Trypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary. Methodology/Principal Findings The “antigen capsid-incorporation” strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His6 were incorporated into the Ad serotype 5 (Ad5) vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83). This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies. Conclusions/Significance This data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses