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Sample records for adenovirus expressing transforming

  1. Expression of Viral DNA in Adenovirus Type 12-Transformed Cells, in Tumor Cells, and in Revertants

    PubMed Central

    Schirm, Sabine; Doerfler, Walter

    1981-01-01

    The expression as cytoplasmic RNA of integrated human adenovirus type 12 (Ad12) DNA in transformed and tumor cell lines and in revertants was investigated. The transformed and tumor cells contained multiple copies of the viral genome, 3 to 22 copies per cell in different cell lines. The integrated Ad12 DNA molecules persisted intact or nearly intact and in most cases colinear with the virion DNA. In the revertant cell lines, which were derived from cell line T637 (22 copies of Ad12 DNA per cell), all of the Ad12 DNA molecules were lost (line F10) or only one copy and a fraction of a second copy persisted (line TR12). The size classes and map locations of Ad12-specific cytoplasmic RNAs in three Ad12-transformed hamster cell lines (T637, HA12/7, and A2497-3), in two revertant lines (F10 and TR12), in one Ad12-induced hamster (CLAC3), and in one rat brain tumor line (RBT12/3) were determined. Cytoplasmic RNA from uninfected B3 hamster cells and from human KB cells productively infected with Ad12 served as controls. In the latter control experiments, the RNA was isolated early or late postinfection. With respect to the amounts of Ad12-specific RNAs detected in cytoplasmic RNA from various Ad12-transformed or Ad12-induced tumor cell lines, we could not establish any correlations to the number of Ad12 genome copies integrated into the cellular DNAs. Thus, the expression of the integrated viral genomes in these lines was regulated by mechanisms more complicated than simple gene dosage effects. Using cloned fragments of Ad12 DNA as hybridization probes, we analyzed the cytoplasmic RNAs from the cell lines mentioned by electrophoresis on agarose gels, blotting, and DNA-RNA hybridization. For each transformed and tumor cell line, except for the revertants, several size classes of Ad12-specific cytoplasmic RNA were detected for the early E1, E2, and E4 regions of Ad12 DNA. Some of these size classes were similar but not identical to those observed in cytoplasmic RNA isolated

  2. Adenovirus Vector Pseudotyping in Fiber-Expressing Cell Lines: Improved Transduction of Epstein-Barr Virus-Transformed B Cells

    PubMed Central

    Von Seggern, Dan J.; Huang, Shuang; Fleck, Shonna Kaye; Stevenson, Susan C.; Nemerow, Glen R.

    2000-01-01

    While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.βgal.ΔF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.βgal.ΔF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types. PMID:10590124

  3. “Hit-and-Run” Transformation by Adenovirus Oncogenes

    PubMed Central

    Nevels, Michael; Täuber, Birgitt; Spruss, Thilo; Wolf, Hans; Dobner, Thomas

    2001-01-01

    According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the “hit-and-run” hypothesis claims that viruses can mediate cellular transformation through an initial “hit,” while maintenance of the transformed state is compatible with the loss (“run”) of viral molecules. It is well established that the adenovirus E1A and E1B gene products can cooperatively transform primary human and rodent cells to a tumorigenic phenotype and that these cells permanently express the viral oncogenes. Additionally, recent studies have shown that the adenovirus E4 region encodes two novel oncoproteins, the products of E4orf6 and E4orf3, which cooperate with the viral E1A proteins to transform primary rat cells in an E1B-like fashion. Unexpectedly, however, cells transformed by E1A and either E4orf6 or E4orf3 fail to express the viral E4 gene products, and only a subset contain E1A proteins. In fact, the majority of these cells lack E4- and E1A-specific DNA sequences, indicating that transformation occurred through a hit-and-run mechanism. We provide evidence that the unusual transforming activities of the adenoviral oncoproteins may be due to their mutagenic potential. Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy. PMID:11238835

  4. Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

    PubMed Central

    Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

    1991-01-01

    Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

  5. Transformation Potentials of the Noninfectious (Defective) Component in Pools of Adenoviruses Type 12 and Simian Adenovirus 7

    PubMed Central

    Schaller, J. P.; Yohn, D. S.

    1974-01-01

    Pools of adenovirus 12 and simian adenovirus 7 were separated into four or five fractions by density gradient centrifugation in cesium chloride. Each fraction was analyzed for total in vitro infectivity units, total transformation activity, and for total virus particle (VP) content. Two major subpopulations were separated with mean densities of 1.30 ± 0.02 and 1.34 ± 0.02 g/ml, respectively. Virions in the 1.34 g/ml range were highly infectious (102 to 103 VP per infectivity unit) in contrast to virions at 1.30 g/ml density (104 to 105 VP per infectivity units). Transformation capacity was evenly distributed throughout fractions of both viruses, indicating that genetically incomplete or defective virus particles were not deficient in their ability to induce transformation. The average VP per transformation unit for adenovirus 12 (2.85 × 106) and for simian adenovirus 7 (4.00 × 106) did not vary significantly from fraction to fraction. These values were obtained with optimal input multiplicities of 16 to 64 VP per cell. At higher multiplicities the apparent increase in VP per transformation unit was attributable to the viral cytocidal effect on hamster cells. These studies revealed that quantitation of in vitro transformation based on VP multiplicities was more reliable than on the basis of infectious units. These estimates were independent of method of virus production, extraction, and purification. Images PMID:4211167

  6. Periluminal expression of a secreted transforming growth factor-β type II receptor inhibits in-stent neointima formation following adenovirus-mediated stent-based intracoronary gene transfer.

    PubMed

    Appleby, Clare E; Ranjzad, Parisa; Williams, Paul D; Kakar, Salik J; Driessen, Anita; Tijsma, Edze; Fernandes, Brian; Heagerty, Anthony M; Kingston, Paul A

    2014-05-01

    Transforming growth factor-β1 (TGF-β1) has been shown unequivocally to enhance neointima formation in carotid and ileo-femoral arteries. In our previous studies, however, TGF-β1 expression in coronary arteries actually reduced neointima formation without affecting luminal loss postangioplasty, while expression of a TGF-β1 antagonist (RIIs) in balloon-injured coronary arteries reduced luminal loss without affecting neointima formation. These observed effects may be a consequence of the mode of coronary artery gene transfer employed, but they may also represent differences in the modes of healing of coronary, carotid, and ileo-femoral arteries after endoluminal injury. To help clarify whether a gene therapy strategy to antagonize TGF-β might have application within the coronary vasculature, we have investigated the effect of high-level periluminal expression of RIIs using stent-based adenovirus-mediated intracoronary gene transfer. Porcine coronary arteries were randomized to receive a custom-made CoverStent preloaded with saline only, or with 1×10(9) infectious units of adenovirus expressing RIIs or β-galactosidase (lacZ). Vessels were analyzed 28 days poststenting, at which time angiographic in-stent diameter was significantly greater in RIIs-treated arteries, and in-stent luminal loss significantly reduced. Computerized morphometric minimum in-stent lumen area was ~300% greater in RIIs-exposed vessels than in lacZ or saline-only groups. This was because of significantly reduced neointima formation in the RIIs group. RIIs had no demonstrable effect on cellular proliferation or apoptosis, but greater normalized neointimal/medial collagen content was observed in RIIs-exposed arteries. These data highlight the qualitatively similar effect of TGF-β antagonism on neointima formation in injured coronary and noncoronary arteries, and suggest that since cellular proliferation is unaffected, TGF-β1 antagonism might prevent in-stent restenosis without the delayed

  7. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    PubMed Central

    Krömmelbein, Natascha; Wiebusch, Lüder; Schiedner, Gudrun; Büscher, Nicole; Sauer, Caroline; Florin, Luise; Sehn, Elisabeth; Wolfrum, Uwe; Plachter, Bodo

    2016-01-01

    The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production. PMID:26848680

  8. Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes

    PubMed Central

    Speiseder, Thomas; Hofmann-Sieber, Helga; Rodríguez, Estefanía; Schellenberg, Anna; Akyüz, Nuray; Dierlamm, Judith; Spruss, Thilo; Lange, Claudia

    2016-01-01

    derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells. PMID:27795433

  9. Effect of adenovirus infection on expression of human histone genes.

    PubMed Central

    Flint, S J; Plumb, M A; Yang, U C; Stein, G S; Stein, J L

    1984-01-01

    The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA

  10. Adenovirus infection stimulates the Raf/MAPK signaling pathway and induces interleukin-8 expression.

    PubMed Central

    Bruder, J T; Kovesdi, I

    1997-01-01

    Previous studies have shown that airway administration of adenovirus or adenovirus vectors results in a dose-dependent inflammatory response which limits the duration of transgene expression. We explored the possibility that adenovirus infection triggers signal transduction pathways that induce the synthesis of cytokines and thus contribute to the early inflammatory response. Since stimulation of the Raf/mitogen-activated protein kinase (MAPK) pathway activates transcription factors that control the expression of inflammatory cytokines, we examined the activation of this pathway following adenovirus infection. Adenovirus infection induced the rapid activation of Raf-1 and a transient increase in the tyrosine phosphorylation and activation of p42mapk at early times postinfection. Activation of the Raf/MAPK pathway by adenovirus is likely triggered by the infection process, since it occurred rapidly and with various mutant adenoviruses and adenovirus vectors. Moreover, interleukin-8 (IL-8) mRNA accumulation was evident at 20 min postinfection and was induced even in the presence of cycloheximide. Both MAPK activation and IL-8 production were inhibited by forskolin, a potent inhibitor of Raf-1. These results suggest that adenovirus-induced Raf/MAPK activation contributes to IL-8 production. Adenovirus-induced activation of the Raf/MAPK signaling pathway and IL-8 production may play critical roles in the inflammation observed following in vivo administration of adenovirus vectors for gene therapy. PMID:8985363

  11. Effect of CD4 gene expression on adenovirus replication.

    PubMed Central

    Hotta, J; Shi, L; Ginsberg, H S

    1994-01-01

    The gene encoding the CD4 receptor was introduced into KB cells to establish the KBT4 cell line, a cell line susceptible to infection with human immunodeficiency virus type 1. Adenovirus replication was found to be significantly less in these cells than in the parental KB cells. Similar decreased adenovirus type 5 (Ad5) replication occurred in HeLaT4 cells compared with the original HeLa cells. The presence of CD4 did not alter the cell surface population of KB cell adenovirus receptors, since viral adsorption was similar in the two cell lines. Moreover, addition of soluble CD4 did not reduce viral replication in either KB or KBT4 infected cells. Uncoating of viral DNA was also unchanged in KBT4 cells compared with the parental KB cells. In contrast, migration to or entrance of viral DNA into nuclei and synthesis of early viral RNAs was delayed and reduced in KBT4 cells. These effects were more pronounced for Ad7 than for Ad5. The yields of infectious viruses were the same in both cell lines, however, after transfection of naked viral DNAs to initiate infection. These results imply that the expression of the CD4 gene in KBT4 cells interfered with passage of uncoated virus across endosomal vesicles and/or transfer of uncoated core viral DNA into the nucleus. Images PMID:7933112

  12. Effect of CD4 gene expression on adenovirus replication.

    PubMed

    Hotta, J; Shi, L; Ginsberg, H S

    1994-11-01

    The gene encoding the CD4 receptor was introduced into KB cells to establish the KBT4 cell line, a cell line susceptible to infection with human immunodeficiency virus type 1. Adenovirus replication was found to be significantly less in these cells than in the parental KB cells. Similar decreased adenovirus type 5 (Ad5) replication occurred in HeLaT4 cells compared with the original HeLa cells. The presence of CD4 did not alter the cell surface population of KB cell adenovirus receptors, since viral adsorption was similar in the two cell lines. Moreover, addition of soluble CD4 did not reduce viral replication in either KB or KBT4 infected cells. Uncoating of viral DNA was also unchanged in KBT4 cells compared with the parental KB cells. In contrast, migration to or entrance of viral DNA into nuclei and synthesis of early viral RNAs was delayed and reduced in KBT4 cells. These effects were more pronounced for Ad7 than for Ad5. The yields of infectious viruses were the same in both cell lines, however, after transfection of naked viral DNAs to initiate infection. These results imply that the expression of the CD4 gene in KBT4 cells interfered with passage of uncoated virus across endosomal vesicles and/or transfer of uncoated core viral DNA into the nucleus.

  13. Fluctuating expression of microRNAs in adenovirus infected cells.

    PubMed

    Zhao, Hongxing; Chen, Maoshan; Tellgren-Roth, Christian; Pettersson, Ulf

    2015-04-01

    The changes in cellular microRNA (miRNA) expression during the course of an adenovirus type 2 infection in human lung fibroblast were studied by deep RNA sequencing. Expressions of 175 miRNAs with over 100 transcripts per million nucleotides were changed more than 1.5-fold. The expression patterns of these miRNAs changed dramatically during the course of the infection, from upregulation of the miRNAs known as tumor suppressors (such as miR-22, miR-320, let-7, miR-181b, and miR-155) and down-regulation of oncogenic miRNAs (such as miR-21 and miR-31) early to downregulation of tumor suppressor miRNAs (such as let-7 family, mir-30 family, 23/27 cluster) and upregulation of oncogenic miRNAs (include miR-125, miR-27, miR-191) late after infection. The switch in miRNA expression pattern occurred when adenovirus DNA replication started. Furthermore, deregulation of cellular miRNA expression was a step-wise and special sets of miRNAs were deregulated in different phases of infection.

  14. Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes.

    PubMed

    Speiseder, Thomas; Hofmann-Sieber, Helga; Rodríguez, Estefanía; Schellenberg, Anna; Akyüz, Nuray; Dierlamm, Judith; Spruss, Thilo; Lange, Claudia; Dobner, Thomas

    2017-01-01

    embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells. Copyright © 2016 American Society for Microbiology.

  15. Transformation of Hamster Embryo Cells and Tumor Induction in Newborn Hamsters by Simian Adenovirus SV11

    PubMed Central

    Casto, Bruce C.

    1969-01-01

    Simian adenovirus, SV11, readily transformed hamster embryo cell cultures in vitro and produced tumors in vivo when inoculated into newborn hamsters. Foci consisting of small, loosely attached, rounded cells could be seen as early as 7 days postinoculation. Many of these cells contained several nuclei or the nucleus was multilobed. The cells grew without extensive cell to cell contact or formed small chains or clusters when passaged in vitro. This pattern of cell morphology and growth has not been reported with other simian or human adenovirus-transformed cells. Linearity of foci formation with virus dilution was observed when the virus multiplicity was less than 3 plaque-forming units (PFU)/cell. The PFU to focus-forming units ratio for SV11 was found to be 2 × 104 to 4 × 104, which is approximately 5- to 10-fold and 50- to 100-fold lower than those reported for simian adenovirus, SA7, and human adenovirus type 12, respectively. Cells transformed by SV11: (i) produced tumors when inoculated into young hamsters, (ii) contained tumor antigen which reacts with serum obtained from hamsters bearing SV11 passaged tumors, and (iii) could be propagated in vitro through an indefinite number of generations. Images PMID:5786181

  16. Human chromosome 11 suppresses the tumorigenicity of adenovirus transformed baby rat kidney cells: involvement of the Wilms' tumor 1 gene.

    PubMed

    Menke, A L; van Ham, R C; Sonneveld, E; Shvarts, A; Stanbridge, E J; Miyagawa, K; van der Eb, A J; Jochemsen, A G

    1995-09-27

    Human chromosome 11 was introduced into adenovirus-transformed baby rat kidney (BRK) cells by microcell-mediated chromosome transfer. The resulting microcell hybrids (MCHs) showed a reduced ability to form tumors upon s.c. injection into athymic mice. Further analysis, with the use of defined deletion chromosomes of 11p, indicated that the presence of region 11p13-p12 is necessary for the suppression of tumorigenicity. In contrast, the presence of region 11p15-14.1 appeared to increase the rate of tumor growth. Expression studies on the human Wilms' tumor I (WTI) and the insulin-like growth factor II (IGF-II) genes, which lie in regions 11p13 and 11p15, respectively, suggested the involvement of both genes in determining the degree of suppression of tumorigenicity. Finally, stable expression of a murine WTI protein in the adenovirus-transformed cells resulted in almost complete suppression of tumorigenicity, establishing the WTI protein as a tumor suppressor in this cell system.

  17. Adenovirus Type 5 E4 Open Reading Frame 4 Protein Induces Apoptosis in Transformed Cells

    PubMed Central

    Shtrichman, Ronit; Kleinberger, Tamar

    1998-01-01

    Adenovirus type 5 E4 open reading frame 4 (E4orf4) protein has been previously shown to counteract transactivation of the junB and c-fos genes by cyclic AMP plus E1A protein and to interact with protein phosphatase 2A (PP2A). Here, we show that the wild-type E4orf4 protein induces apoptosis in the E1A-expressing 293 cells, in NIH 3T3 cells transformed with v-Ras, and in the lung carcinoma cell line H1299. The induction of apoptosis is not accompanied by enhanced levels of p53 in 293 cells and occurs in the absence of p53 in H1299 cells, indicating involvement of a p53-independent pathway. A mutant E4orf4 protein that had lost the ability to induce apoptosis also lost its ability to bind PP2A. We suggest that E4orf4 antagonizes continuous signals to proliferate, like those given by E1A or v-Ras, and that the conflicting signals lead to the induction of cell death. PMID:9525619

  18. Tumorigenicity and adenovirus-transformed cells: Collagen interaction and cell surface laminin are controlled by the serotype origin of the E1A and E1B genes

    SciTech Connect

    Bober, F.J.; Birk, D.E.; Raska, K. Jr. ); Shenk, T. )

    1988-02-01

    A library of cells transformed with recombinant adenoviruses was used to study tumorigenicity and interaction with extracellular matrix. Cells expressing the complete E1 region of highly oncogenic adenovirus type 12 (Ad12) are tumorigenic, adhere preferentially to type IV collagen, and express cell surface laminin. Weakly tumorigenic cells, which express the E1A oncogene of Ad12 and the E1B genes of Ad5, also attach preferentially to type IV collagen but do not contain laminin on their surface. Cells which express the E1A oncogene of Ad5 and the E1B genes of Ad12 are nontumorigenic and do not preferentially attach to type IV versus type I collagen but have laminin on their surface. There is no significant difference in the amounts of laminin secreted into the culture medium among cells expressing the E1B genes of Ad5 or Ad12. In vitro assays show that cells which express the E1B genes of Ad12, irrespective of the origin of the E1A genes, can bind three times more exogenously added {sup 125}I-laminin than cells expressing the E1B genes of nononcogenic Ad5. The interaction of adenovirus-transformed cells with collagen is controlled by the serotype origin of the E1A oncogene, whereas cell surface laminin is controlled by the serotype origin of the E1B genes.

  19. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  20. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  1. Adenovirus-mediated gene delivery of interleukin-10, but not transforming growth factor β, ameliorates the induction of Graves’ hyperthyroidism in BALB/c mice

    PubMed Central

    Saitoh, O; Mizutori, Y; Takamura, N; Yamasaki, H; Kita, A; Kuwahara, H; Nagayama, Y

    2005-01-01

    Interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) are well known anti-inflammatory cytokines. We have studied the effect of adenovirus-mediated IL-10 and TGF-β gene delivery on the induction of Graves’ hyperthyroidism in our mouse model that involves repeated injections of adenovirus expressing the thyrotropin receptor A subunit (AdTSHR). We first constructed adenoviruses encoding the two cytokines (AdIL10 and AdTGFβ) and confirmed expression by in vitro infection of COS cells. Susceptible BALB/c mice were injected twice with AdTSHR alone or together with AdIL10 or AdTGFβ, and bled two weeks after the second immunization. Significantly elevated serum thyroxine levels were seen in 26% of mice immunized with AdTSHR and AdIL10 versus 61% with AdTSHR alone. Levels of thyroid stimulating antibody, but not nonstimulating antibody, were also decreased, and TSHR-specific splenocyte secretion of interferon-γ in recall assays was impaired in mice treated with AdIL10. In contrast, AdTGFβ had little effect on hyperthyroidism. Overall, our findings demonstrate that gene delivery of IL-10, but not TGF-β, suppresses the induction of Graves’ hyperthyroidism in a mouse model. However, the effect of IL-10 is less powerful than we observed previously with T helper type 2-inducers including adenovirus expressing IL-4, Shistosoma mansoni infection or α-galactosylceramide. PMID:16045729

  2. Adenovirus type 2 expresses fiber in monkey-human hybrids and reconstructed cells

    SciTech Connect

    Zorn, G.A.; Anderson, C.W.

    1981-02-01

    Adenovirus type 2 protein expression was measured by indirect immunofluorescence in monkey-human hybrids and in cells reconstructed from monkey and human cell karyoplasts and cytoplasts. Monkey-human hybrid clones infected with adenovirus type 2 expressed fiber protein, whereas infected monkey cells alone did not. Hybrids constructed after the parental monkey cells were infected with adenovirus type 2 demonstrated that fiber synthesis in these cells could be rescued by fusion to uninfected human cells. Thus, human cells contain a dominant factor that acts in trans and overcomes the inability of monkey cells to synthesize fiber. These results are consistent with the hypothesis that the block to adenovirus replication in monkey cells involves a nuclear event that prevents the formation of functional mRNA for some late viral proteins including fiber polypeptide.

  3. Hamster embryo cells transformed by herpes simplex virus: reactions with adeno-associated satellite virus (AAV) and its adenovirus helpers.

    PubMed

    Mayor, H D; Jordan, L E; Gorman, C

    1977-01-01

    We have studied the reactions of hamster embryo cells transformed by ultraviolet-inactivated herpes simplex type 2 (333-8-9 T cells) to infections with adeno-associated satellite virus (AAV) and its adenovirus helpers. Resident HSV structural antigens were not detectable in early or late passage of 333-8-9 T cells. AAV structural antigens were not detected in these cells unless the cells were coinfected with a helper adenovirus. In early passage 333-8-9 T cells were permissive to infections with simian adenovirus SV15 whereas normal hamster cell line LSH was nonpermissive. In some late passages of 333-8-9 T cells infections with SV15 adenovirus led to the production of viruslike particles whose morphology was identical with reoviruses.

  4. Transformation of human diploid cells by adenovirus type 4 irradiated with ultraviolet light.

    PubMed

    Hozoc, M; Nastac, E; Suru, M; Stoian, M; Bercovici, S; Cajal, N

    1983-01-01

    Inoculation of ultraviolet (UV)-irradiated adenovirus type 4 (Ad4) led to in vitro transformation of human diploid cells (HDC). Two transformed cell lines could be established: cell line H 1418, from HDC inoculated with the 10(-3) dilution of Ad4 UV-irradiated for 20 min at a distance of 20 cm, co-cultivated with uninfected HDC, and cell line H 1557, from HDC inoculated with the 10(-2) dilution of Ad4 irradiated at the same distance for 12 min. Both transformed cell lines were resistant to superinfection with homologous virus. Virus-specific antigen could be made evident by the indirect immunofluorescence technique in the nuclei of both H 1418 and H 1557 cells.

  5. Transforming Region of Group A, B, and C Adenoviruses: DNA Homology Studies with Twenty-Nine Human Adenovirus Serotypes

    PubMed Central

    Mackey, Jesse K.; Wold, William S. M.; Rigden, Patricia; Green, Maurice

    1979-01-01

    The 31 human adenovirus (Ad) serotypes form five groups based upon DNA genome homologies: group A (Ad12, 18, 31), group B (Ad3, 7, 11, 14, 16, 21), group C (Ad1, 2, 5, 6), group D (Ad8, 9, 10, 13, 15, 17, 19, 20, 22-30), and group E (Ad4) (M. Green, J. Mackey, W. Wold, and P. Rigden, Virology, in press). Group A Ads are highly oncogenic in newborn hamsters, group B Ads are weakly oncogenic, and other Ads are nononcogenic. However, most or all Ads transform cultured cells. We have studied the homology of Ad5, Ad7, and Ad12 transforming restriction endonuclease DNA fragments with DNAs of 29 Ad types. Ad5 HindIII-G (map position 0-7.3), Ad7 XhoI-C (map position 0-10.8), and Ad12 (strain Huie) EcoRI-C (map position 0-16) and SalI-C (map position 0-10.6) fragments were purified, labeled in vitro (nick translation), and annealed with DNAs of Ad1 to Ad16, Ad18 to Ad24, and Ad26 to Ad31. Hybrids were assayed by using hydroxylapatite. Ad5 HindIII-G hybridized 98 to 100% with DNAs of group C Ads, but only 1 to 15% with DNAs of other types. Ad7 XhoI-C fragment hybridized 85 to 99% with DNAs of group B Ads, but only 6 to 21% with DNAs of other types. Ad12 (Huie) EcoRI-C hybridized 53 to 68% with DNAs of five other Ad12 strains, 53% with Ad18 DNA, 56% with Ad31 DNA, but only 3 to 13% with DNAs of other types. In vitro-labeled Ad12 (Huie) SalI-C hybridized 35 to 71% with DNAs of 6 other Ad12 strains, 44% with Ad18 DNA, 52% with Ad31 DNA, but only 2 to 7% with DNAs Ad7, Ad2, Ad26, or Ad4. When assayed using S-1 nuclease, SalI-C annealed 17 to 44% with DNAs of group A Ads. The melting temperatures of the hybrids of Ad5 HindIII-G with all group C Ad DNAs were 84°C in 0.12 M sodium phosphate (pH 6.8). The melting temperature of the Ad12 (Huie) EcoRI-C hybrid with Ad12 (Huie) DNA was 83°C, but was only 71 to 77°C with DNAs of other group A Ads. Thus, group C and group B Ads both have very homologous transforming regions that are not represented in DNAs of non-group C Ads or non

  6. Role of coxsackievirus and adenovirus receptor (CAR) expression and viral load of adenovirus and enterovirus in patients with dilated cardiomyopathy.

    PubMed

    Sharma, Mirnalini; Mishra, Baijayantimala; Saikia, Uma Nahar; Bahl, Ajay; Ratho, Radha Kanta; Talwar, Kewal Kishan

    2016-01-01

    Enteroviruses (EVs) and adenoviruses (AdVs) are two important etiological agents of viral myocarditis and dilated cardiomyopathy (DCM). Both these viruses share a common receptor, the coxsackievirus and adenovirus receptor (CAR), for their infection. However, the role of viral load and CAR expression in disease severity has not yet been completely elucidated. The present study aimed to determine viral load of EV and AdV in DCM patients and correlate them with the level of CAR expression in these patients. Sixty-three DCM cases and 30 controls, each of whom died of heart disease other than DCM and non-cardiac disease respectively, were included. Viral load was determined by TaqMan real-time PCR using primers and probes specific for the AdV hexon gene and the 5'UTR region of EV. The CAR mRNA level was semi-quantitated by RT-PCR, and antigen expression was studied by immunohistochemistry. A significantly high AdV load (p < 0.05) and CAR expression (p < 0.05) were observed in DCM cases versus controls, whereas the EV load showed no significant difference. The data suggests a clinical threshold of 128 AdV copies/500 ng of DNA for DCM, with 66.7 % sensitivity and 65 % specificity. A positive correlation between AdV load and CAR expression (p < 0.001) was also observed in DCM cases. The high adenoviral load and increased CAR expression in DCM and their association with adverse disease outcome indicates role of both virus and receptor in disease pathogenesis. Thus, the need for targeting both the virus and the receptor for treatment of viral myocarditis and early DCM requires further confirmation with larger studies.

  7. Infectivity and expression of the early adenovirus proteins are important regulators of wild-type and DeltaE1B adenovirus replication in human cells.

    PubMed

    Steegenga, W T; Riteco, N; Bos, J L

    1999-09-09

    An adenovirus mutant lacking the expression of the large E1B protein (DeltaE1B) has been reported to replicate selectively in cells lacking the expression of functionally wild-type (wt) p53. Based on these results the DeltaE1B or ONYX-015 virus has been proposed to be an oncolytic virus which might be useful to treat p53-deficient tumors. Recently however, contradictory results have been published indicating that p53-dependent cell death is required for productive adenovirus infection. Since there is an urgent need for new methods to treat aggressive, mutant p53-expressing primary tumors and their metastases we carefully examined adenovirus replication in human cells to determine whether or not the DeltaE1B virus can be used for tumor therapy. The results we present here show that not all human tumor cell lines take up adenovirus efficiently. In addition, we observed inhibition of the expression of adenovirus early proteins in tumor cells. We present evidence that these two factors rather than the p53 status of the cell determine whether adenovirus infection results in lytic cell death. Furthermore, the results we obtained by infecting a panel of different tumor cell lines show that viral spread of the DeltaE1B is strongly inhibited in almost all p53-proficient and -deficient cell lines compared to the wt virus. We conclude that the efficiency of the DeltaE1B virus to replicate efficiently in tumor cells is determined by the ability to infect cells and to express the early adenovirus proteins rather than the status of p53.

  8. Adenovirus E4orf4 Protein Downregulates MYC Expression through Interaction with the PP2A-B55 Subunit▿

    PubMed Central

    Ben-Israel, Haggit; Sharf, Rakefet; Rechavi, Gideon; Kleinberger, Tamar

    2008-01-01

    The adenovirus E4 open reading frame 4 (E4orf4) protein is a multifunctional viral regulator that is involved in the temporal regulation of viral gene expression by modulating cellular and viral genes at the transcription and translation levels and by controlling alternative splicing of adenoviral late mRNAs. When expressed individually, E4orf4 induces apoptosis in transformed cells. Using oligonucleotide microarray analysis, validated by quantitative real time PCR, we found that MYC (also known as c-Myc) is downregulated early after the induction of E4orf4 expression. As a result, Myc protein levels are reduced in E4orf4-expressing cells. MYC downregulation is observed both when E4orf4 is expressed individually and within the context of viral infection. E4orf4 reduces MYC transcription but does not affect transcriptional elongation or RNA stability. An interaction with the PP2A-B55 subunit is required for the downregulation of MYC by E4orf4. Since Myc overexpression was previously shown to inhibit adenovirus replication, the downregulation of Myc by E4orf4 would contribute to efficient virus infection. PMID:18653458

  9. Adenovirus E4orf4 protein downregulates MYC expression through interaction with the PP2A-B55 subunit.

    PubMed

    Ben-Israel, Haggit; Sharf, Rakefet; Rechavi, Gideon; Kleinberger, Tamar

    2008-10-01

    The adenovirus E4 open reading frame 4 (E4orf4) protein is a multifunctional viral regulator that is involved in the temporal regulation of viral gene expression by modulating cellular and viral genes at the transcription and translation levels and by controlling alternative splicing of adenoviral late mRNAs. When expressed individually, E4orf4 induces apoptosis in transformed cells. Using oligonucleotide microarray analysis, validated by quantitative real time PCR, we found that MYC (also known as c-Myc) is downregulated early after the induction of E4orf4 expression. As a result, Myc protein levels are reduced in E4orf4-expressing cells. MYC downregulation is observed both when E4orf4 is expressed individually and within the context of viral infection. E4orf4 reduces MYC transcription but does not affect transcriptional elongation or RNA stability. An interaction with the PP2A-B55 subunit is required for the downregulation of MYC by E4orf4. Since Myc overexpression was previously shown to inhibit adenovirus replication, the downregulation of Myc by E4orf4 would contribute to efficient virus infection.

  10. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.

    PubMed Central

    Henry, L J; Xia, D; Wilke, M E; Deisenhofer, J; Gerard, R D

    1994-01-01

    The adenovirus fiber protein is used for attachment of the virus to a specific receptor on the cell surface. Structurally, the protein consists of a long, thin shaft that protrudes from the vertex of the virus capsid and terminates in a globular domain termed the knob. To verify that the knob is the domain which interacts with the cellular receptor, we have cloned and expressed the knob from adenovirus type 5 together with a single repeat of the shaft in Escherichia coli. The protein was purified by conventional chromatography and functionally characterized for its interaction with the adenovirus receptor. The recombinant knob domain bound about 4,700 sites per HeLa cell with an affinity of 3 x 10(9) M-1 and blocked adenovirus infection of human cells. Antibodies raised against the knob also blocked virus infection. By gel filtration and X-ray diffraction analysis of protein crystals, the knob was shown to consist of a homotrimer of 21-kDa subunits. The results confirm that the trimeric knob is the ligand for attachment to the adenovirus receptor. Images PMID:8035520

  11. Helper-Dependent Adenovirus is Superior to First-Generation Adenovirus for Expressing Transgenes in Atherosclerosis-Prone Arteries

    PubMed Central

    Jiang, Bo; Qian, Kun; Du, Liang; Luttrell, Ian; Chitaley, Kanchan; Dichek, David A.

    2011-01-01

    Objective- Vascular gene transfer is a powerful tool for investigating and treating vascular diseases; however, its utility is limited by brevity of transgene expression and vector-associated inflammation. Helper-dependent adenovirus (HDAd), an advanced-generation Ad that lacks all viral genes, is superior to first-generation Ad (FGAd) in normal rabbit arteries. We compared HDAd to FGAd in arteries of cholesterol-fed rabbits, a model of early atherogenesis in which transgene expression might be decreased, and inflammation increased. Methods and Results- Carotid arteries of chow- and cholesterol-fed rabbits were infused with FGAd, HDAd, or medium. HDAd expressed a transgene at least as well in arteries of cholesterol-fed rabbits as in arteries of chow-fed rabbits and expressed more durably than FGAd. In arteries of cholesterol-fed rabbits, HDAd stimulated less intimal growth, lipid deposition, and inflammation than FGAd. Neither vector affected phenylephrine-induced contraction or nitroprusside-mediated relaxation; however, both vectors decreased maximal acetylcholine-stimulated vasorelaxation. Relative absence of intimal growth in HDAd arteries could interfere with the utility of this model for testing atheroprotective genes; however, both co-infusion of FGAd and extension of cholesterol feeding yielded larger intimal lesions, on which atheroprotective genes could be tested. Conclusions- HDAd is superior to FGAd for expression of transgenes in atherosclerosis-prone arteries. PMID:21454808

  12. Evaluation of adenovirus capsid labeling versus transgene expression

    PubMed Central

    2010-01-01

    Adenoviral vectors have been utilized for a variety of gene therapy applications. Our group has incorporated bioluminescent, fluorographic reporters, and/or suicide genes within the adenovirus genome for analytical and/or therapeutic purposes. These molecules have also been incorporated as capsid components. Recognizing that incorporations at either locale yield potential advantages and disadvantages, our report evaluates the benefits of transgene incorporation versus capsid incorporation. To this end, we have genetically incorporated firefly luciferase within the early region 3 or at minor capsid protein IX and compared vector functionality by means of reporter readout. PMID:20102632

  13. Enhanced therapeutic efficacy of an adenovirus-PEI-bile-acid complex in tumors with low coxsackie and adenovirus receptor expression.

    PubMed

    Lee, Cho-Hee; Kasala, Dayananda; Na, Youjin; Lee, Min Sang; Kim, Sung Wan; Jeong, Ji Hoon; Yun, Chae-Ok

    2014-07-01

    Adenovirus (Ad) is a potential vehicle for cancer gene therapy. However, cells that express low levels of the coxsackie and adenovirus receptor (CAR) demonstrate poor Ad infection efficiency. We developed a bile acid-conjugated poly(ethyleneimine) (DA3)-coated Ad complex (Ad/DA3) to enhance Ad transduction efficiency. The size distribution and zeta potential of Ad/DA3 increased to 324 ± 3.08 nm and 10.13 ± 0.21 mV, respectively, compared with those of naked Ad (108 ± 2.26 nm and -17.7 ± 1.5 mV). The transduction efficiency of Ad/DA3 increased in a DA3 polymer concentration-dependent manner. Enhanced gene transfer by Ad/DA3 was more evident in CAR-moderate and CAR-negative cancer cells. Competition assays with a CAR-specific antibody revealed that internalization of Ad/DA3 was not mediated primarily by CAR but involved clathrin-, caveolae-, and macropinocytosis-mediated endocytosis. Cancer cell death was significantly increased when oncolytic Ad and DA3 were complexed (RdB-KOX/DA3) compared to that of naked oncolytic Ad and was inversely proportional to CAR levels. Importantly, RdB-KOX/DA3 significantly enhanced apoptosis, reduced angiogenesis, reduced proliferation, and increased active viral replication in human tumor xenografts compared to that of naked Ad. These results demonstrate that a hybrid vector system can increase the efficacy of oncolytic Ad virotherapy, particularly in CAR-limited tumors.

  14. Adenovirus type 2 expresses fiber in monkey-human hybrids and reconstructed cells.

    PubMed Central

    Zorn, G A; Anderson, C W

    1981-01-01

    Adenovirus type 2 protein expression was measured by indirect immunofluorescence in monkey-human hybrids and in cells reconstructed from monkey and human cell karyoplasts and cytoplasts. Monkey-human hybrid clones infected with adenovirus type 2 expressed fiber protein, whereas infected monkey cells alone did not. Hybrids constructed after the parental monkey cells were infected with adenovirus type 2 demonstrated that fiber synthesis in these cells could be rescued by fusion to uninfected human cells. Thus, human cells contain a dominant factor that acts in trans and overcomes the inability of monkey cells to synthesize fiber. Cells reconstructed from infected human karyoplasts and monkey cytoplasts expressed fiber, whereas cells reconstructed from infected monkey karyoplasts and human cytoplasts did not. These results are consistent with the hypothesis that the block to adenovirus replication in monkey cells involves a nuclear event that prevents the formation of functional mRNA for some late viral proteins including fiber polypeptide. Furthermore, they suggest that the translational apparatus of monkey cells is competent to translate functional fiber mRNA synthesized in human cells. Images PMID:7218436

  15. The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha.

    PubMed Central

    White, E; Sabbatini, P; Debbas, M; Wold, W S; Kusher, D I; Gooding, L R

    1992-01-01

    The adenovirus E1A and E1B proteins are required for transformation of primary rodent cells. When expressed in the absence of the 19,000-dalton (19K) E1B protein, however, the E1A proteins are acutely cytotoxic and induce host cell chromosomal DNA fragmentation and cytolysis, analogous to cells undergoing programmed cell death (apoptosis). E1A alone can efficiently initiate the formation of foci which subsequently undergo abortive transformation whereby stimulation of cell growth is counteracted by continual cell death. Cell lines with an immortalized growth potential eventually arise with low frequency. Coexpression of the E1B 19K protein with E1A is sufficient to overcome abortive transformation to produce high-frequency transformation. Like E1A, the tumoricidal cytokine tumor necrosis factor alpha (TNF-alpha) evokes a programmed cell death response in many tumor cell lines by inducing DNA fragmentation and cytolysis. Expression of the E1B 19K protein by viral infection, by transient expression, or in transformed cells completely and specifically blocks this TNF-alpha-induced DNA fragmentation and cell death. Cosegregation of 19K protein transforming activity with protection from TNF-alpha-mediated cytolysis demonstrates that both activities are likely the consequence of the same function of the protein. Therefore, we propose that by suppressing an intrinsic cell death mechanism activated by TNF-alpha or E1A, the E1B 19K protein enhances the transforming activity of E1A and enables adenovirus to evade TNF-alpha-dependent immune surveillance. Images PMID:1317006

  16. Transforming genes among three different oncogenic subgroups of human adenoviruses have similar replicative functions.

    PubMed Central

    Brusca, J S; Chinnadurai, G

    1981-01-01

    We have examined the functional similarity of the transforming genes for replicative functions among three different subgroups of human adenoviruses (A, B, and C), using mutant complementation as an assay. A host range deletion mutant (dl201.2) of Ad2 (nononcogenic subgroup C) lacking about 5% of the viral DNA covering two early gene blocks (E1a and E1b) involved in cellular transformation was isolated and tested for its ability to replicate in nonpermissive KB cells in the presence of Ad7 (weakly oncogenic group B) or ad12 (highly oncogenic group A). The complementation of the mutant defect was demonstrated by cleaving the viral DNA extracted from mixed infected cells or the DNA extracted from purified virions from mixed infected cells with restriction endonuclease BamHI, which produces a different cleavage pattern with the DNA of each serotype. It was found that the defects in E1a plus E1b of dl201.2 could be complemented by Ad7 and Ad12, indicating that these genes in Ad2, Ad7, and Ad12 have similar functions during productive infection. Images PMID:7277578

  17. Oncolytic adenovirus expressing soluble TGFβ receptor II-Fc-mediated inhibition of established bone metastases: a safe and effective systemic therapeutic approach for breast cancer.

    PubMed

    Hu, Zebin; Gerseny, Helen; Zhang, Zhenwei; Chen, Yun-Ju; Berg, Arthur; Zhang, Zhiling; Stock, Stuart; Seth, Prem

    2011-09-01

    In recent years, oncolytic adenoviruses have shown some promise as a novel class of antitumor agents. However, their utility in targeting bone metastases is relatively less studied. We have examined whether the systemic therapy of oncolytic adenoviruses expressing the soluble form of transforming growth factor-β (TGFβ) receptor II fused with human immunoglobulin G1 can be developed for the treatment of established breast cancer bone metastases. MDA-MB-231-luc2 human breast cancer cells were injected in the left heart ventricle of nude mice to establish bone metastasis. Mice with hind limb tumors were administered (on days 8 and 11) oncolytic adenoviruses-Ad.sTβRFc or mhTERTAd.sTβRFc. Skeletal tumor growth was monitored weekly by bioluminescence imaging (BLI) and radiography. At the termination time on day 28, hind limb bones were analyzed for tumor burden, synchrotron micro-computed tomography, and osteoclast activation. Intravenous delivery of Ad.sTβRFc and mhTERTAd.sTβRFc induced significant inhibition of tumor growth, reduction of tumor burden, osteoclast activation, and increased animals' survival. Oncolytic adenoviruses were safer than dl309, a wild-type virus. A slight elevation of liver enzyme activity was observed after Ad.sTβRFc administration; this subsided with time. Based on these studies, we believe that Ad.sTβRFc and mhTERTAd.sTβRFc can be developed as a safe and effective approach for the treatment of established bone metastasis.

  18. Expression of the gene encoding secreted placental alkaline phosphatase (SEAP) by a nondefective adenovirus vector.

    PubMed

    Doronin, K K; Zakharchuk, A N; Grinenko, N F; Yurov, G K; Krougliak, V A; Naroditsky, B S

    1993-04-30

    A nondefective recombinant human adenovirus 5 (Ad5) carrying the SEAP gene, encoding human secreted placental alkaline phosphatase, in the E3 region of the Ad5 genome was obtained. The expression of SEAP at the early and late stages of Ad5 infection was demonstrated in permissive and semi-permissive cell cultures. The amount of SEAP in the culture medium of the 293 cells was 13.6% of the total protein.

  19. Adenovirus-mediated GDF-5 promotes the extracellular matrix expression in degenerative nucleus pulposus cells*

    PubMed Central

    Luo, Xu-wei; Liu, Kang; Chen, Zhu; Zhao, Ming; Han, Xiao-wei; Bai, Yi-guang; Feng, Gang

    2016-01-01

    Objective: To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 (GDF-5) gene, investigate the biological effects of adenovirus-mediated GDF-5 (Ad-GDF-5) on extracellular matrix (ECM) expression in human degenerative disc nucleus pulposus (NP) cells, and explore a candidate gene therapy method for intervertebral disc degeneration (IDD). Methods: Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan (sGAG), deoxyribonucleic acid (DNA) and hydroxyproline (Hyp), synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results: The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium (BM) or Ad-green fluorescent protein (GFP) groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions: The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP cells. PMID:26739524

  20. Inducible Overexpression of a Toxic Protein by an Adenovirus Vector with a Tetracycline-Regulatable Expression Cassette†

    PubMed Central

    Massie, Bernard; Couture, France; Lamoureux, Linda; Mosser, Dick D.; Guilbault, Claire; Jolicoeur, Pierre; Bélanger, François; Langelier, Yves

    1998-01-01

    We have constructed two new adenovirus expression cassettes that expand both the range of genes which can be expressed with adenovirus vectors (AdV) and the range of cells in which high-level expression can be attained. By inclusion of a tetracycline-regulated promoter in the transfer vector pAdTR5, it is now possible to generate recombinant adenoviruses expressing proteins that are either cytotoxic or that interfere with adenovirus replication. We have used this strategy to generate a recombinant adenovirus encoding a deletion in the R1 subunit [R1(Δ2-357)] of the herpes simplex virus type 2 ribonucleotide reductase. Cell lines expressing the tetracycline-regulated transactivator (tTA) from an integrated vector or following infection with an AdV expressing tTA are able to produce ΔR1 protein at a level approaching 10% total cell protein (TCP) when infected with Ad5TR5ΔR1 before they subsequently die. To our knowledge, this is the first report of the overexpression of a toxic gene product with AdV. We have also constructed a new constitutive adenovirus expression cassette based on an optimized cytomegalovirus immediate-early promoter-enhancer that allows the expression of recombinant proteins at a level greater than 20% TCP in nonpermissive cell lines. Together, these new expression cassettes significantly improve the utility of the adenovirus system for high-level expression of recombinant proteins in animal cells and will undoubtedly find useful applications in gene therapy. PMID:9499088

  1. Receptor-targeted recombinant adenovirus conglomerates: a novel molecular conjugate vector with improved expression characteristics.

    PubMed Central

    Schwarzenberger, P; Hunt, J D; Robert, E; Theodossiou, C; Kolls, J K

    1997-01-01

    To develop improved strategies for gene transfer to hematopoietic cells, we have explored targeted gene transfer using molecular conjugate vectors (MCVs). MCVs are constructed by condensing plasmid DNA containing the gene of interest with polylysine (PL), PL linked to a replication-incompetent adenovirus (endosomolytic agent), and PL linked to streptavidin for targeting with biotinylated ligands. In this report, we compare gene transfer to K562 cells by using the previously described transferrin-targeted MCV (Trans-MCV) to a novel transferrin-targeted MCV. In the novel MCV, the transferred gene (luciferase) is in the genome of recombinant replication-incompetent adenovirus (recMCV), which also acts as the endosomolytic agent. The level of luciferase gene expression was fivefold higher in K562 cells transfected with Trans-recMCV than in cells transfected with Trans-MCV. Furthermore, targeted transfection with recMCV resulted in prolonged luciferase expression that declined 14 to 20 days after transfection, in comparison with Trans-MCV, where luciferase expression declined by 4 to 8 days. Moreover, targeted transfection of K562 cells with the Trans-recMCV resulted in persistent luciferase gene expression for 6 months. Analysis of luciferase gene expression in K562 single-cell clones that were subcloned 5 weeks after transfection with Trans-recMCV showed that 35 to 50% of the single-cell clones had intermediate to high levels of luciferase gene expression that was stable for 6 months, with the remaining clones showing low or no luciferase gene expression. Stable gene expression was associated with integration of adenovirus sequences into genomic DNA. PMID:9343214

  2. Cutting edge: adenovirus E19 has two mechanisms for affecting class I MHC expression.

    PubMed

    Bennett, E M; Bennink, J R; Yewdell, J W; Brodsky, F M

    1999-05-01

    Viral strategies for immune evasion include inhibition of various steps in the class I MHC assembly pathway. Here, we demonstrate that adenovirus produces one gene product with a dual function in this regard. It is well established that adenovirus E19 binds class I molecules and retains them in the endoplasmic reticulum (ER). However, E19 also delays the expression of class I alleles to which it cannot tightly bind. Here, we show that E19 binds TAP and acts as a tapasin inhibitor, preventing class I/TAP association. DeltaE19, an E19 mutant lacking the ER-retention signal, delays maturation of class I molecules, indicating that E19's inhibition of class I/TAP interaction is sufficient to delay class I expression. These data identify tapasin inhibition as a novel mechanism of viral immune evasion and suggest that, through this secondary mechanism, adenovirus can affect Ag presentation by MHC alleles that it can only weakly affect by direct retention.

  3. Expression of the Coxsackievirus and Adenovirus Receptor in Cultured Human Umbilical Vein Endothelial Cells: Regulation in Response to Cell Density

    PubMed Central

    Carson, Steven D.; Hobbs, Justin T.; Tracy, Steven M.; Chapman, Nora M.

    1999-01-01

    Primary cultures of human umbilical vein endothelial cells (HUVEC) express the human coxsackievirus and adenovirus receptor (HCAR). Whereas HCAR expression in HeLa cells was constant with respect to cell density, HCAR expression in HUVEC increased with culture confluence. HCAR expression in HUVEC was not quantitatively altered by infection with coxsackievirus B. PMID:10400813

  4. Construction and characterization of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus capsid proteins of Indian vaccine strain, O/IND/R2/75

    PubMed Central

    Kumar, Ramesh; Sreenivasa, B. P.; Tamilselvan, R. P.

    2015-01-01

    Aim: Generation of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus (FMDV) capsid protein genes along with full-length 2B, 3B and 3Cpro and its characterization. Materials and Methods: FMD viral RNA isolation, cDNA synthesis, and polymerase chain reaction were performed to synthesize expression cassettes (P1-2AB3BCwt and P1-2AB3BCm) followed by cloning in pShuttle-CMV vector. Chemically competent BJ5183-AD-1 cells were transformed with the recombinant pShuttle-CMV to produce recombinant adenoviral plasmids. HEK-293 cells were transfected with the recombinant adenoviral plasmids to generate recombinant adenoviruses (hAd5/P1-2AB3BCwt and hAd5/P1-2AB3BCm). Expression of the target proteins was analyzed by sandwich ELISA and indirect immunofluorescence assay. The recombinant adenoviruses were purified and concentrated by CsCl density gradient ultracentrifugation. Growth kinetics and thermostability of the recombinant adenoviruses were compared with that of non-recombinant replication-defective adenovirus (dAd5). Results: The recombinant adenoviruses containing capsid protein genes of the FMDV O/IND/R2/75 were generated and amplified in HEK-293 cells. The titer of the recombinant adenoviruses was approximately 108, 109.5 and 1011 TCID50/ml in supernatant media, cell lysate and CsCl purified preparation, respectively. Expression of the FMDV capsid protein was detectable in sandwich ELISA and confirmed by immunofluorescence assay. Growth kinetics of the recombinant adenoviruses did not reveal a significant difference when compared with that of dAd5. A decrement of up to 10-fold at 4°C and 21-fold at 37°C was recorded in the virus titers during 60 h incubation period and found to be statistically significant (p<0.01). Conclusion: Recombinant adenoviruses expressing capsid proteins of the FMDV O/IND/R2/75 were constructed and produced in high titers. In vitro expression of the target proteins in the adenovirus vector system was detected by

  5. Nucleotide sequence of the transforming early region E1b of adenovirus type 12 DNA: structure and gene organization, and comparison with those of adenovirus type 5 DNA.

    PubMed Central

    Kimura, T; Sawada, Y; Shinawawa, M; Shimizu, Y; Shiroki, K; Shimojo, H; Sugisaki, H; Takanami, M; Uemizu, Y; Fujinaga, K

    1981-01-01

    The nucleotide sequence of the entire transforming early region of E1b of the highly oncogenic adenovirus type 12 (Ad12) DNA has been determined. The total sequence (3860 base pairs) encompasses the entire transforming early region E1 of Ad12 DNA. From the sequence for the E1b region of Ad12, and the transcription map of the E1b region (1, 2, 3, and this paper) the structure and gene organization of the early region E1b of Ad12 DNA were analyzed and compared with those of the E1b region in the non-oncogenic Ad5 DNA (4, 5). Most of the sequences in the E1b region of Ad12 was highly homologous to that of Ad5. It is predicted that the Ad12 region E1b codes for polypeptides of 53.9, 19.1, and 8.9 kd. This situation is identical with that of the Ad5 region E1b which codes for polypeptides of 54.9, 20.6, and 8.3 kd. The function of these predicted polypeptides encoded by the E1b regions in cell transformation is discussed. PMID:6275367

  6. Identification and purification of a protein encoded by the human adenovirus type 2 transforming region.

    PubMed Central

    Green, M; Brackmann, K H; Cartas, M A; Matsuo, T

    1982-01-01

    The human adenovirus type 2 (Ad2) transforming genes are located in early regions E1a (map position 1.3 to 4.5) and E1b (map position 4.6 to 11.2). We have identified and purified to near homogeneity a major 20,000-molecular-weight (20K) protein and have shown that it is coded by E1b. Using an Ad2-transformed cell antiserum which contained antibody to E1b-coded proteins, we immunoprecipitated 53K and 19K proteins from the nucleoplasm and 53K, 19K, and 20K proteins from the cytoplasmic S-100 fraction of Ad2 productively infected and Ad2-transformed cells. The 19K protein was present in both the nucleoplasm and the cytoplasm, whereas the 20K protein was found only in the cytoplasm. The 53K and 19K proteins are known Ad2 E1b-coded proteins. The 20K protein was purified to near homogeneity in 20 to 50% yields by sequential DEAE-Sephacel chromatography and reverse-phase high-performance liquid chromatography. Purified 20K protein shares most of its methionine-labeled tryptic peptides with E1b-53K, as shown by reverse-phase high-performance liquid chromatography, and therefore is closely related to the 53K protein. The 19K protein does not appear to share tryptic peptides with either 20K or 53K protein. To provide more direct evidence that 20K protein is virus-coded, we translated E1b-specific mRNA in vitro. Both immunoprecipitation analysis and high-performance liquid chromatography purification of the translated product identified a 20K protein that has the same tryptic peptides as the 20K protein isolated from infected and from transformed cells. These findings suggest that the Ad2 20K protein is a primary translation product of an Ad2 E1b mRNA. Images PMID:7045392

  7. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

    PubMed

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B; Iozzo, Renato V; Seth, Prem

    2015-12-01

    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases.

  8. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases

    PubMed Central

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R.; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B.; Iozzo, Renato V.; Seth, Prem

    2015-01-01

    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases. PMID:26467629

  9. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

    PubMed Central

    Stunnenberg, H G; Lange, H; Philipson, L; van Miltenburg, R T; van der Vliet, P C

    1988-01-01

    Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system. Images PMID:3362670

  10. Expression of coxsackievirus and adenovirus receptor and its cellular localization in myocardial tissues of dilated cardiomyopathy

    PubMed Central

    Kaur, Tripta; Mishra, Baijayantimala; Saikia, Uma Nahar; Sharma, Mirnalini; Bahl, Ajay; Ratho, Radha Kanta

    2012-01-01

    BACKGROUND: Myocarditis and dilated cardiomyopathy (DCM) are common causes of morbidity and mortality in children and adults. Recently, the human coxsackievirus and adenovirus receptor (CAR), a common receptor for coxsackieviruses and adenoviruses, was discovered and its increased expression has been reported in patients with DCM and myocarditis. OBJECTIVE: To measure the expression of CAR in myocardial tissues of patients with DCM and its cellular localization in DCM cases. METHODS: Formalin-fixed myocardial tissues collected during autopsy from 26 cases of DCM, and 20 cases each of noncardiac disease and cardiac disease other than DCM were included as the test group, and control groups A and B, respectively. Expression of CAR was studied using immunohistochemical staining of myocardial tissue with a CAR-specific rabbit polyclonal antibody. CAR messenger RNA was semiquantified by reverse transcription polymerase chain reaction followed by agarose gel analysis and measurement of band intensity. RESULTS: CAR positivity in DCM cases was found to be 96% (25 of 26) compared with 30% in control group A and 40% in control group B. CAR was found to be expressed in myocytes, endothelial and interstitial cells; however, positivity in myocytes was significantly higher than in other cells in all groups. The site of CAR expression was predominantly the sarcolemma along with cytoplasm in cardiomyocytes. CONCLUSIONS: The present study highlighted the increased expression of CAR in DCM cases, with localization in myocytes and endothelial cells. PMID:23592932

  11. Expression of Coxsackie-Adenovirus receptor (CAR) in the developing mouse olfactory system.

    PubMed

    Venkatraman, Giri; Behrens, Maik; Pyrski, Martina; Margolis, Frank L

    2005-09-01

    Interest in manipulating gene expression in olfactory sensory neurons (OSNs) has led to the use of adenoviruses (AdV) as gene delivery vectors. OSNs are the first order neurons in the olfactory system and the initial site of odor detection. They are highly susceptible to adenovirus infection although the mechanism is poorly understood. The Coxsackie-Adenovirus receptor (CAR) and members of the integrin family have been implicated in the process of AdV infection in various systems. Multiple serotypes of AdV efficiently bind to the CAR, leading to entry and infection of the host cell by a mechanism that can also involve integrins. Cell lines that do not express CAR are relatively resistant, but not completely immune to AdV infection, suggesting that other mechanisms participate in mediating AdV attachment and entry. Using in situ hybridization and western blot analyses, we show that OSNs and olfactory bulbs (OB) of mice express abundant CAR mRNA at embryonic and neonatal stages, with progressive diminution during postnatal development. By contrast to the olfactory epithelium (OE), CAR mRNA is still present in the adult mouse OB. Furthermore, despite a similar postnatal decline, CAR protein expression in the OE and OB of mice continues into adulthood. Our results suggest that the robust AdV infection observed in the postnatal olfactory system is mediated by CAR and that expression of even small amounts of CAR protein as seen in the adult rodent, permits efficient AdV infection and entry. CAR is an immunoglobulin domain-containing protein that bears homology to cell-adhesion molecules suggesting the possibility that it may participate in organization of the developing olfactory system.

  12. A Recombinant Adenovirus Expressing Ovine Interferon Tau Prevents Influenza Virus-Induced Lethality in Mice

    PubMed Central

    Pascual, E.; Avia, M.; Rangel, G.; de Molina, A.; Alejo, A.; Sevilla, N.

    2016-01-01

    Ovine interferon tau (IFN-τ) is a unique type I interferon with low toxicity and a broad host range in vivo. We report the generation of a nonreplicative recombinant adenovirus expressing biologically active IFN-τ. Using the B6.A2G-Mx1 mouse model, we showed that single-dose intranasal administration of recombinant Ad5-IFN-τ can effectively prevent lethality and disease induced by highly virulent hv-PR8 influenza virus by activating the interferon response and preventing viral replication. PMID:26739058

  13. The role of Cajal bodies in the expression of late phase adenovirus proteins.

    PubMed

    James, Nicola J; Howell, Gareth J; Walker, John H; Blair, G Eric

    2010-04-10

    Cajal bodies (CBs) are subnuclear structures involved in RNA metabolism. Here we show that, following infection of HeLa cells by adenovirus type 5 (Ad5), CBs fragment and form ordered structures, which we have termed "rosettes". Formation of CB rosettes was prevented by inhibition of viral DNA synthesis and preceded expression of the L4-33K protein. CB rosettes localised to the periphery of E2A-72K-containing replication centers and to the edges of ASF/SF2 and hnRNP A1 ring structures that demarcate sites of viral transcription and splicing. At later times of infection, CB rosettes were undetectable. Furthermore, knock-down of p80-coilin (the major structural protein of CBs) by RNA interference reduced the yield of infectious Ad5 and expression of the late proteins IIIa (from L1), hexon (from L3) and fiber (from L5), whereas the E2A-72K protein was unaffected. We conclude that CBs have an important role in the expression of adenovirus major late gene products.

  14. Revertants of Adenovirus Type 12-Transformed Hamster Cell Line T637 as Tools in the Analysis of Integration Patterns

    PubMed Central

    Eick, Dirk; Stabel, Silvia; Doerfler, Walter

    1980-01-01

    Spontaneously arising morphological revertants of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 had been previously isolated, and it had been demonstrated that in these revertants varying amounts of the integrated Ad12 genome were eliminated from the host genome. In this report, the patterns of persistence of the viral genome in the revertants were analyzed in detail. In some of the revertant cell lines, F10, TR3, and TR7, all copies of Ad12 DNA integrated in line T637 were lost. In lines TR1, -2, -4 to -6, -8 to -10, and -13 to -16, only the right-hand portion of one Ad12 genome was preserved; it consisted of the intact right segment of Ad12 DNA and was integrated at the same site as in line T637. In revertant lines G12, TR11, and TR12, one Ad12 DNA and varying parts of a second viral DNA molecule persisted in the host genome. These patterns of persistence of Ad12 DNA molecules in different revertants supported a model for a mode of integration of Ad12 DNA in T637 hamster cells in which multiple (20 to 22) copies of the entire Ad12 DNA were serially arranged, separated from each other by stretches of cellular DNA. The occurrence of such revertants demonstrated that foreign DNA sequences could not only be acquired but could also be lost from eucaryotic genomes. There was very little, if any, expression of Ad12-specific DNA sequences in the revertant lines TR7 and TR12. Moreover, Ad12 DNA sequences which were found to be undermethylated in line T637 were completely methylated in the revertant cell lines G12, TR11, TR12, and TR2. These findings were consistent with the absence of T antigen from the revertant lines reported earlier. Hence it was conceivable that the expression of integrated viral DNA sequences was somehow dependent on their positions in the cellular genome. In cell line TR637, the early segments of Ad12 DNA were expressed and undermethylated; conversely, in the revertant lines G12, TR11, TR12, and TR2, the same segments appeared to

  15. Oncolytic adenovirus expressing interleukin-18 improves antitumor activity of dacarbazine for malignant melanoma

    PubMed Central

    Yang, Chunhua; Cao, Hang; Liu, Ning; Xu, Kai; Ding, Meng; Mao, Li-jun

    2016-01-01

    Conditionally replicating adenoviruses have emerged as novel therapeutic agents for cancer. This study aimed to evaluate synergistic antitumor activity of replication-competent adenovirus armed with interleukin (IL)-18 (ZD55-IL-18) and dacarbazine (DTIC) against melanoma. Melanoma A375 cells or nude mouse tumor xenografts were treated with ZD55-IL-18 alone or together with DTIC. The results showed that ZD55-IL-18 competently replicated in A375 cells and expressed IL-18, and these were not affected by DTIC. ZD55-IL-18 enhanced the cytotoxicity of DTIC accompanied by increased apoptosis. Moreover, ZD55-IL-18 and DTIC synergistically inhibited the growth but promoted the apoptosis of A375 xenografts and inhibited vascular endothelial growth factor expression and lung metastasis in xenografts of nude mice. In conclusion, this is the first study to show synergistic anticancer activity of ZD55-IL-18 and DTIC for malignant melanoma. Our results provide evidence that chemo-gene-viro therapeutic approach has greater potential for malignant cancers than conventional chemotherapy or gene therapy. PMID:27895465

  16. Oncolytic adenovirus expressing interleukin-18 improves antitumor activity of dacarbazine for malignant melanoma.

    PubMed

    Yang, Chunhua; Cao, Hang; Liu, Ning; Xu, Kai; Ding, Meng; Mao, Li-Jun

    2016-01-01

    Conditionally replicating adenoviruses have emerged as novel therapeutic agents for cancer. This study aimed to evaluate synergistic antitumor activity of replication-competent adenovirus armed with interleukin (IL)-18 (ZD55-IL-18) and dacarbazine (DTIC) against melanoma. Melanoma A375 cells or nude mouse tumor xenografts were treated with ZD55-IL-18 alone or together with DTIC. The results showed that ZD55-IL-18 competently replicated in A375 cells and expressed IL-18, and these were not affected by DTIC. ZD55-IL-18 enhanced the cytotoxicity of DTIC accompanied by increased apoptosis. Moreover, ZD55-IL-18 and DTIC synergistically inhibited the growth but promoted the apoptosis of A375 xenografts and inhibited vascular endothelial growth factor expression and lung metastasis in xenografts of nude mice. In conclusion, this is the first study to show synergistic anticancer activity of ZD55-IL-18 and DTIC for malignant melanoma. Our results provide evidence that chemo-gene-viro therapeutic approach has greater potential for malignant cancers than conventional chemotherapy or gene therapy.

  17. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  18. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  19. Chromosomal damage induced by human adenovirus type 12 requires expression of the E1B 55-kilodalton viral protein.

    PubMed Central

    Schramayr, S; Caporossi, D; Mak, I; Jelinek, T; Bacchetti, S

    1990-01-01

    Infection of human embryonic kidney cells with adenovirus type 12 results in the induction of damage at specific (17q21-22, 1p36, 1q21, and 1q42-43) and random sites in the cellular chromosomes. A previous study by Durnam et al. (D. M. Durnam, P. P. Smith, J. C. Menninger, and J. K. McDougall, Cancer Cells 4:349-354, 1986) indicated that the expression of viral early region 1 (E1) is sufficient for the induction of damage at band 17q21-22. In the present report we used an adenovirus type 12-adenovirus type 5 recombinant with E1A hybrid sequences as well as viruses with mutations in the adenovirus type 12 E1B genes to map adenovirus type 12 E1 functions involved in the induction of genetic damage. Our results show that the expression of the E1A proteins is not sufficient for this effect. On the other hand, mutations within the E1B 55-kilodalton protein but not the E1B 19-kilodalton protein affect the ability of the virus to induce both specific and random chromosomal damage. Images PMID:2325204

  20. Impact of Adenovirus E4-ORF3 Oligomerization and Protein Localization on Cellular Gene Expression.

    PubMed

    Vink, Elizabeth I; Zheng, Yueting; Yeasmin, Rukhsana; Stamminger, Thomas; Krug, Laurie T; Hearing, Patrick

    2015-05-13

    The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation.

  1. Impact of Adenovirus E4-ORF3 Oligomerization and Protein Localization on Cellular Gene Expression

    PubMed Central

    Vink, Elizabeth I.; Zheng, Yueting; Yeasmin, Rukhsana; Stamminger, Thomas; Krug, Laurie T.; Hearing, Patrick

    2015-01-01

    The Adenovirus E4-ORF3 protein facilitates virus replication through the relocalization of cellular proteins into nuclear inclusions termed tracks. This sequestration event disrupts antiviral properties associated with target proteins. Relocalization of Mre11-Rad50-Nbs1 proteins prevents the DNA damage response from inhibiting Ad replication. Relocalization of PML and Daxx impedes the interferon-mediated antiviral response. Several E4-ORF3 targets regulate gene expression, linking E4-ORF3 to transcriptional control. Furthermore, E4-ORF3 was shown to promote the formation of heterochromatin, down-regulating p53-dependent gene expression. Here, we characterize how E4-ORF3 alters cellular gene expression. Using an inducible, E4-ORF3-expressing cell line, we performed microarray experiments to highlight cellular gene expression changes influenced by E4-ORF3 expression, identifying over four hundred target genes. Enrichment analysis of these genes suggests that E4-ORF3 influences factors involved in signal transduction and cellular defense, among others. The expression of mutant E4-ORF3 proteins revealed that nuclear track formation is necessary to induce these expression changes. Through the generation of knockdown cells, we demonstrate that the observed expression changes may be independent of Daxx and TRIM33 suggesting that an additional factor(s) may be responsible. The ability of E4-ORF3 to manipulate cellular gene expression through the sequestration of cellular proteins implicates a novel role for E4-ORF3 in transcriptional regulation. PMID:25984715

  2. Loss of coxsackie and adenovirus receptor expression in human colorectal cancer: A potential impact on the efficacy of adenovirus-mediated gene therapy in Chinese Han population.

    PubMed

    Ma, Ying-Yu; Wang, Xiao-Jun; Han, Yong; Li, Gang; Wang, Hui-Ju; Wang, Shi-Bing; Chen, Xiao-Yi; Liu, Fan-Long; He, Xiang-Lei; Tong, Xiang-Min; Mou, Xiao-Zhou

    2016-09-01

    The coxsackie and adenovirus receptor (CAR) is considered a tumor suppressor and critical factor for the efficacy of therapeutic strategies that employ the adenovirus. However, data on CAR expression levels in colorectal cancer are conflicting and its clinical relevance remains to be elucidated. Immunohistochemistry was performed on tissue microarrays containing 251 pairs of colon cancer and adjacent normal tissue samples from Chinese Han patients to assess the expression levels of CAR. Compared with healthy mucosa, decreased CAR expression (40.6% vs. 95.6%; P<0.001) was observed in colorectal cancer samples. The CAR immunopositivity in tumor tissues was not significantly associated with gender, age, tumor size, differentiation, TNM stage, lymph node metastasis or distant metastasis in patients with colon cancer. However, expression of CAR is present in 83.3% of the tumor tissues from patient with colorectal liver metastasis, which was significantly higher than those without liver metastasis (39.6%; P=0.042). At the plasma membrane, CAR was observed in 29.5% normal mucosa samples, which was significantly higher than in colorectal cancer samples (4.0%; P<0.001). In addition, the survival analysis demonstrated that the expression level of CAR has no association with the prognosis of colorectal cancer. CAR expression was observed to be downregulated in colorectal cancer, and it exerts complex effects during colorectal carcinogenesis, potentially depending on the stage of the cancer development and progression. High CAR expression may promote liver metastasis. With regard to oncolytic therapy, CAR expression analysis should be performed prior to adenoviral oncolytic treatment to stratify Chinese Han patients for treatment.

  3. Loss of coxsackie and adenovirus receptor expression in human colorectal cancer: A potential impact on the efficacy of adenovirus-mediated gene therapy in Chinese Han population

    PubMed Central

    Ma, Ying-Yu; Wang, Xiao-Jun; Han, Yong; Li, Gang; Wang, Hui-Ju; Wang, Shi-Bing; Chen, Xiao-Yi; Liu, Fan-Long; He, Xiang-Lei; Tong, Xiang-Min; Mou, Xiao-Zhou

    2016-01-01

    The coxsackie and adenovirus receptor (CAR) is considered a tumor suppressor and critical factor for the efficacy of therapeutic strategies that employ the adenovirus. However, data on CAR expression levels in colorectal cancer are conflicting and its clinical relevance remains to be elucidated. Immunohistochemistry was performed on tissue microarrays containing 251 pairs of colon cancer and adjacent normal tissue samples from Chinese Han patients to assess the expression levels of CAR. Compared with healthy mucosa, decreased CAR expression (40.6% vs. 95.6%; P<0.001) was observed in colorectal cancer samples. The CAR immunopositivity in tumor tissues was not significantly associated with gender, age, tumor size, differentiation, TNM stage, lymph node metastasis or distant metastasis in patients with colon cancer. However, expression of CAR is present in 83.3% of the tumor tissues from patient with colorectal liver metastasis, which was significantly higher than those without liver metastasis (39.6%; P=0.042). At the plasma membrane, CAR was observed in 29.5% normal mucosa samples, which was significantly higher than in colorectal cancer samples (4.0%; P<0.001). In addition, the survival analysis demonstrated that the expression level of CAR has no association with the prognosis of colorectal cancer. CAR expression was observed to be downregulated in colorectal cancer, and it exerts complex effects during colorectal carcinogenesis, potentially depending on the stage of the cancer development and progression. High CAR expression may promote liver metastasis. With regard to oncolytic therapy, CAR expression analysis should be performed prior to adenoviral oncolytic treatment to stratify Chinese Han patients for treatment. PMID:27485384

  4. Integrin alpha(v) and coxsackie adenovirus receptor expression in clinical bladder cancer.

    PubMed

    Sachs, Markus D; Rauen, Katherine A; Ramamurthy, Meera; Dodson, Jennifer L; De Marzo, Angelo M; Putzi, Mathew J; Schoenberg, Mark P; Rodriguez, Ronald

    2002-09-01

    To evaluate the expression of the coxsackie and adenovirus receptor (CAR) and alpha(v) integrins in clinical specimens of bladder cancer to determine the susceptibility to adenoviral gene therapy. Efficient adenovirus-based gene therapy requires binding of the virus to CAR and involves the alpha(v) integrins. Studies on bladder cancer cell lines have shown that low adenoviral transduction rates were associated with low-level expression of CAR. Integrin alpha(v) expression increases in various tumors suggest its importance in differentiation, proliferation, and migration. CAR is structurally a member of the Ig-type superfamily of cell-cell adhesion molecules, suggesting that its expression may also be related to the state of tumor differentiation. We performed immunohistochemistry for CAR and integrin alpha(v) expression in bladder cancer specimens in 50 paraffin-embedded tumor-normal pairs and confirmed the results by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of 11 separate bladder tumors and 4 separate normal bladder controls. Immunochemistry demonstrated a stage and grade-dependent decrease in CAR expression (90.0%, 83.3%, and 31.3% of normal urothelium and superficial and invasive transitional cell carcinoma [TCC] and 83.3% and 39.5% of low and high-grade TCC, respectively). Furthermore, we found a stage and grade-dependent increase in alpha(v) integrin expression (13.3%, 46.0%, and 56.3% of normal urothelium, superficial TCC, and invasive TCC and 25% and 52.6% of low and high-grade TCC, respectively). Quantitative RT-PCR analysis confirmed a downregulation at the CAR gene expression level. This down-regulation may have a major impact on developing adenoviral-based gene therapy modalities. In addition, we propose that loss of CAR expression decreases rigid cell adhesion, possibly increasing the migratory potential. Loss of CAR expression correlates with the invasive phenotype in our analysis of bladder cancer. Simultaneously

  5. Heat shock protein 72 expression allows permissive replication of oncolytic adenovirus dl1520 (ONYX-015) in rat glioblastoma cells

    PubMed Central

    Madara, Jonathan; Krewet, James A; Shah, Maulik

    2005-01-01

    In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus. PMID:15762988

  6. Induction of apoptosis by adenovirus E4orf4 protein is specific to transformed cells and requires an interaction with protein phosphatase 2A

    PubMed Central

    Shtrichman, Ronit; Sharf, Rakefet; Barr, Haim; Dobner, Thomas; Kleinberger, Tamar

    1999-01-01

    We previously have shown that adenovirus type 5 E4orf4 protein associates with protein phosphatase 2A (PP2A) and induces apoptosis in transformed cells in a p53-independent manner. Here we show that the interaction between E4orf4 and PP2A is required for induction of apoptosis by the viral protein. This conclusion is supported by a mutation analysis of E4orf4 protein, showing a correlation between the ability to bind PP2A and to induce apoptosis, and by the observation that transfection of an antisense construct of the PP2A-B55 subunit reduces expression of the PP2A-B55 subunit and inhibits induction of apoptosis by E4orf4, but not by p53. The mutant analysis also indicates that even a low level of interaction with PP2A is sufficient to initiate the E4orf4 apoptotic pathway. In addition, E4orf4 inhibits cellular transformation by various oncogenes, and this function is coupled to its ability to induce apoptosis. Furthermore, expression of oncogenes in primary cell cultures sensitizes these cells to induction of apoptosis by E4orf4. Our results suggest that E4orf4 is a potentially useful tool for cancer gene therapy. PMID:10468565

  7. Room-temperature, atmospheric plasma needle reduces adenovirus gene expression in HEK 293A host cells

    NASA Astrophysics Data System (ADS)

    Xiong, Z.; Lu, X.; Cao, Y.; Ning, Q.; Ostrikov, K.; Lu, Y.; Zhou, X.; Liu, J.

    2011-12-01

    Room-temperature, atmospheric-pressure plasma needle treatment is used to effectively minimize the adenovirus (AdV) infectivity as quantified by the dramatic reduction of its gene expression in HEK 293A primary human embryonic kidney cells studied by green fluorescent protein imaging. The AdV titer is reduced by two orders of magnitude within only 8 min of the plasma exposure. This effect is due to longer lifetimes and higher interaction efficacy of the plasma-generated reactive species in confined space exposed to the plasma rather than thermal effects commonly utilized in pathogen inactivation. This generic approach is promising for the next-generation anti-viral treatments and imunotherapies.

  8. Intramuscular delivery of adenovirus serotype 5 vector expressing humanized protective antigen induces rapid protection against anthrax that may bypass intranasally originated preexisting adenovirus immunity.

    PubMed

    Wu, Shipo; Zhang, Zhe; Yu, Rui; Zhang, Jun; Liu, Ying; Song, Xiaohong; Yi, Shaoqiong; Liu, Ju; Chen, Jianqin; Yin, Ying; Xu, Junjie; Hou, Lihua; Chen, Wei

    2014-02-01

    Developing an effective anthrax vaccine that can induce a rapid and sustained immune response is a priority for the prevention of bioterrorism-associated anthrax infection. Here, we developed a recombinant replication-deficient adenovirus serotype 5-based vaccine expressing the humanized protective antigen (Ad5-PAopt). A single intramuscular injection of Ad5-PAopt resulted in rapid and robust humoral and cellular immune responses in Fisher 344 rats. Animals intramuscularly inoculated with a single dose of 10⁸ infectious units of Ad5-PAopt achieved 100% protection from challenge with 10 times the 50% lethal dose (LD₅₀) of anthrax lethal toxin 7 days after vaccination. Although preexisting intranasally induced immunity to Ad5 slightly weakened the humoral and cellular immune responses to Ad5-PAopt via intramuscular inoculation, 100% protection was achieved 15 days after vaccination in Fisher 344 rats. The protective efficacy conferred by intramuscular vaccination in the presence of preexisting intranasally induced immunity was significantly better than that of intranasal delivery of Ad5-PAopt and intramuscular injection with recombinant PA and aluminum adjuvant without preexisting immunity. As natural Ad5 infection often occurs via the mucosal route, the work here largely illuminates that intramuscular inoculation with Ad5-PAopt can overcome the negative effects of immunity induced by prior adenovirus infection and represents an efficient approach for protecting against emerging anthrax.

  9. Loss of Coxsackie and adenovirus receptor downregulates alpha-catenin expression.

    PubMed

    Stecker, K; Koschel, A; Wiedenmann, B; Anders, M

    2009-11-03

    The Coxsackie and adenovirus receptor (CAR) has been shown to inhibit cancer cell proliferation, migration, and invasion. The underlying mechanisms, however, are poorly understood. The differential gene expression in the human colon cancer cell line DLD1 on RNAi-mediated functional CAR knockdown was analysed using oligo-array technology. Expression of alpha-catenin was determined by quantitative RT-PCR and western blotting. Proliferation, migration, and invasion after CAR knockdown were assessed by in vitro assays, and cell morphology in a three-dimensional context was evaluated using matrigel. Oligo-array technology identified alpha-catenin as the strongest downregulated gene after CAR knockdown. Western blotting and quantitative RT-PCR confirmed a reduced alpha-catenin expression after CAR knockdown in DLD1 cells and in the rat intestinal cell line IEC-6. Functionally, both cell lines showed a marked increase in proliferation, migration, and invasion on CAR knockdown. In matrigel, both cell lines formed amorphous cell clusters in contrast to well-organised three-dimensional structures of CAR-expressing vector controls. Ectopic 're'-expression of alpha-catenin in DLD1 and IEC-6 CAR knockdown cells reversed these functional and morphological effects. These data suggest that an interaction of CAR and alpha-catenin mediates the impact of CAR on cell proliferation, migration, invasion, and morphology.

  10. Adenovirus type 5 early encoded proteins of the E1 and E4 regions induce oncogenic transformation of primary rabbit cells.

    PubMed

    Wimmer, Peter; Täuber, Birgit; Spruss, Thilo; Dobner, Thomas

    2010-07-01

    Analysis of the molecular mechanisms of viral-mediated oncogenesis has contributed enormously to the understanding of the basic principles of normal/malignant cell growth. Transformation by human adenoviruses is a multi-step process involving the modulation of numerous cellular pathways, leading to inhibition of apoptosis and growth arrest. However, the molecular mechanism of how the adenovirus oncogenes facilitate transformation of rodent cells, while concurrently failing to do so for human cells, remains elusive. In this report, we demonstrate for the first time that the transformation capabilities of adenovirus type 5 oncogenes are not restricted to rodent cells, but include cells of the related mammalian order Lagomorpha, inducing considerable morphological alterations, enhanced cell growth and tumour induction in vivo. Furthermore, the established cell lines may represent a suitable tool for further development to generate E4-mutated adenoviruses, which has so far been difficult as mutations within the E4 region often prove to be lethal without a helper-cell system.

  11. Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation.

    PubMed Central

    Ames, R S; Holskin, B; Mitcho, M; Shalloway, D; Chen, M J

    1990-01-01

    We have previously shown that expression of the adenovirus E1A 12S or 13S products in NIH 3T3 fibroblasts induces susceptibility to the cytotoxic actions of tumor necrosis factor alpha (TNF alpha). A large number of studies have mapped the multiple biological functions of the 12S and 13S products to three highly conserved regions (CR) within the E1A sequence. Here we used plasmids coding for E1A deletion and point mutants in these regions to generate target cell lines for TNF alpha cytotoxicity assays to determine which regions and functions are necessary for the induction of TNF alpha sensitivity. Expression of CR1 was required for the induction of TNF alpha sensitivity. This finding did not reflect a requirement for transforming or transcriptional repression activity, since some mutants that were defective in both of these properties were able to induce TNF alpha sensitivity. CR2 transformation-defective point mutants, but not a CR2/3 region deletion mutant, were also able to induce sensitivity. In addition, NIH 3T3 cells expressing the retroviral transcription activators tat from human immunodeficiency virus type 1 and tax from human T-lymphotropic virus type I were not sensitive to TNF alpha. However, the possibility that E1A-mediated transcriptional activation can augment the induction of TNF alpha sensitivity is not excluded. Comparison of data from previous biological studies with the TNF alpha cytotoxicity assays presented here suggested that the mechanism by which E1A induces sensitivity to TNF alpha in NIH 3T3 cells is independent of many of the known E1A biological functions, including transformation in cooperation with ras, immortalization, induction of DNA synthesis in quiescent cells, and transcriptional repression. A novel E1A-mediated effect may be involved, although our data do not exclude the possibility that sensitization to TNF alpha is mediated through E1A binding to cellular proteins. Images PMID:2143540

  12. Distinct temporal changes in host cell lncRNA expression during the course of an adenovirus infection

    SciTech Connect

    Zhao, Hongxing; Chen, Maoshan; Lind, Sara Bergström; Pettersson, Ulf

    2016-05-15

    The deregulation of cellular long non-coding RNA (lncRNA) expression during a human adenovirus infection was studied by deep sequencing. Expression of lncRNAs increased substantially following the progression of the infection. Among 645 significantly expressed lncRNAs, the expression of 398 was changed more than 2-fold. More than 80% of them were up-regulated and 80% of them were detected during the late phase. Based on the genomic locations of the deregulated lncRNAs in relation to known mRNAs and miRNAs, they were predicted to be involved in growth, structure, apoptosis and wound healing in the early phase, cell proliferation in the intermediate phase and protein synthesis, modification and transport in the late phase. The most significant functions of cellular RNA-binding proteins, previously shown to interact with the deregulated lncRNAs identified here, are involved in RNA splicing, nuclear export and translation events. We hypothesize that adenoviruses exploit the lncRNA network to optimize their reproduction. - Highlights: • The expression of 398 lncRNAs showed a distinct temporal pattern during Ad2 infection. • 80% of the deregulated lncRNAs were up-regulated during the late phase of infection. • The deregulated lncRNAs potentiallyinteract with 33 cellular RNA binding proteins. • These RBPs are involved in RNA splicing, nuclear export and translation. • Adenovirus exploits the cellular lncRNA network to optimize its replication.

  13. Human coxsackie adenovirus receptor (CAR) expression in transgenic mouse prostate tumors enhances adenoviral delivery of genes.

    PubMed

    Bao, Yunhua; Peng, Weidan; Verbitsky, Amy; Chen, Jiping; Wu, Lily; Rauen, Katherine A; Sawicki, Janet A

    2005-09-01

    Transgenic mice that recapitulate the progression of human diseases are potentially useful models for testing the effectiveness of new therapeutic strategies. Their use in pre-clinical testing of adenovirally-delivered gene therapies, however, is limited because of restricted cell surface expression of Coxsackie adenovirus receptor (CAR) in mice. To develop a more suitable transgenic mouse model for testing adenoviral-based gene therapies for prostate cancer, we generated prostate specific antigen/human CAR (PSA/hCAR) transgenic mice in which a chimeric enhancer/promoter sequence of the human PSA gene drives expression of a functional hCAR coding sequence. Expression of an adenovirally-delivered luciferase reporter gene in prostate tumor cells in bigenic mice (PSA/hCAR + TRAMP) was enhanced compared to the level in tumor cells lacking the PSA/hCAR transgene. Breeding PSA/hCAR mice to existing transgenic mouse models for prostate cancer (e.g., TRAMP) results in improved mouse models for testing adenovirally-delivered therapeutic genes. Copyright 2005 Wiley-Liss, Inc.

  14. Coxsackie–adenovirus receptor expression is enhanced in pancreas from patients with type 1 diabetes

    PubMed Central

    Hodik, M; Anagandula, M; Fuxe, J; Krogvold, L; Dahl-Jørgensen, K; Hyöty, H; Sarmiento, L; Frisk, G

    2016-01-01

    Objectives One of the theories connecting enterovirus (EV) infection of human islets with type 1 diabetes (T1D) is the development of a fertile field in the islets. This implies induction of appropriate proteins for the viral replication such as the coxsackie–adenovirus receptor (CAR). The aim of this study was to investigate to what extent CAR is expressed in human islets of Langerhans, and what conditions that would change the expression. Design Immunohistochemistry for CAR was performed on paraffin-embedded pancreatic tissue from patients with T1D (n=9 recent onset T1D, n=4 long-standing T1D), islet autoantibody-positive individuals (n=14) and non-diabetic controls (n=24) individuals. The expression of CAR was also examined by reverse transcription PCR on microdissected islets (n=5), exocrine tissue (n=5) and on explanted islets infected with EV or exposed to chemokines produced by EV-infected islet cells. Results An increased frequency of patients with T1D and autoantibody-positive individuals expressed CAR in the pancreas (p<0.039). CAR staining was detected more frequently in pancreatic islets from patients with T1D and autoantibody-positive subjects (15/27) compared with (6/24) non-diabetic controls (p<0.033). Also in explanted islets cultured in UV-treated culture medium from coxsackievirus B (CBV)-1-infected islets, the expression of the CAR gene was increased compared with controls. Laser microdissection of pancreatic tissue revealed that CAR expression was 10-fold higher in endocrine compared with exocrine cells of the pancreas. CAR was also expressed in explanted islets and the expression level decreased with time in culture. CBV-1 infection of explanted islets clearly decreased the expression of CAR (p<0.05). In contrast, infection with echovirus 6 did not affect the expression of CAR. Conclusions CAR is expressed in pancreatic islets of patients with T1D and the expression level of CAR is increased in explanted islets exposed to proinflammatory

  15. Coxsackie-adenovirus receptor expression is enhanced in pancreas from patients with type 1 diabetes.

    PubMed

    Hodik, M; Anagandula, M; Fuxe, J; Krogvold, L; Dahl-Jørgensen, K; Hyöty, H; Sarmiento, L; Frisk, G

    2016-01-01

    One of the theories connecting enterovirus (EV) infection of human islets with type 1 diabetes (T1D) is the development of a fertile field in the islets. This implies induction of appropriate proteins for the viral replication such as the coxsackie-adenovirus receptor (CAR). The aim of this study was to investigate to what extent CAR is expressed in human islets of Langerhans, and what conditions that would change the expression. Immunohistochemistry for CAR was performed on paraffin-embedded pancreatic tissue from patients with T1D (n=9 recent onset T1D, n=4 long-standing T1D), islet autoantibody-positive individuals (n=14) and non-diabetic controls (n=24) individuals. The expression of CAR was also examined by reverse transcription PCR on microdissected islets (n=5), exocrine tissue (n=5) and on explanted islets infected with EV or exposed to chemokines produced by EV-infected islet cells. An increased frequency of patients with T1D and autoantibody-positive individuals expressed CAR in the pancreas (p<0.039). CAR staining was detected more frequently in pancreatic islets from patients with T1D and autoantibody-positive subjects (15/27) compared with (6/24) non-diabetic controls (p<0.033). Also in explanted islets cultured in UV-treated culture medium from coxsackievirus B (CBV)-1-infected islets, the expression of the CAR gene was increased compared with controls. Laser microdissection of pancreatic tissue revealed that CAR expression was 10-fold higher in endocrine compared with exocrine cells of the pancreas. CAR was also expressed in explanted islets and the expression level decreased with time in culture. CBV-1 infection of explanted islets clearly decreased the expression of CAR (p<0.05). In contrast, infection with echovirus 6 did not affect the expression of CAR. CAR is expressed in pancreatic islets of patients with T1D and the expression level of CAR is increased in explanted islets exposed to proinflammatory cytokines/chemokines produced by infected

  16. Effects of adeno-associated virus on adenovirus replication and gene expression during coinfection.

    PubMed

    Timpe, Jennifer M; Verrill, Kristin C; Trempe, James P

    2006-08-01

    Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

  17. Effects of Adeno-Associated Virus on Adenovirus Replication and Gene Expression during Coinfection

    PubMed Central

    Timpe, Jennifer M.; Verrill, Kristin C.; Trempe, James P.

    2006-01-01

    Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis. PMID:16873238

  18. Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses.

    PubMed

    Kuate, Seraphin; Stefanou, Daniela; Hoffmann, Dennis; Wildner, Oliver; Uberla, Klaus

    2004-11-01

    The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.

  19. Production of Viral mRNA in Adenovirus-Transformed Cells by the Post-Transcriptional Processing of Heterogeneous Nuclear RNA Containing Viral and Cell Sequences

    PubMed Central

    Wall, R.; Weber, J.; Gage, Z.; Darnell, J. E.

    1973-01-01

    Adenovirus 2-transformed cells contain virus-specific sequences which are covalently linked to cell-specific RNA sequences in heterogeneous nuclear RNA (HnRNA) molecules larger than 45S. Virus sequences are identified by hybridization to viral DNA, and the cell sequences are detected by hybridization to cellular DNA under conditions where hybridization only occurs to reiterated sites in cell DNA. Such large composite viral-cell HnRNA molecules presumably arise through the uninterrupted transcription of host sequences and integrated viral DNA. Adenovirus-specific polysomal RNA from these cells sediments as three discrete species at 16, 20, and 26S. These specific classes of viral mRNA do not contain rapidly hybridizing host-specific RNA sequences. Both virus-specific HnRNA and mRNA contain polyadenylic acid sequences since they bind to polyU columns at levels characteristics of other polyA-terminated HnRNA and mRNA. Thus, the discrete species of virus-specific mRNA in adenovirus 2 transformed cells appear to be derived from high-molecular-weight virus-specific HnRNA through a series of post-transcriptional modifications involving polyA addition. Subsequently the HnRNA is cleaved so that the cell-specific RNA sequences that originate from the reiterated sites in cell DNA do not accompany the adenovirus mRNA to the cytoplasm. These events for the adenovirus-specific mRNA appear, therefore, to be similar to the stages in the biogenesis of the majority of mRNA in eukaryotic cells. PMID:4736534

  20. Genetic modification of human embryonic stem cells with adenoviral vectors: differences of infectability between lines and correlation of infectability with expression of the coxsackie and adenovirus receptor.

    PubMed

    Brokhman, Irina; Pomp, Oz; Fishman, Lital; Tennenbaum, Tamar; Amit, Michal; Itzkovitz-Eldor, Joseph; Goldstein, Ronald S

    2009-04-01

    Adenovirus is an efficient vector for expression of transgenes in dividing and nondividing cells. However, very few studies of human embryonic stem cells (hESCs) have utilized adenoviral vectors. We examine here the ability of adenovirus to infect naive hESCs and the differentiated derivatives of multiple hESC lines. We found a striking variation in adenovirus infection rates between lines. The variability in infection rates was positively correlated with the expression of the coxsackievirus and adenovirus receptor, but not that of alpha(nu)-integrin. Adenoviral infection did not interfere with the expression of pluripotency markers, even after passaging. In addition, infection did not affect differentiation of hESC-derived neural precursors in vitro. We also found that green fluorescent protein expression mediated by adenovirus can be a useful marker for tracking hESC in xenografts. We conclude that adenovirus is a practical vector for genetic modification of naive hESC from most, but not all lines, but may be more generally useful for gene transfer into differentiated derivatives of hESC lines.

  1. Intratumoral Injection of an Adenovirus Expressing Interleukin 2 Induces Regression and Immunity in a Murine Breast Cancer Model

    NASA Astrophysics Data System (ADS)

    Addison, Christina L.; Braciak, Todd; Ralston, Robert; Muller, William J.; Gauldie, Jack; Graham, Frank L.

    1995-08-01

    Rodent tumor cells engineered to secrete cytokines such as interleukin 2 (IL-2) or IL-4 are rejected by syngeneic recipients due to an enhanced antitumor host immune response. An adenovirus vector (AdCAIL-2) containing the human IL-2 gene has been constructed and shown to direct secretion of high levels of human IL-2 in infected tumor cells. AdCAIL-2 induces regression of tumors in a transgenic mouse model of mammary adenocarcinoma following intratumoral injection. Elimination of existing tumors in this way results in immunity against a second challenge with tumor cells. These findings suggest that adenovirus vectors expressing cytokines may form the basis for highly effective immunotherapies of human cancers.

  2. A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation

    PubMed Central

    Arbelo-Roman, Christina; Hartl, Barbara; Kremmer, Elisabeth; Speiseder, Thomas; Meier, Chris; Dobner, Thomas

    2013-01-01

    Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies. PMID:23555268

  3. A ubiquitin-specific protease possesses a decisive role for adenovirus replication and oncogene-mediated transformation.

    PubMed

    Ching, Wilhelm; Koyuncu, Emre; Singh, Sonia; Arbelo-Roman, Christina; Hartl, Barbara; Kremmer, Elisabeth; Speiseder, Thomas; Meier, Chris; Dobner, Thomas

    2013-03-01

    Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies.

  4. Noninvasive, repetitive, quantitative measurement of gene expression from a bicistronic message by positron emission tomography, following gene transfer with adenovirus.

    PubMed

    Liang, Qianwa; Gotts, Jeff; Satyamurthy, Nagichettiar; Barrio, Jorge; Phelps, Michael E; Gambhir, Sanjiv S; Herschman, Harvey R

    2002-07-01

    Gene therapy protocols are hampered by the inability to monitor the location, magnitude, and duration of ectopic gene expression following DNA delivery. Consequently, it is difficult to establish quantitative correlations and/or causal relationships between therapeutic gene expression and phenotypic responses in treated individuals. One approach to monitor "therapeutic gene" expression indirectly is to incorporate reporter genes that can be imaged in vivo into bicistronic transcription units, along with the therapeutic genes. Expression of the dopamine D2 receptor (D2R) and herpes simplex virus thymidine kinase (HSV1-TK) can both be monitored, in vivo, by positron-emission tomography (PET). We created ad.DTm, an adenovirus containing a cytomegalovirus (CMV) early promoter-driven transcription unit, in which the D2R gene is placed proximal to an encephalomyocarditis virus internal ribosomal entry site (IRES) and a modified HSV1-tk gene is placed distal to the IRES. Following intravenous ad.DTm injection into mice, correlated hepatic D2R and HSV1-sr39tk PET reporter gene expression was demonstrated. Repeated microPET scanning quantitated both D2R-dependent sequestration of a positron-emitting ligand and HSV1-TK-dependent sequestration of a positron-emitting product. It is possible, in living mice, to investigate noninvasively and to measure quantitatively and repeatedly correlated expression of two coding regions from a bicistronic transcription unit over a 3-month period following adenovirus delivery.

  5. Imaging of viral thymidine kinase gene expression by replicating oncolytic adenovirus and prediction of therapeutic efficacy.

    PubMed

    Kim, Eun-Jung; Yoo, Ji Young; Choi, Young-Hwan; Ahn, Keun-Jae; Lee, Jong-Doo; Yun, Chae-Ok; Yun, Mijin

    2008-10-31

    We have used a genetically attenuated adenoviral vector which expresses HSVtk to assess the possible additive role of suicidal gene therapy for enhanced oncolytic effect of the virus. Expression of TK was measured using a radiotracer-based molecular counting and imaging system. Replication-competent recombinant adenoviral vector (Ad-DeltaE1B19/55) was used in this study, whereas replication-incompetent adenovirus (Ad-DeltaE1A) was generated as a control. Both Ad-DeltaE1B19/55-TK and Ad-DeltaE1A-TK comprise the HSVtk gene inserted into the E3 region of the viruses. YCC-2 cells were infected with the viruses and incubated with 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (I-131 FIAU) to measure amount of radioactivity. The cytotoxicity of the viruses was determined, and gamma ray imaging of HSVtk gene was performed. MTT assay was also performed after GCV treatment. On gamma counter-analyses, counts/ minute (cpm)/microg of protein showed MOIs dependency with DeltaE1B19/55-TK infection. On MTT assay, Ad-DeltaE1B19/55-TK led to more efficient cell killing than Ad-DeltaE1A-TK. On plate imaging by gamma camera, both Ad-DeltaE1B19/55-TK and Ad-DeltaE1A-TK infected cells showed increased I-131 FIAU uptake in a MOI dependent pattern, and with GCV treatment, cell viability of DeltaE1B19/55-TK infection was remarkably reduced compared to that of Ad-DeltaE1A-TK infection. Replicating Ad-DeltaE1B19/55-TK showed more efficient TK expression even in the presence of higher-cancer cell killing effects compared to non-replicating Ad-DeltaE1A-TK. Therefore, GCV treatment still possessed an additive role to oncolytic effect of Ad-DeltaE1B19/55-TK. The expression of TK by oncolytic viruses could rapidly be screened using a radiotracer-based counting and imaging technique.

  6. Recombinant GABAC receptors expressed in rat hippocampal neurons after infection with an adenovirus containing the human ρ1 subunit

    PubMed Central

    Filippova, Natalia; Sedelnikova, Anna; Tyler, William J; Whitworth, Terri L; Fortinberry, Henry; Weiss, David S

    2001-01-01

    A recombinant adenovirus was generated with the human ρ1 GABAC receptor subunit (adeno-ρ). Patch-clamp and antibody staining were employed to confirm functional expression of recombinant ρ1 receptors after infection of human embryonic kidney cells (HEK293 cell line), human embryonic retinal cells (911 cell line), dissociated rat hippocampal neurons and cultured rat hippocampal slices. Standard whole-cell recording and Western blot analysis using ρ1 GABAC receptor antibodies revealed that recombinant ρ1 receptors were expressed in HEK293 and 911 cells after adeno-ρ infection and exhibited properties similar to those of ρ1 receptors after standard transfection. Cultured rat hippocampal neurons (postnatal day (P)3-P5) did not show a native GABAC-like current. After adeno-ρ infection, however, a GABAC-like current appeared in 70-90 % of the neurons. Five days after infection, expression of GABAC receptors in hippocampal neurons significantly decreased native GABAA receptor currents from 1200 ± 300 to 150 ± 70 pA (n = 10). The native glutamate-activated current was unchanged. Hippocampal slices (P8) did not show a native GABAC-like current, although recombinant ρ1 receptors could be expressed in cultured hippocampal slices after adeno-ρ infection. These data indicate that an adenovirus can be used to express recombinant GABAC receptors in hippocampal neurons. This finding could represent an important step towards the gene therapy of CNS receptor-related diseases. PMID:11507165

  7. Waterborne adenovirus.

    PubMed

    Mena, Kristina D; Gerba, Charles P

    2009-01-01

    Adenoviruses are associated with numerous disease outbreaks, particularly those involving d-cares, schools, children's camps, hospitals and other health care centers, and military settings. In addition, adenoviruses have been responsible for many recreational water outbreaks, including a great number of swimming pool outbreaks than any other waterborne virus (Gerba and Enriquez 1997). Two drinking water outbreaks have been documented for adenovirus (Divizia et al. 2004; Kukkula et al. 1997) but none for food. Of the 51 known adenovirus serotypes, one third are associated with human disease, while other infections are asymptomatic. Human disease associated with adenovirus infections include gastroenteritis, respiratory infections, eye infections, acute hemorrhagic cystitis, and meningoencephalitis (Table 2). Children and the immunocompromised are more severely impacted by adenovirus infections. Subsequently, adenovirus is included in the EPA's Drinking Water Contaminant Candidate List (CCL), which is a list of unregulated contaminants found in public water systems that may pose a risk to public health (National Research Council 1999). Adenoviruses have been detected in various waters worldwide including wastewater, river water, oceans, and swimming pools (Hurst et al. 1988; Irving and Smith 1981; Pina et al. 1998). Adenoviruses typically outnumber the enteroviruses, when both are detected in surface waters. Chapron et al. (2000) found that 38% of 29 surface water samples were positive for infectious Ad40 and Ad41. Data are lacking regarding the occurrence of adenovirus in water in the US, particularly for groundwater and drinking water. Studies have shown, however, that adenoviruses survive longer in water than enteroviruses and hepatitis A virus (Enriquez et al. 1995), which may be due to their double-stranded DNA. Risk assessments have been conducted on waterborne adenovirus (Crabtree et al. 1997; van Heerden et al. 2005c). Using dose-response data for inhalation

  8. Construction of human BMP2-IRES-HIF1αmu adenovirus expression vector and its expression in mesenchymal stem cells.

    PubMed

    Liu, Danping; Hu, Liang; Zhang, Zheng; Li, Quan Ying; Wang, Guoxian

    2013-02-01

    The present study aimed to construct a novel recombinant adenovirus expression vector Ad-BMP2-IRES-HIF1αmu that expresses human bone morphogenetic protein (BMP2) and mutant hypoxia-inducible factor 1α, and investigated its effects in promoting neogenesis of bone and angiogenesis. The recombinant adenovirus BMP2, HIF1αmu and pIRES2-EGFP expression vectors were constructed and transfected into HEK293A cells. The groups were divided into group A, transfection with Ad-BMP2-IRES-HIF1αmu; group B, transfection with Ad-HIF1αmu-IRES-hrGFP-1; group C, transfection with Ad-BMP2-IRES-hrGFP-1; group D, transfection with Ad-IRES-hrGFP-1; group E, not transfected. Adenovirus liquid was transferred into rabbit mesenchymal stem cells (MSCs) pretreated with dexamethasone at the best multiplicity of infection (MOI). The mRNA and protein expression of BMP2 and HIF1α were detected by RT-PCR and western blot analysis. Adenovirus was successfully packaged. The expression level of HIF1α mRNA in group A and B was markedly higher than that in groups C, D and E, showing a significant difference (P<0.01). There was a significant difference in the expression level of BMP2 mRNA between group A and C (P<0.05) and this was markedly higher than that in groups B, D and E (P<0.01). The protein expression level of HIF1α in group A and B was markedly higher than that in groups C, D and E (P<0.01). The protein expression level of BMP2 in group A and C was markedly higher than that in groups B, D and E (P<0.01). The human BMP2-IRES-HIF1αmu adenovirus expression vector was successfully constructed and the experimental groups formed bone and blood vessels prior to the positive and negative control groups.

  9. Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

    PubMed

    Liu, Youhong; Chen, Lin; Gong, Zhicheng; Shen, Liangfang; Kao, Chinghai; Hock, Janet M; Sun, Lunquan; Li, Xiong

    2015-02-20

    Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future.

  10. Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells

    PubMed Central

    Gong, Zhicheng; Shen, Liangfang; Kao, Chinghai; Hock, Janet M.; Sun, Lunquan; Li, Xiong

    2015-01-01

    Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future. PMID:25605010

  11. Expression of Coxsackievirus and Adenovirus Receptor Separates Hematopoietic and Cardiac Progenitor Cells in Fetal Liver Kinase 1-Expressing Mesoderm

    PubMed Central

    Tashiro, Katsuhisa; Hirata, Nobue; Okada, Atsumasa; Yamaguchi, Tomoko; Takayama, Kazuo; Mizuguchi, Hiroyuki

    2015-01-01

    In developing embryos or in vitro differentiation cultures using pluripotent stem cells (PSCs), such as embryonic stem cells and induced pluripotent stem cells, fetal liver kinase 1 (Flk1)-expressing mesodermal cells are thought to be a heterogeneous population that includes hematopoietic progenitors, endothelial progenitors, and cardiac progenitors. However, information on cell surface markers for separating these progenitors in Flk1+ cells is currently limited. In the present study, we show that distinct types of progenitor cells in Flk1+ cells could be separated according to the expression of coxsackievirus and adenovirus receptor (CAR, also known as CXADR), a tight junction component molecule. We found that mouse and human PSC- and mouse embryo-derived Flk1+ cells could be subdivided into Flk1+CAR+ cells and Flk1+CAR− cells. The progenitor cells with cardiac potential were almost entirely restricted to Flk1+CAR+ cells, and Flk1+CAR− cells efficiently differentiated into hematopoietic cells. Endothelial differentiation potential was observed in both populations. Furthermore, from the expression of CAR, Flk1, and platelet-derived growth factor receptor-α (PDGFRα), Flk1+ cells could be separated into three populations (Flk1+PDGFRα−CAR− cells, Flk1+PDGFRα−CAR+ cells, and Flk1+PDGFRα+CAR+ cells). Flk1+PDGFRα+ cells and Flk1+PDGFRα− cells have been reported as cardiac and hematopoietic progenitor cells, respectively. We identified a novel population (Flk1+PDGFRα−CAR+ cells) with the potential to differentiate into not only hematopoietic cells and endothelial cells but also cardiomyocytes. Our findings indicate that CAR would be a novel and prominent marker for separating PSC- and embryo-derived Flk1+ mesodermal cells with distinct differentiation potentials. PMID:25762001

  12. “Stealth” Adenoviruses Blunt Cell-Mediated and Humoral Immune Responses against the Virus and Allow for Significant Gene Expression upon Readministration in the Lung

    PubMed Central

    Croyle, Maria A.; Chirmule, Narendra; Zhang, Yi; Wilson, James M.

    2001-01-01

    Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy. PMID:11312351

  13. Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

    PubMed Central

    Klessig, D F; Brough, D E; Cleghon, V

    1984-01-01

    The DNA-binding protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central role in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by DBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DBP is toxic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50- to 200-fold, and within 8 h its synthesis from the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells. Images PMID:6542172

  14. Potent antitumor activity of oncolytic adenovirus expressing Beclin-1 via induction of autophagic cell death in leukemia

    PubMed Central

    Liu, Hui; Li, Lu; Meng, Haitao; Qian, Qijun

    2013-01-01

    An attractive strategy among adenovirus-based oncolytic systems is to design adenoviral vectors to express pro-apoptotic genes, in which this gene-virotherapy approach significantly enhances tumor cell death by activating apoptotic pathways. However, the existence of cancer cells with apoptotic defects is one of the major obstacles in gene-virotherapy. Here, we investigated whether a strategy that combines the oncolytic effects of an adenoviral vector with simultaneous expression of Beclin-1, an autophagy gene, offers a therapeutic advantage for leukemia. A Beclin-1 cDNA was cloned in an oncolytic adenovirus with chimeric Ad5/11 fiber (SG511-BECN). SG511-BECN treatment induced significant autophagic cell death, and resulted in enhanced cell killing in a variety of leukemic cell lines and primary leukemic blasts. SG511-BECN effects were seen in chronic myeloid leukemia and acute myeloid leukemia with resistance to imatinib or chemotherapy, but exhibited much less cytotoxicity on normal cells. The SG511-BECN-induced autophagic cell death could be partially reversed by RNA interference knockdown of UVRAG, ATG5, and ATG7. We also showed that SG511-BECN strongly inhibited the growth of leukemic progenitors in vitro. In murine leukemia models, SG511-BECN prolonged the survival and decreased the xenograft tumor size by inducing autophagic cell death. Our results suggest that infection of leukemia cells with an oncolytic adenovirus overexpressing Beclin-1 can induce significant autophagic cell death and provide a new strategy for the elimination of leukemic cells via a unique mechanism of action distinct from apoptosis. PMID:23765161

  15. The Adenovirus L4-33K Protein Regulates both Late Gene Expression Patterns and Viral DNA Packaging

    PubMed Central

    Wu, Kai; Guimet, Diana

    2013-01-01

    The adenovirus (Ad) L4-33K protein has been linked to disparate functions during infection. L4-33K is a virus-encoded alternative RNA splicing factor which activates splicing of viral late gene transcripts that contain weak 3′ splice sites. Additionally, L4-33K has been indicated to play a role in adenovirus assembly. We generated and characterized an Ad5 L4-33K mutant virus to further explore its function(s) during infection. Infectivity, viral genome replication, and most viral gene expression of the L4-33K mutant virus are comparable to those of the wild-type virus, except for a prominent decrease in the levels of the late proteins IIIa and pVI. The L4-33K mutant virus produces only empty capsids, indicating a defect in viral DNA packaging. We demonstrate that L4-33K does not preferentially bind to viral packaging sequences in vivo, and mutation of L4-33K does not interfere with the binding of the known viral packaging proteins IVa2, L4-22K, L1-52/55K, and IIIa to the packaging sequences in vivo. Collectively, these results demonstrate that the phenotype of an Ad5 L4-33K mutant virus is complex. The L4-33K protein regulates the accumulation of selective Ad late gene mRNAs and is involved in the proper transition of gene expression during the late phase of infection. The L4-33K protein also plays a role in adenovirus morphogenesis by promoting the packaging of the viral genome into the empty capsid. These results demonstrate the multifunctional nature of the L4-33K protein and its involvement in several different and critical aspects of viral infection. PMID:23552425

  16. Adenovirus-mediated gene transfer and expression of human beta-glucuronidase gene in the liver, spleen, and central nervous system in mucopolysaccharidosis type VII mice.

    PubMed

    Ohashi, T; Watabe, K; Uehara, K; Sly, W S; Vogler, C; Eto, Y

    1997-02-18

    Mucopolysaccharidosis type VII (Sly syndrome) is a lysosomal storage disease caused by inherited deficiency of the lysosomal enzyme beta-glucuronidase. A murine model of this disorder has been well characterized and used to study a number of forms of experimental therapies, including gene therapy. We produced recombinant adenovirus that expresses human beta-glucuronidase and administered this recombinant adenovirus to beta-glucuronidase-deficient mice intravenously. The beta-glucuronidase activities in liver and spleen were elevated to 40% and 20%, respectively, of the heterozygote enzymatic level at day 16. Expression persisted for at least 35 days. Pathological abnormalities of these tissues were also improved, and the elevated levels of urinary glycosaminoglycans were reduced in treated mice. However, the beta-glucuronidase activity in kidney and brain was not significantly increased. After administration of the recombinant adenovirus directly into the lateral ventricles of mutant mice, the beta-glucuronidase activity in crude brain homogenates increased to 30% of heterozygote activity. Histochemical demonstration of beta-glucuronidase activity in brain revealed that the enzymatic activity was mainly in ependymal cells and choroid. However, in some regions, the adenovirus-mediated gene expression was also evident in brain parenchyma associated with vessels and in the meninges. These results suggest that adenovirus-mediated gene delivery might improve the central nervous system pathology of mucopolysaccharidosis in addition to correcting visceral pathology.

  17. Adenovirus-mediated gene transfer and expression of human β-glucuronidase gene in the liver, spleen, and central nervous system in mucopolysaccharidosis type VII mice

    PubMed Central

    Ohashi, Toya; Watabe, Kazuhiko; Uehara, Keiko; Sly, William S.; Vogler, Carole; Eto, Yoshikatsu

    1997-01-01

    Mucopolysaccharidosis type VII (Sly syndrome) is a lysosomal storage disease caused by inherited deficiency of the lysosomal enzyme β-glucuronidase. A murine model of this disorder has been well characterized and used to study a number of forms of experimental therapies, including gene therapy. We produced recombinant adenovirus that expresses human β-glucuronidase and administered this recombinant adenovirus to β-glucuronidase-deficient mice intravenously. The β-glucuronidase activities in liver and spleen were elevated to 40% and 20%, respectively, of the heterozygote enzymatic level at day 16. Expression persisted for at least 35 days. Pathological abnormalities of these tissues were also improved, and the elevated levels of urinary glycosaminoglycans were reduced in treated mice. However, the β-glucuronidase activity in kidney and brain was not significantly increased. After administration of the recombinant adenovirus directly into the lateral ventricles of mutant mice, the β-glucuronidase activity in crude brain homogenates increased to 30% of heterozygote activity. Histochemical demonstration of β-glucuronidase activity in brain revealed that the enzymatic activity was mainly in ependymal cells and choroid. However, in some regions, the adenovirus-mediated gene expression was also evident in brain parenchyma associated with vessels and in the meninges. These results suggest that adenovirus-mediated gene delivery might improve the central nervous system pathology of mucopolysaccharidosis in addition to correcting visceral pathology. PMID:9037045

  18. Systemic delivery of oncolytic adenoviruses targeting transforming growth factor-β inhibits established bone metastasis in a prostate cancer mouse model.

    PubMed

    Hu, Zebin; Gupta, Janhavi; Zhang, Zhenwei; Gerseny, Helen; Berg, Arthur; Chen, Yun Ju; Zhang, Zhiling; Du, Hongyan; Brendler, Charles B; Xiao, Xianghui; Pienta, Kenneth J; Guise, Theresa; Lee, Chung; Stern, Paula H; Stock, Stuart; Seth, Prem

    2012-08-01

    We have examined whether Ad.sTβRFc and TAd.sTβRFc, two oncolytic viruses expressing soluble transforming growth factor-β receptor II fused with human Fc (sTGFβRIIFc), can be developed to treat bone metastasis of prostate cancer. Incubation of PC-3 and DU-145 prostate tumor cells with Ad.sTβRFc and TAd.sTβRFc produced sTGFβRIIFc and viral replication; sTGFβRIIFc caused inhibition of TGF-β-mediated SMAD2 and SMAD3 phosphorylation. Ad(E1-).sTβRFc, an E1(-) adenovirus, produced sTGFβRIIFc but failed to replicate in tumor cells. To examine the antitumor response of adenoviral vectors, PC-3-luc cells were injected into the left heart ventricle of nude mice. On day 9, mice were subjected to whole-body bioluminescence imaging (BLI). Mice bearing hind-limb tumors were administered viral vectors via the tail vein on days 10, 13, and 17 (2.5×10(10) viral particles per injection per mouse, each injection in a 0.1-ml volume), and subjected to BLI and X-ray radiography weekly until day 53. Ad.sTβRFc, TAd.sTβRFc, and Ad(E1-).sTβRFc caused significant inhibition of tumor growth; however, Ad.sTβRFc was the most effective among all the vectors. Only Ad.sTβRFc and TAd.sTβRFc inhibited tumor-induced hypercalcemia. Histomorphometric and synchrotron micro-computed tomographic analysis of isolated bones indicated that Ad.sTβRFc induced significant reduction in tumor burden, osteoclast number, and trabecular and cortical bone destruction. These studies suggest that Ad.sTβRFc and TAd.sTβRFc can be developed as potential new therapies for prostate cancer bone metastasis.

  19. Systemic Delivery of Oncolytic Adenoviruses Targeting Transforming Growth Factor-β Inhibits Established Bone Metastasis in a Prostate Cancer Mouse Model

    PubMed Central

    Hu, Zebin; Gupta, Janhavi; Zhang, Zhenwei; Gerseny, Helen; Berg, Arthur; Chen, Yun Ju; Zhang, Zhiling; Du, Hongyan; Brendler, Charles B.; Xiao, Xianghui; Pienta, Kenneth J.; Guise, Theresa; Lee, Chung; Stern, Paula H.; Stock, Stuart

    2012-01-01

    Abstract We have examined whether Ad.sTβRFc and TAd.sTβRFc, two oncolytic viruses expressing soluble transforming growth factor-β receptor II fused with human Fc (sTGFβRIIFc), can be developed to treat bone metastasis of prostate cancer. Incubation of PC-3 and DU-145 prostate tumor cells with Ad.sTβRFc and TAd.sTβRFc produced sTGFβRIIFc and viral replication; sTGFβRIIFc caused inhibition of TGF-β-mediated SMAD2 and SMAD3 phosphorylation. Ad(E1-).sTβRFc, an E1– adenovirus, produced sTGFβRIIFc but failed to replicate in tumor cells. To examine the antitumor response of adenoviral vectors, PC-3-luc cells were injected into the left heart ventricle of nude mice. On day 9, mice were subjected to whole-body bioluminescence imaging (BLI). Mice bearing hind-limb tumors were administered viral vectors via the tail vein on days 10, 13, and 17 (2.5×1010 viral particles per injection per mouse, each injection in a 0.1-ml volume), and subjected to BLI and X-ray radiography weekly until day 53. Ad.sTβRFc, TAd.sTβRFc, and Ad(E1-).sTβRFc caused significant inhibition of tumor growth; however, Ad.sTβRFc was the most effective among all the vectors. Only Ad.sTβRFc and TAd.sTβRFc inhibited tumor-induced hypercalcemia. Histomorphometric and synchrotron micro-computed tomographic analysis of isolated bones indicated that Ad.sTβRFc induced significant reduction in tumor burden, osteoclast number, and trabecular and cortical bone destruction. These studies suggest that Ad.sTβRFc and TAd.sTβRFc can be developed as potential new therapies for prostate cancer bone metastasis. PMID:22551458

  20. A Multi Targeting Conditionally Replicating Adenovirus Displays Enhanced Oncolysis while Maintaining Expression of Immunotherapeutic Agents

    PubMed Central

    Dobbins, G. Clement; Ugai, Hideyo; Curiel, David T.; Gillespie, G. Yancey

    2015-01-01

    Studies have demonstrated that oncolytic adenoviruses based on a 24 base pair deletion in the viral E1A gene (D24) may be promising therapeutics for treating a number of cancer types. In order to increase the therapeutic potential of these oncolytic viruses, a novel conditionally replicating adenovirus targeting multiple receptors upregulated on tumors was generated by incorporating an Ad5/3 fiber with a carboxyl terminus RGD ligand. The virus displayed full cytopathic effect in all tumor lines assayed at low titers with improved cytotoxicity over Ad5-RGD D24, Ad5/3 D24 and an HSV oncolytic virus. The virus was then engineered to deliver immunotherapeutic agents such as GM-CSF while maintaining enhanced heterogenic oncolysis. PMID:26689910

  1. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    DOE PAGES

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; ...

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle tomore » establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.« less

  2. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    SciTech Connect

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; Kaul, Karen; Brendler, Charles; Iozzo, Renato V.; Seth, Prem

    2014-12-11

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.

  3. An adenovirus with enhanced infectivity mediates molecular chemotherapy of ovarian cancer cells and allows imaging of gene expression.

    PubMed

    Hemminki, A; Belousova, N; Zinn, K R; Liu, B; Wang, M; Chaudhuri, T R; Rogers, B E; Buchsbaum, D J; Siegal, G P; Barnes, M N; Gomez-Navarro, J; Curiel, D T; Alvarez, R D

    2001-09-01

    The adenovirus (Ad) is a useful vector for cancer gene therapy due to its unparalleled gene transfer efficiency to dividing and quiescent cells. Primary cancer cells, however, often have highly variable or low levels of the requisite coxsackie-adenovirus receptor (CAR). Also, assessment of gene transfer and vector persistence has been logistically difficult in human clinical trials. We describe here two novel bicistronic adenoviral (Ad) vectors, AdTKSSTR and RGDTKSSTR, which contain the herpes simplex virus thymidine kinase gene (TK) for molecular chemotherapy and bystander effect. In addition, the viruses contain the human somatostatin receptor subtype-2 gene (SSTR2), the expression of which can be noninvasively imaged. We enhanced the infectivity of RGDTKSSTR by genetically incorporating the RGD-4C motif into the HI-loop of the fiber. This allows the virus to circumvent CAR deficiency by binding to alpha(v)beta(3) and alpha(v)beta(5) integrins, which are highly expressed on most ovarian cancers. The expanded tropism of RGDTKSSTR results in increased infectivity of purified primary ovarian cancer cells and allows enhanced gene transfer in the presence of malignant ascites containing anti-Ad antibodies. RGDTKSSTR may be a useful agent for treating ovarian cancer in clinical trials.

  4. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer.

    PubMed

    Xu, W; Neill, T; Yang, Y; Hu, Z; Cleveland, E; Wu, Y; Hutten, R; Xiao, X; Stock, S R; Shevrin, D; Kaul, K; Brendler, C; Iozzo, R V; Seth, P

    2015-03-01

    In an effort to develop a new therapy for prostate cancer (PCa) bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β-catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels and inhibited tumor cell migration. To examine the antitumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum tartrate-resistant acid phosphatase 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for PCa bone metastasis.

  5. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

    PubMed Central

    Xu, Weidong; Neill, Thomas; Yang, Yuefeng; Hu, Zebin; Cleveland, Elyse; Wu, Ying; Hutten, Ryan; Xiao, Xianghui; Stock, Stuart R.; Shevrin, Daniel; Kaul, Karen; Brendler, Charles; Iozzo, Renato V.; Seth, Prem

    2014-01-01

    In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β-catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis. PMID:25503693

  6. Toxic activity of the CdtB component of Haemophilus ducreyi cytolethal distending toxin expressed from an adenovirus 5 vector.

    PubMed

    Wising, Catharina; Magnusson, Maria; Ahlman, Karin; Lindholm, Leif; Lagergård, Teresa

    2010-02-01

    The Haemophilus ducreyi cytolethal distending toxin (HdCDT) catalytic subunit CdtB has DNase-like activity and mediates DNA damage after its delivery into target cells. We constructed a replication-deficient adenovirus type 5 (Ad5) vector expressing CdtB and investigated the toxic properties of this vector on HeLa cells. Ad5CdtB caused loss of cell viability, morphologic changes, and cell cycle arrest, findings similar to HdCDT intoxication. This confirmed that CdtB is responsible for the toxicity of the holotoxin when expressed in cells following transduction by an adenoviral vector, and indicated a possible potential of this novel strategy in studies of activity of intracellular products and in gene therapy of cancer.

  7. Inhibition of NF-κB Activation in Combination with Bcl-2 Expression Allows for Persistence of First-Generation Adenovirus Vectors in the Mouse Liver

    PubMed Central

    Lieber, André; He, Chen-Yi; Meuse, Leonard; Himeda, Charis; Wilson, Christopher; Kay, Mark A.

    1998-01-01

    NF-κB is a key regulator of the innate antiviral immune response, due in part to its transcriptional activation of cytokines and adhesion molecules, which, in turn, function in chemotaxis and activation of inflammatory cells. We reported earlier that viral gene expression in hepatocytes transduced with first-generation (E1-deleted) adenoviruses induced NF-κB activation, elevation of serum cytokines, and hepatocellular apoptosis during the first days postinfusion. These events did not occur in mice infused with an adenovirus vector deleted for E1, E2, E3, and late gene expression. In the present study, we used an adenovirus expressing an IκBα supersuppressor (Ad.IκBM) and bcl-2 transgenic mice to unravel the role of virus-induced NF-κB activation and apoptosis in the clearance of recombinant adenovirus vectors from the liver. The combined action of IκBM and Bcl-2 allowed for vector persistence in livers of C57BL/6 × C3H mice. In the absence of Bcl-2, IκBM expression in mouse livers significantly reduced NF-κB activation, cytokine expression, leukocyte infiltration, and the humoral immune response against the transgene product; however, this was not sufficient to prevent the decline of vector DNA in transduced cells. Infusion of Ad.IκBM caused extended apoptosis predominantly in periportal liver regions, indicating that NF-κB activation may protect transduced hepatocytes from apoptosis induced by adenovirus gene products. To confer vector persistence, bcl-2 transgene expression was required to block virus-induced apoptosis if NF-κB protection was inactivated by IκBM. Expression of gene products involved in early stages of apoptotic pathways was up-regulated in response to virus infusion in bcl-2 transgenic mice, which may represent a compensatory effect. Our study supports the idea that the suppression of innate defense mechanisms improves vector persistence. PMID:9765474

  8. Vaccination with recombinant adenoviruses expressing the peste des petits ruminants virus F or H proteins overcomes viral immunosuppression and induces protective immunity against PPRV challenge in sheep.

    PubMed

    Rojas, José M; Moreno, Héctor; Valcárcel, Félix; Peña, Lourdes; Sevilla, Noemí; Martín, Verónica

    2014-01-01

    Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants caused by the Morbillivirus peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenoviruses serotype 5 (Ad5) expressing either the highly immunogenic fusion protein (F) or hemagglutinin protein (H) from PPRV were used to vaccinate sheep by intramuscular inoculation. Both recombinant adenovirus vaccines elicited PPRV-specific B- and T-cell responses. Thus, neutralizing antibodies were detected in sera from immunized sheep. In addition, we detected a significant antigen specific T-cell response in vaccinated sheep against two different PPRV strains, indicating that the vaccine induced heterologous T cell responses. Importantly, no clinical signs and undetectable virus shedding were observed after virulent PPRV challenge in vaccinated sheep. These vaccines also overcame the T cell immunosuppression induced by PPRV in control animals. The results indicate that these adenovirus constructs could be a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.

  9. A complex adenovirus vector that delivers FASL-GFP with combined prostate-specific and tetracycline-regulated expression.

    PubMed

    Rubinchik, S; Wang, D; Yu, H; Fan, F; Luo, M; Norris, J S; Dong, J Y

    2001-11-01

    Cell-type-restricted transgene expression delivered by adenovirus vectors is highly desirable for gene therapy of cancer, as it can limit cytotoxic gene expression to tumor cells. However, many tumor- and tissue-specific promoters are weaker than the constitutively active promoters and are thus less effective. To combine cell-type specificity with high-level regulated transgene expression, we have developed a complex adenoviral vector. We have placed the tetracycline transactivator gene under the control of a prostate-specific ARR2PB promoter, and a mouse Tnfsf6 (encoding FASL)-GFP fusion gene under the control of the tetracycline responsive promoter. We have incorporated both expression cassettes into a single construct. We show that FASL-GFP expression from this vector is essentially restricted to prostate cancer cells, in which it can be regulated by doxycycline. Higher levels of prostate-specific FASL-GFP expression were generated by this approach than by driving the FASL-GFP expression directly with ARR2PB. More FASL-GFP expression correlated with greater induction of apoptosis in prostate cancer LNCaP cells. Mouse studies confirmed that systemic delivery of both the prostate-specific and the prostate-specific/tet-regulated vectors was well tolerated at doses that were lethal for FASL-GFP vector with CMV promoter. This strategy should be able to improve the safety and efficacy of cancer gene therapy using other cytotoxic genes as well.

  10. Expression of an Antigenic Adenovirus Epitope in a Group B Coxsackievirus

    PubMed Central

    Höfling, Katja; Tracy, Steven; Chapman, Nora; Kim, Kyung-Soo; Smith Leser, J.

    2000-01-01

    Group B coxsackieviruses (CVB) cause human myocarditis, while human adenovirus type 2 (Ad2) is implicated as an agent of this disease. The L1 loop of the Ad2 hexon protein has been demonstrated to be antigenic in rabbits. To evaluate the feasibility of a multivalent vaccine strain against the CVB and Ad2, we cloned the sequence encoding the Ad2 hexon L1 loop, flanked by dissimilar sequences encoding the protease 2A (2Apro) recognition sites, into the genome of an attenuated strain of CVB type 3 (CVB3/0) at the junction of 2Apro and the capsid protein 1D. Progeny virus (CVB3-PL2-Ad2L1) was obtained following transfection of the construct into HeLa cells. Replication of CVB3-PL2-Ad2L1 in diverse cell cultures demonstrated that the yield of the chimeric virus was between 0.5 to 2 log units less than the parental strain. Western blot analyses of the CVB3 capsid protein 1D in CVB3-PL2-Ad2L1-infected HeLa cells demonstrated production of the expected capsid protein. Viral proteins were detected earlier and in approximately fourfold greater amounts in CVB3-PL2-Ad2L1-infected HeLa cells than in CVB3/0-infected cells. Cleavage of the CVB3-PL2-Ad2L1 polyprotein by 2Apro was slowed, accompanied by an accumulation of the fusion 1D-L1 loop protein. Reverse transcription-PCR sequence analysis of CVB3-PL2-Ad2L1 RNA demonstrated that the Ad2 hexon polypeptide coding sequence was maintained in the chimeric viral genome through at least 10 passages in HeLa cells. Mice inoculated with CVB3-PL2-Ad2L1 demonstrated a brief viremia with no replication detectable in the heart but prolonged replication of virus in the pancreas in the absence of pathologic changes in either organ. CVB3-PL2-Ad2L1 induced binding and neutralizing anti-Ad2 antibodies, in addition to antibodies against CVB3 in mice. CVB3-PL2-Ad2L1 was used to challenge mice previously inoculated with CVB3/0 and with preexisting anti-CVB3 neutralizing-antibody titers; anti-Ad2 neutralizing and binding antibodies were induced in

  11. Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle

    USDA-ARS?s Scientific Manuscript database

    Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O,A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutral...

  12. 11R-P53 and GM-CSF Expressing Oncolytic Adenovirus Target Cancer Stem Cells with Enhanced Synergistic Activity

    PubMed Central

    Lv, Sai-qun; Ye, Zhen-long; Liu, Pin-yi; Huang, Yao; Li, Lin-fang; Liu, Hui; Zhu, Hai-li; Jin, Hua-jun; Qian, Qi-jun

    2017-01-01

    Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells. PMID:28243324

  13. PED/PEA-15 modulates coxsackievirus-adenovirus receptor expression and adenoviral infectivity via ERK-mediated signals in glioma cells.

    PubMed

    Botta, Ginevra; Perruolo, Giuseppe; Libertini, Silvana; Cassese, Angela; Abagnale, Antonella; Beguinot, Francesco; Formisano, Pietro; Portella, Giuseppe

    2010-09-01

    Glioblastoma multiforme (GBM) is the most aggressive human brain tumor, and is highly resistant to chemo- and radiotherapy. Selectively replicating oncolytic viruses represent a novel approach for the treatment of neoplastic diseases. Coxsackievirus-adenovirus receptor (CAR) is the primary receptor for adenoviruses, and loss or reduction of CAR greatly decreases adenoviral entry. Understanding the mechanisms regulating CAR expression and localization will contribute to increase the efficacy of oncolytic adenoviruses. Two glioma cell lines (U343MG and U373MG) were infected with the oncolytic adenovirus dl922-947. U373MG cells were more susceptible to cell death after viral infection, compared with U343MG cells. The enhanced sensitivity was paralleled by increased adenoviral entry and CAR mRNA and protein levels in U373MG cells. In addition, U373MG cells displayed a decreased ERK1/2 (extracellular signal-regulated kinase-1/2) nuclear-to-cytosolic ratio, compared with U343MG cells. Intracellular content of PED/PEA-15, an ERK1/2-interacting protein, was also augmented in these cells. Both ERK2 overexpression and genetic silencing of PED/PEA-15 by antisense oligonucleotides increased ERK nuclear accumulation and reduced CAR expression and adenoviral entry. Our data indicate that dl922-947 could represent an useful tool for the treatment of GBM and that PED/PEA-15 modulates CAR expression and adenoviral entry, by sequestering ERK1/2.

  14. MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2.

    PubMed

    Machitani, Mitsuhiro; Sakurai, Fuminori; Wakabayashi, Keisaku; Nakatani, Kosuke; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2017-06-15

    Recent studies have reported that host microRNAs (miRNAs) regulate infections by several types of viruses via various mechanisms and that inhibition of the miRNA processing factors enhances or prevents viral infection. However, it has not been clarified whether these effects of miRNAs extend to adenovirus (Ad) infection. Here we show that miR-27a and -b efficiently inhibit infection with an Ad via the downregulation of SNAP25 and TXN2, which are members of the SNARE proteins and the thioredoxin family, respectively. Approximately 80% reductions in Ad genomic copy number were found in cells transfected with miR-27a/b mimics, whereas there were approximately 2.5- to 5-fold larger copy numbers of the Ad genome following transfection with miR-27a/b inhibitors. Microarray gene expression analysis and in silico analysis demonstrated that SNAP25 and TXN2 are target genes of miR-27a/b. A reporter assay using plasmids containing the 3' untranslated regions of the SNAP25 and TXN2 genes showed that miR-27a/b directly suppressed SNAP25 and TXN2 expression through posttranscriptional gene silencing. Knockdown of SNAP25 led to a significant inhibition of Ad entry into cells. Knockdown of TXN2 induced cell cycle arrest at G1 phase, leading to a reduction in Ad replication. In addition, overexpression of Ad-encoded small noncoding RNAs (VA-RNAs) restored the miR-27a/b-mediated reduction in infection level with a VA-RNA-lacking Ad mutant due to the VA-RNA-mediated inhibition of miR-27a/b expression. These results indicate that miR-27a and -b suppress SNAP25 and TXN2 expression via posttranscriptional gene silencing, leading to efficient suppression of Ad infection.IMPORTANCE Adenovirus (Ad) is widely used as a platform for replication-incompetent Ad vectors (Adv) and replication-competent oncolytic Ad (OAd) in gene therapy and virotherapy. Regulation of Ad infection is highly important for efficient gene therapies using both Adv and OAd. In this study, we demonstrate that miR-27a

  15. Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

    NASA Astrophysics Data System (ADS)

    Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

    2013-12-01

    First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

  16. Ocular transfer of retinal glial cells transduced ex vivo with adenovirus expressing viral IL-10 or CTLA4-Ig inhibits experimental autoimmune uveoretinitis.

    PubMed

    Verwaerde, C; Naud, M-C; Delanoye, A; Wood, M; Thillaye-Goldenberg, B; Auriault, C; de Kozak, Y

    2003-11-01

    Gene transfer using immunomodulatory molecules is a promising tool for in vivo regulation of immune responses. Experimental autoimmune uveitis (EAU), which serves as a model for human ocular inflammation, is induced by systemic immunization with autoantigens, but its expression is restricted to the eye. Previously, we reported protection of rodents against EAU by intravenous or/and periocular injection of vIL-10-expressing adenovirus. Here, the expression of vIL-10 was targeted into the rat Lewis eye, by intravitreal injection of either the free virus or ex vivo transfected retinal Müller glial cells (RMG-vIL-10). As shown using GFP-expressing adenovirus, a longer expression of transgene was observed in the eye after transfer of transfected syngeneic RMG cells than was seen after injection of free virus. Intravitreal injection of RMG-vIL-10 led to significant decrease in ocular pathological manifestations, compared to control RMG cells. This was observed when cells were injected simultaneously with autoantigen, but also after a delayed administration of transfected cells. Finally, injection of RMG cells transfected with adenovirus expressing CTLA4 had a strongly protective effect. In conclusion, inhibition of antigen presentation at the site of expression of the autoimmune disorders represents an attractive alternative to treat ocular inflammation, and the transfer of ex vivo genetically modified cells provides a promising method to target the factor of interest into the eye.

  17. Adenovirus type 5 early region 1B 55-kDa oncoprotein can promote cell transformation by a mechanism independent from blocking p53-activated transcription.

    PubMed

    Härtl, B; Zeller, T; Blanchette, P; Kremmer, E; Dobner, T

    2008-06-12

    Inhibition of p53-activated transcription is an integral part of the mechanism by which early region 1B 55K oncoprotein (E1B-55K) from adenovirus type 5 (Ad5) contributes to complete cell transformation in combination with Ad E1A. In addition, more recent data suggest that the mode of action of the Ad protein during transformation may involve additional functions and other protein interactions. In the present study, we performed a comprehensive mutational analysis to assign further transforming functions of Ad5 E1B-55K to distinct domains within the viral polypeptide. Results from these studies show that the functions required for transformation are encoded within several patches of the 55K primary sequence, including several clustered cysteine and histidine residues, some of which match the consensus for zinc fingers. In addition, two amino-acid substitutions (C454S/C456S) created a 55K mutant protein, which had substantially reduced transforming activity. Interestingly, the same mutations neither affected binding to p53 nor inhibition of p53-mediated transactivation. Therefore, an activity necessary for efficient transformation of primary rat cells can be separated from functions required for inhibition of p53-stimulated transcription. Our data indicate that this activity is linked to the ability of the Ad5 protein to bind to components of the Mre11/Rad50/NBS1 DNA double-strand break repair complex, and/or its ability to assemble multiprotein aggregates in the cytoplasm and nucleus of transformed rat cells. These results introduce a new function for Ad5 E1B-55K and suggest that the viral protein contributes to cell transformation through p53 transcription-dependent and -independent pathways.

  18. Protection of guinea pigs and swine by a recombinant adenovirus expressing O serotype of foot-and-mouth disease virus whole capsid and 3C protease.

    PubMed

    Lu, Zengjun; Bao, Huifang; Cao, Yimei; Sun, Pu; Guo, Jianhun; Li, Pinghua; Bai, Xingwen; Chen, Yingli; Xie, Baoxia; Li, Dong; Liu, Zaixin; Xie, Qingge

    2008-12-19

    Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1-3 x 10(8)TCID(50) Ad-P12x3C recombinant adenovirus were completely protected against 10,000GID(50) homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12x3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1x10(9)TCID(50) Ad-P12x3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID(50)) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12x3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine.

  19. Stable In Vivo Transgene Expression in Endothelial Cells with Helper-Dependent Adenovirus: Roles of Promoter and Interleukin-10.

    PubMed

    Dronadula, Nagadhara; Wacker, Bradley K; Van Der Kwast, Reginald; Zhang, Jingwan; Dichek, David A

    2017-03-01

    Our long-term goal is to prevent or reverse atherosclerosis by delivering gene therapy from stably transduced endothelial cells (EC). We previously reported that EC-directed gene therapy with a helper-dependent adenovirus (HDAd) expressing apolipoprotein A-I (apo A-I) retarded development of atherosclerosis in rabbit carotid arteries over a 1-month interval. However, a 70% decline in apo A-I expression during this time raised concerns about long-term efficacy of this approach. Here we report use of several approaches aimed either at preventing this decline or at increasing apo A-I expression from HDAd at all time points: codon optimization, deletion of 3' untranslated sequences, substitution of a synthetic mammalian-based promoter (4XETE) for the cytomegalovirus (CMV) promoter, and co-transduction with an HDAd expressing interleukin-10. We tested these approaches using plasmid transfection of cultured EC and in vivo transduction of rabbit carotid artery EC. Codon optimization did not increase apo A-I expression. Deletion of 3' untranslated sequences extinguished apo A-I expression. Both substitution of 4XETE for the CMV promoter and expression of interleukin-10 stabilized apo A-I expression in vivo, although at the cost of lower early (3-day) expression levels. Surprisingly, both interventions stabilized apo A-I expression without altering the rate at which HDAd genomes were lost. These data establish that transgene expression from HDAd in EC is inherently stable in vivo and suggest that the early decline of CMV promoter-driven expression from HDAd-transduced EC is due neither to active downregulation of transcription nor to loss of HDAd genomes. Instead, apparent loss of expression from the CMV promoter appears to be a consequence of early (3-day) upregulation of CMV promoter activity via inflammatory pathways. Our results yield new paradigms to explain the early loss of genomes and transgene expression after in vivo gene transfer. These new paradigms will

  20. Transgene Expression and Host Cell Responses to Replication-Defective, Single-Cycle, and Replication-Competent Adenovirus Vectors

    PubMed Central

    Crosby, Catherine M.; Barry, Michael A.

    2017-01-01

    Most adenovirus (Ad) vectors are E1 gene deleted replication defective (RD-Ad) vectors that deliver one transgene to the cell and all expression is based on that one gene. In contrast, E1-intact replication-competent Ad (RC-Ad) vectors replicate their DNA and their transgenes up to 10,000-fold, amplifying transgene expression markedly higher than RD-Ad vectors. While RC-Ad are more potent, they run the real risk of causing adenovirus infections in vector recipients and those that administer them. To gain the benefits of transgene amplification, but avoid the risk of Ad infections, we developed “single cycle” Ad (SC-Ad) vectors. SC-Ads amplify transgene expression and generated markedly stronger and more persistent immune responses than RD-Ad as expected. However, they also unexpectedly generated stronger immune responses than RC-Ad vectors. To explore the basis of this potency here, we compared gene expression and the cellular responses to infection to these vectors in vitro and in vivo. In vitro, in primary human lung epithelial cells, SC- and RC-Ad amplified their genomes more than 400-fold relative to RD-Ad with higher replication by SC-Ad. This replication translated into higher green fluorescent protein (GFP) expression for 48 h by SC- and RC-Ad than by RD-Ad. In vitro, in the absence of an immune system, RD-Ad expression became higher by 72 h coincident with cell death mediated by SC- and RC-Ad and release of transgene product from the dying cells. When the vectors were compared in human THP-1 Lucia- interferon-stimulated gene (ISG) cells, which are a human monocyte cell line that have been modified to quantify ISG activity, RC-Ad6 provoked significantly stronger ISG responses than RD- or SC-Ad. In mice, intravenous or intranasal injection produced up to 100-fold genome replication. Under these in vivo conditions in the presence of the immune system, luciferase expression by RC and SC-Ad was markedly higher than that by RD-Ad. In immunodeficient mice, SC

  1. Inhalation of Nebulized Perfluorochemical Enhances Recombinant Adenovirus and Adeno-Associated Virus-Mediated Gene Expression in Lung Epithelium

    PubMed Central

    Beckett, Travis; Bonneau, Laura; Howard, Alan; Blanchard, James; Borda, Juan; Weiner, Daniel J.; Wang, Lili; Gao, Guang Ping; Kolls, Jay K.; Bohm, Rudolf; Liggitt, Denny

    2012-01-01

    Abstract Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Recombinant adenoviral vector was administered by transtracheal puncture at selected times afterward and mice were killed 3 days after vector administration to assess transgene expression. Mice tolerated the nebulized perflubron without obvious ill effects. Vector administration 6 hr after nebulized perflubron exposure resulted in an average 540% increase in gene expression in airway and alveolar epithelium, compared with that with vector alone or saline plus vector control (p<0.05). However, vector administration 1 hr, 1 day, or 3 days after perflubron exposure was not different from either nebulized saline with vector or vector alone and a 60-min exposure to nebulized perflubron is required. In parallel pilot studies in macaques, inhalation of nebulized perflubron enhanced recombinant AAV2/5 vector expression throughout the lung. Serial chest radiographs, bronchoalveolar lavages, and results of complete blood counts and serum biochemistries demonstrated no obvious adverse effects of nebulized perflubron. Further, one macaque receiving nebulized perflubron only was monitored for 1 year with no obvious adverse effects of exposure. These results demonstrate that inhalation of nebulized perflubron, a simple, clinically more feasible technique than intratracheal administration of liquid perflubron, safely enhances lung gene expression. PMID:22568624

  2. Development of a novel recombinant adenovirus containing gfp-zeocin fusion expression cassette for conditional replication in p53-deficient human tumor cells.

    PubMed

    Hu, Baoli; Joshua, Mallam Nock; Dong, Changyuan; Qi, Yipeng

    2004-05-01

    Two obstacles limiting the efficacy of nearly all cancer gene therapy trails are low gene transduction efficiency and the lack of tumor specificity. Fortunately, a replication-competent, E1B-deficient adenovirus (dl1520) was developed that could overcome these limitations, because it was capable of efficiently and selectively destroying tumor cells lacking functional p53. In an attempt to appraise the efficiency and safety of this approach, a novel recombinant adenovirus, r3/Ad, containing a gfp-zeocin expression cassette was constructed in this work. The study in vitro demonstrated that r3/Ad has the ability to replicate in and lyse only the p53-deficient human tumor cells such as the human glioblastoma cells (U251) and human bladder cells (EJ) but not in the human fibroblast cells (MRC-5) with functional p53. Importantly, this gfp-zeocin fusion gene driven by the bipromoter (CMV and EM-7) could be used as an effective selective marker and reporter in prokaryotic and eukaryotic cells; and also zeocin as a selective marker could minimize contamination of the recombinant virus by the wt-Ad5. Additionally, it was found that the r3/Ad could be useful for studying the selective replication of E1B-deficient adenovirus in vivo, it could be used as a "guide" to study the ability of the recombinant adenovirus to spread and to infect distant tumor cells in any tumor bearing animal model by GFP as a reporter. This may help determine the safety of using any E1B-deficient adenovirus in cancer gene therapy.

  3. Vaccination of rabbits with an adenovirus vector expressing the papillomavirus E2 protein leads to clearance of papillomas and infection.

    PubMed

    Brandsma, Janet L; Shlyankevich, Mark; Zhang, Lixin; Slade, Martin D; Goodwin, Edward C; Peh, Woei; Deisseroth, Albert B

    2004-01-01

    Cervical cancer arises from lesions caused by infection with high-risk types of human papillomavirus (HPV). Therefore, vaccination against HPV could prevent carcinogenesis by preventing HPV infection or inducing lesion regression. HPV E2 protein is an attractive candidate for vaccine development because it is required for papilloma formation, is involved in all stages of the virus life cycle, and is expressed in all premalignant lesions as well as some cancers. This study reports vaccination against E2 protein using a rabbit model of papillomavirus infection. A recombinant adenovirus (Ad) vector expressing the E2 protein of cottontail rabbit papillomavirus (CRPV) was tested for therapeutic efficacy in CRPV-infected rabbits. Primary immunization with the Ad-E2 vaccine, compared to immunization with a control Ad vector, reduced the number of papilloma-forming sites from 17 of 45 to 4 of 45. After booster immunization, vaccinated rabbits formed no new papillomas versus an additional 23 papillomas in rabbits that received the control vector. Papillomas in the Ad-E2 vaccinees were significantly smaller than those in the control rabbits, and all four papillomas in the Ad-E2 vaccinated rabbits regressed. No CRPV DNA was detected either in the regression sites or in sites that did not form papillomas, indicating that the vaccination led to clearance of CRPV from all infected sites.

  4. A New Group B Adenovirus Receptor Is Expressed at High Levels on Human Stem and Tumor Cells▿

    PubMed Central

    Tuve, Sebastian; Wang, Hongjie; Ware, Carol; Liu, Ying; Gaggar, Anuj; Bernt, Kathrin; Shayakhmetov, Dmitry; Li, ZongYi; Strauss, Robert; Stone, Daniel; Lieber, André

    2006-01-01

    CD46 is used by human group B adenoviruses (Ads) as a high-affinity attachment receptor. Here we show evidence that several group B Ads utilize an additional receptor for infection of human cells, which is different from CD46. We tentatively named this receptor receptor X. Competition studies with unlabeled and labeled Ads, recombinant Ad fiber knobs, and soluble CD46 and CD46 antibodies revealed three different subgroups of group B Ads, in terms of their receptor usage. Group I (Ad16, -21, -35, and -50) nearly exclusively uses CD46. Group II (Ad3, -7p, and -14) utilizes receptor X and not CD46. Group III (Ad11p) uses both CD46 and the alternative receptor X. Interaction of group II and III Ads with receptor X occurs via the fiber knob. Receptor X is an abundantly expressed glycoprotein that interacts with group II and III Ads at relatively low affinity in a Ca2+-dependent manner. This receptor is expressed at high levels on human mesenchymal and undifferentiated embryonic stem cells, as well as on human cancer cell lines. These findings have practical implications for stem cell and gene therapy. PMID:17020944

  5. Prevention of hepatic ischemia-reperfusion injury by pre-administration of catalase-expressing adenovirus vectors.

    PubMed

    Ushitora, Masahiro; Sakurai, Fuminori; Yamaguchi, Tomoko; Nakamura, Shin-ichiro; Kondoh, Masuo; Yagi, Kiyohito; Kawabata, Kenji; Mizuguchi, Hiroyuki

    2010-03-19

    Liver ischemia/reperfusion (I/R) injury, which is mainly caused by the generation of reactive oxygen species (ROS) during the reperfusion, remains an important clinical problem associated with liver transplantation and major liver surgery. Therefore, ROS should be detoxified to prevent hepatic I/R-induced injury. Delivery of antioxidant genes into liver is considered to be promising for prevention of hepatic I/R injury; however, therapeutic effects of antioxidant gene transfer to the liver have not been fully examined. The aim of this study was to examine whether adenovirus (Ad) vector-mediated catalase gene transfer in the liver is an effective approach for scavenging ROS and preventing hepatic I/R injury. Intravenous administration of Ad vectors expressing catalase, which is an antioxidant enzyme scavenging H(2)O(2), resulted in a significant increase in catalase activity in the liver. Pre-injection of catalase-expressing Ad vectors dramatically prevented I/R-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and hepatic necrosis. The livers were also protected in another liver injury model, CCl(4)-induced liver injury, by catalase-expressing Ad vectors. Furthermore, the survival rates of mice subjected to both partial hepatectomy and I/R treatment were improved by pre-injection of catalase-expressing Ad vectors. On the other hand, control Ad vectors expressing beta-galactosidase did not show any significant preventive effects in the liver on the models of I/R-induced or CCl(4)-induced hepatic injury described above. These results indicate that hepatic delivery of the catalase gene by Ad vectors is a promising approach for the prevention of oxidative stress-induced liver injury. (c) 2009. Published by Elsevier B.V. All rights reserved.

  6. Hepatic Expression of Adenovirus 36 E4ORF1 Improves Glycemic Control and Promotes Glucose Metabolism Through AKT Activation.

    PubMed

    McMurphy, Travis B; Huang, Wei; Xiao, Run; Liu, Xianglan; Dhurandhar, Nikhil V; Cao, Lei

    2017-02-01

    Considering that impaired proximal insulin signaling is linked with diabetes, approaches that enhance glucose disposal independent of insulin signaling are attractive. In vitro data indicate that the E4ORF1 peptide derived from human adenovirus 36 (Ad36) interacts with cells from adipose tissue, skeletal muscle, and liver to enhance glucose disposal, independent of proximal insulin signaling. Adipocyte-specific expression of Ad36E4ORF1 improves hyperglycemia in mice. To determine the hepatic interaction of Ad36E4ORF1 in enhancing glycemic control, we expressed E4ORF1 of Ad36 or Ad5 or fluorescent tag alone by using recombinant adeno-associated viral vector in the liver of three mouse models. In db/db or diet-induced obesity (DIO) mice, hepatic expression of Ad36E4ORF1 but not Ad5E4ORF1 robustly improved glycemic control. In normoglycemic wild-type mice, hepatic expression of Ad36E4ORF1 lowered nonfasting blood glucose at a high dose of expression. Of note, Ad36E4ORF1 significantly reduced insulin levels in db/db and DIO mice. The improvement in glycemic control was observed without stimulation of the proximal insulin signaling pathway. Collectively, these data indicate that Ad36E4ORF1 is not a typical sensitizer, mimetic, or secretagogue of insulin. Instead, it may have insulin-sparing action, which seems to reduce the need for insulin and, hence, to reduce insulin levels. © 2017 by the American Diabetes Association.

  7. Lac-regulated system for generating adenovirus 5 vaccine vectors expressing cytolytic human immunodeficiency virus 1 genes.

    PubMed

    Zhao, Chunxia; Crews, Charles Jefferson; Derdeyn, Cynthia A; Blackwell, Jerry L

    2009-09-01

    Adenovirus (Ad) vectors have been developed as human immunodeficiency-1 (HIV-1) vaccine vectors because they consistently induce immune responses in preclinical animal models and human trials. Strong promoters and codon-optimization are often used to enhance vaccine-induced HIV-1 gene expression and immunogenicity. However, if the transgene is inherently cytotoxic in the cell line used to produce the vector, and is expressed at high levels, it is difficult to rescue a stable Ad HIV-1 vaccine vector. Therefore we hypothesized that generation of Ad vaccine vectors expressing cytotoxic genes, such as HIV-1 env, would be more efficient if expression of the transgene was down-regulated during Ad rescue. To test this hypothesis, a Lac repressor-operator system was applied to regulate expression of reporter luciferase and HIV-1 env transgenes during Ad rescue. The results demonstrate that during Ad rescue, constitutive expression of the Lac repressor in 293 cells reduced transgene expression levels to approximately 5% of that observed in the absence of regulation. Furthermore, Lac-regulation translated into more efficient Ad rescue compared to traditional 293 cells. Importantly, Ad vectors rescued with this system showed high levels of transgene expression when transduced into cells that lack the Lac repressor protein. The Lac-regulated system also facilitated the rescue of modified Ad vectors that have non-native receptor tropism. These tropism-modified Ad vectors infect a broader range of cell types than the unmodified Ad, which could increase their effectiveness as a vaccine vector. Overall, the Lac-regulated system described here (i) is backwards compatible with Ad vector methods that employ bacterial-mediated homologous recombination, (ii) is adaptable for the engineering of tropism-modified Ad vectors, and (iii) does not require co-expression of regulatory genes from the vector or the addition of exogenous chemicals to induce or repress transgene expression. This

  8. An adenoviral vector-based expression and delivery system for the inhibition of wild-type adenovirus replication by artificial microRNAs

    PubMed Central

    Ibrišimović, Mirza; Kneidinger, Doris; Lion, Thomas; Klein, Reinhard

    2013-01-01

    Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections (i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude. PMID:23127366

  9. An adenoviral vector-based expression and delivery system for the inhibition of wild-type adenovirus replication by artificial microRNAs.

    PubMed

    Ibrišimović, Mirza; Kneidinger, Doris; Lion, Thomas; Klein, Reinhard

    2013-01-01

    Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections (i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude.

  10. Adenovirus structure.

    PubMed

    Rux, John J; Burnett, Roger M

    2004-12-01

    Structural studies continue to play an essential role as the focus of adenovirus research shifts in emphasis from basic biology to adenovirus-based vector technologies. A crucial step in developing novel therapeutics for gene replacement, cancer, and vaccines is often to modify the virion. Such engineered changes are designed to retarget the virus, or to reduce the immunological responses to infection. These efforts are far more effective when they are based on detailed structural knowledge. This minireview provides a brief summary of the wealth of information that has been obtained from the combined application of X-ray crystallography and electron microscopy. This knowledge now includes a good working model for the architectural organization of the virion, and atomic resolution molecular structures for all the major capsid proteins, hexon, penton, and fiber. We highlight new developments, which include the structure of the penton base and the discovery that adenovirus has several relatives. We sketch how the structural information can be used to engineer novel virions and conclude with the prospects for future progress.

  11. Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit

    PubMed Central

    Suzuki, Mariko; Kondo, Saki; Yamasaki, Manabu; Matsuda, Norie; Nomoto, Akio; Suzuki, Tetsuro; Saito, Izumu; Kanegae, Yumi

    2017-01-01

    The complicated replication mechanisms of hepatitis B virus (HBV) have impeded HBV studies and anti-HBV therapy development as well. Herein we report efficient genome replication of HBV applying adenovirus vectors (AdVs) showing high transduction efficiency. Even in primary hepatocytes derived from humanized mice the transduction efficiencies using AdVs were 450-fold higher compared than those using plasmids. By using an expression unit consisting of the CMV promoter, 1.03-copy HBV genome and foreign poly(A) signal, we successfully generated an improved AdV (HBV103-AdV) that efficiently provided 58 times more pregenomic RNA than previously reported AdVs. The HBV103-AdV-mediated HBV replication was easily and precisely detected using quantitative real-time PCR in primary hepatocytes as well as in HepG2 cells. Notably, when the AdV containing replication-defective HBV genome of 1.14 copy was transduced, we observed that HBV DNA-containing circular molecules (pseudo-ccc DNA) were produced, which were probably generated through homologous recombination. However, the replication-defective HBV103-AdV hardly yielded the pseudo-ccc, probably because the repeated sequences are vey short. Additionally, the efficacies of entecavir and lamivudine were quantitatively evaluated using this system at only 4 days postinfection with HBV103-AdVs. Therefore, this system offers high production of HBV genome replication and thus could become used widely. PMID:28157182

  12. Midkine promoter-driven suicide gene expression and -mediated adenovirus replication produced cytotoxic effects to immortalised and tumour cells.

    PubMed

    Yu, L; Hamada, K; Namba, M; Kadomatsu, K; Muramatsu, T; Matsubara, S; Tagawa, M

    2004-07-01

    We examined possible application of a regulatory region of midkine (MK) gene, which is frequently upregulated in a number of human tumours but not in normal cells, to cancer gene therapy. We examined transcriptional activity of the MK genomic fragments in paired cell lines, immortalized cells and their parental normal fibroblasts, and found that the MK fragments activated a fused reporter or a suicide gene preferentially in the immortalized cells. Recombinant adenoviruses (Ad), in which the MK fragment was inserted upstream to the E1A gene (AdMK), replicated preferentially in the immortalized cells and were cytotoxie to them. Human hepatocellular carcinoma cells were significantly susceptible to AdMK compared with human normal fibroblasts in vitro and the replication of AdMK was less than that of wild-type Ad in the infected fibroblasts. Hepatocellular carcinoma cells infected with AdMK did not form tumours in immunocompromised mice and intratumoural injection of AdMK into the hepatocellular carcinoma developed in mice retarded the subsequent tumour growth. Expression of E1A and necrosis of tumours were detected in AdMK-injected but not control Ad-injected cases. The MK promoter-driven suicide gene therapy and -mediated replicative Ad can thereby produce cytotoxic effects to immortalized and tumour cells with minimal damage to normal cells.

  13. Targeted suppression of Has2 mRNA in mouse cumulus cell-oocyte complexes by adenovirus-mediated short-hairpin RNA expression

    PubMed Central

    Sugiura, Koji; Su, You-Qiang; Eppig, John J.

    2008-01-01

    RNA interference (RNAi) is an effective tool for studying gene function in oocytes, but no studies have targeted somatic cells of primary cultured cumulus cell-oocyte complexes (COCs). This is probably due to difficulty in introducing RNAi-inducing molecules, such as a short-hairpin RNA (shRNA) gene, into COCs by commonly used transfection reagents. We therefore tested whether a developmental process of intact COCs could be suppressed by adenovirus-mediated shRNA expression. Has2, encoding hyaluronan synthase 2, was selected as the target transcript, because the process of cumulus expansion depends upon expression of Has2 mRNA and this process is easily evaluated in vitro. Intact COCs were infected with replication-incompetent adenoviruses containing an expression sequence of shRNA targeting either Has2 (Has2 shRNA) or a control transcript not expressed in cumulus cells, and the effects on epidermal growth factor (EGF)-stimulated cumulus expansion were determined. Has2 shRNA expression suppressed Has2 mRNA levels in COCs by more than 70%, without affecting expression levels of Ptgs2, Ptx3, Tnfaip6 mRNAs, which are also required for cumulus expansion, or other transcripts not related to expansion. Interestingly, levels of Areg and Ereg mRNAs were decreased in COCs expressing Has2 shRNA when compared with those in controls, while Btc mRNA levels remained unaffected. Furthermore, the degree of cumulus expansion by Has2 shRNA-expressing COCs was significantly less than that of controls. Thus adenovirus-mediated introduction of shRNA produces specific gene silencing and a phenotype in intact COCs, providing proof of principle that this method will be a helpful tool for understanding mechanisms of COC development. PMID:18951380

  14. Neonatal Gene Therapy for Hemophilia B by a Novel Adenovirus Vector Showing Reduced Leaky Expression of Viral Genes.

    PubMed

    Iizuka, Shunsuke; Sakurai, Fuminori; Tachibana, Masashi; Ohashi, Kazuo; Mizuguchi, Hiroyuki

    2017-09-15

    Gene therapy during neonatal and infant stages is a promising approach for hemophilia B, a congenital disorder caused by deficiency of blood coagulation factor IX (FIX). An adenovirus (Ad) vector has high potential for use in neonatal or infant gene therapy for hemophilia B due to its superior transduction properties; however, leaky expression of Ad genes often reduces the transduction efficiencies by Ad protein-mediated tissue damage. Here, we used a novel Ad vector, Ad-E4-122aT, which exhibits a reduction in the leaky expression of Ad genes in liver, in gene therapy studies for neonatal hemophilia B mice. Ad-E4-122aT exhibited significantly higher transduction efficiencies than a conventional Ad vector in neonatal mice. In neonatal hemophilia B mice, a single neonatal injection of Ad-E4-122aT expressing human FIX (hFIX) (Ad-E4-122aT-AHAFIX) maintained more than 6% of the normal plasma hFIX activity levels for approximately 100 days. Sequential administration of Ad-E4-122aT-AHAFIX resulted in more than 100% of the plasma hFIX activity levels for more than 100 days and rescued the bleeding phenotypes of hemophilia B mice. In addition, immunotolerance to hFIX was induced by Ad-E4-122aT-AHAFIX administration in neonatal hemophilia B mice. These results indicated that Ad-E4-122aT is a promising gene delivery vector for neonatal or infant gene therapy for hemophilia B.

  15. Adenovirus-mediated Wnt5a expression inhibits the telogen-to-anagen transition of hair follicles in mice.

    PubMed

    Xing, Yi-Zhan; Wang, Rui-Min; Yang, Ke; Guo, Hai-Ying; Deng, Fang; Li, Yu-Hong; Ye, Ji-Xing; He, Long; Lian, Xiao-Hua; Yang, Tian

    2013-01-01

    The canonical Wnt/β-catenin pathway plays an important role in hair cycle induction. Wnt5a is a non-canonical Wnt family member that generally antagonizes canonical Wnt signaling in other systems. In hair follicles, Wnt5a and canonical Wnt are both expressed in cells in the telogen stage. Wnt5a has been shown to be critical for controlling hair cell fate. However, the role that Wnt5a plays in the transition from the telogen to anagen stage is unknown. In this study, using whole-mount in situ hybridization, we show that Wnt5a is produced by several other cell types, excluding dermal papilla cells, throughout the hair cycle. For example, Wnt5a is expressed in bulge and secondary hair germ cells in the telogen stage. Our studies focused on the depilated 8-week-old mouse as a synchronized model of hair growth. Interestingly, overexpression of adenovirus Wnt5a in the dorsal skin of mice led to the elongation of the telogen stage and inhibition of the initiation of the anagen stage. However, following an extended period of time, four pelage hair types grew from hairless skin that was induced by Wnt5a, and the structure of these new hair shafts was normal. Using microarray analysis and quantitative arrays, we showed that the expression of β-catenin and some target genes of canonical Wnt signaling decreased after Wnt5a treatment. These data demonstrate that Wnt5a may inhibit the telogen stage to maintain a quiescent state of the hair follicle.

  16. Combined Therapy with Cytokine-Induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor Model

    PubMed Central

    Shan, Juanjuan; Shen, Junjie; Liu, Limei; Xu, Yanmin; Xia, Feng; Bie, Ping; Zhang, Xia; Cui, Youhong; Bian, Xiu-wu; Qian, Cheng

    2012-01-01

    Both adoptive immunotherapy and gene therapy hold a great promise for treatment of malignancies. However, these strategies exhibit limited anti-tumor activity, when they are used alone. In this study, we explore whether combination of cytokine-induced killer (CIK) adoptive immunotherapy with oncolytic adenovirus-mediated transfer of human interleukin-12 (hIL-12) gene induce the enhanced antitumor potency. Our results showed that oncolytic adenovirus carrying hIL-12 (AdCN205-IL12) could produce high levels of hIL-12 in liver cancer cells, as compared with replication-defective adenovirus expressing hIL-12 (Ad-IL12). AdCN205-IL12 could specifically induce cytotoxocity to liver cancer cells. Combination of CIK cells with AdCN205-IL12 could induce higher antitumor activity to liver cancer cells in vitro than that induced by either CIK or AdCN205-IL12 alone, or combination of CIK and control vector AdCN205-GFP. Furthermore, treatment of the established liver tumors with the combined therapy of CIK cells and AdCN205-IL12 resulted in tumor regression and long-term survival. High level expression of hIL-12 in tumor tissues could increase traffic of CIK cells to tumor tissues and enhance their antitumor activities. Our study provides a novel strategy for the therapy of cancer by the combination of CIK adoptive immunotherapy with oncolytic adenovirus-mediated transfer of immune stimulatory molecule hIL-12. PMID:23028626

  17. Synthesis in Escherichia coli of human adenovirus type 12 transforming proteins encoded by early region 1A 13S mRNA and 12S mRNA.

    PubMed Central

    Kimelman, D; Lucher, L A; Brackmann, K H; Symington, J S; Ptashne, M; Green, M

    1984-01-01

    Human adenovirus (Ad)-encoded early region 1A (E1A) tumor (T) antigens have been implicated in the positive regulation of viral early genes, the positive and negative regulation of some cellular genes, and cell immortalization and transformation. To further study the Ad E1A T antigens and to facilitate their purification, we have cloned cDNA copies of the Ad12 E1A 13S mRNA and 12S mRNA downstream of a hybrid Escherichia coli trp-lac (tac) promoter. Up to 8% of the protein synthesized in E. coli cells transformed by each of the two different Ad12 E1A cDNA constructs were immunoprecipitated as a Mr 47,000 protein by antibody to a synthetic peptide encoded in the Ad12 E1A DNA sequence. Both proteins produced in E. coli appear to be authentic and complete Ad12 E1A T antigens because they possess (i) the Ad12 E1A NH2-terminal amino acid sequence predicted from the DNA sequence; (ii) the Ad12 E1A COOH-terminal sequence, as shown by immunoprecipitation with anti-peptide antibody; and (iii) a molecular weight and an acidic isoelectric point similar to that of the E1A T antigens synthesized in Ad12-infected and transformed mammalian cells. The T antigens were purified to near homogeneity in yields of 100-200 micrograms per g wet weight of transformed E. coli cells. Images PMID:6387701

  18. Recombinant soluble adenovirus receptor

    DOEpatents

    Freimuth, Paul I.

    2002-01-01

    Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

  19. Vaccination with Recombinant Adenoviruses Expressing the Peste des Petits Ruminants Virus F or H Proteins Overcomes Viral Immunosuppression and Induces Protective Immunity against PPRV Challenge in Sheep

    PubMed Central

    Rojas, José M.; Moreno, Héctor; Valcárcel, Félix; Peña, Lourdes; Sevilla, Noemí; Martín, Verónica

    2014-01-01

    Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants caused by the Morbillivirus peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenoviruses serotype 5 (Ad5) expressing either the highly immunogenic fusion protein (F) or hemagglutinin protein (H) from PPRV were used to vaccinate sheep by intramuscular inoculation. Both recombinant adenovirus vaccines elicited PPRV-specific B- and T-cell responses. Thus, neutralizing antibodies were detected in sera from immunized sheep. In addition, we detected a significant antigen specific T-cell response in vaccinated sheep against two different PPRV strains, indicating that the vaccine induced heterologous T cell responses. Importantly, no clinical signs and undetectable virus shedding were observed after virulent PPRV challenge in vaccinated sheep. These vaccines also overcame the T cell immunosuppression induced by PPRV in control animals. The results indicate that these adenovirus constructs could be a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines. PMID:25013961

  20. Canine adenovirus based rabies vaccines.

    PubMed

    Tordo, N; Foumier, A; Jallet, C; Szelechowski, M; Klonjkowski, B; Eloit, M

    2008-01-01

    Adenovirus based vectors are very attractive candidates for vaccination purposes as they induce in mammalian hosts potent humoral, mucosal and cellular immune responses to antigens encoded by the inserted genes. We have generated E1-deleted and replication-competent recombinant canine type-2 adenoviruses expressing the rabies virus glycoprotein (G). The effectiveness of both vectors to express a native G protein has been characterized in vitro in permissive cell lines. We compared the humoral and cellular immune responses induced in mice by intramuscular injection of the recombinant canine adenovirus vectors with those induced by a human (Ad5) E1-deleted virus expressing the same rabies G protein. Humoral responses specific to the adenoviruses or the rabies glycoprotein antigens were studied. The influence of the mouse strain was observed using replication-competent canine adenovirus. A high level of rabies neutralizing antibody was observed upon i.m. inoculation, and 100% of mice survived lethal challenge. These results are very promising in the perspective of oral vaccine for dog rabies control.

  1. Tight positive regulation of transgene expression by a single adenovirus vector containing the rtTA and tTS expression cassettes in separate genome regions.

    PubMed

    Mizuguchi, Hiroyuki; Xu, Zhi-Li; Sakurai, Fuminori; Mayumi, Tadanori; Hayakawa, Takao

    2003-09-01

    We previously developed single adenovirus (Ad) vectors that contained the components for a tetracycline-regulatable gene-expression system in the E1 and E3 deletion regions, and showed that the Ad vectors containing the tet-on system exhibit a much inferior regulation of transgene expression than those containing the tet-off system. In many cases, the tet-on system may be preferable because of its positive regulation of transgene expression. To this end, in the present study, by introducing the latest generation reverse tetracycline-responsive transcriptional activator (rtTA2s-M2 or rtTA2s-S2) and the tetracycline-controlled transcriptional silencer (tTS) into the original tet-on system, we constructed various modified Ad-mediated tet-on systems. Among them, the novel single Ad vector, which contained three heterologous gene-expression cassettes of the gene of interest, rtTA2s-S2, and tTS in the E1 deletion region, the E3 deletion region, and the region between E4 and 3'ITR, respectively, displayed vastly improved doxycycline-inducible gene expression in terms of low basal expression, high induced expression, and high responsiveness to doxycycline both in vitro and in vivo. These results also suggest that the low responsiveness to doxycycline may explain why the original tet-on system in the context of the Ad vector is not effective in vivo. This is the first report describing the cloning of three heterologous gene-expression cassettes into three separate regions of the E1/E3-deleted Ad genome. This improved Ad-mediated tet-on system might be useful for gene therapy and greatly facilitate the analyses of gene function.

  2. Anti-adenoviral Artificial MicroRNAs Expressed from AAV9 Vectors Inhibit Human Adenovirus Infection in Immunosuppressed Syrian Hamsters.

    PubMed

    Schaar, Katrin; Geisler, Anja; Kraus, Milena; Pinkert, Sandra; Pryshliak, Markian; Spencer, Jacqueline F; Tollefson, Ann E; Ying, Baoling; Kurreck, Jens; Wold, William S; Klopfleisch, Robert; Toth, Karoly; Fechner, Henry

    2017-09-15

    Infections of immunocompromised patients with human adenoviruses (hAd) can develop into life-threatening conditions, whereas drugs with anti-adenoviral efficiency are not clinically approved and have limited efficacy. Small double-stranded RNAs that induce RNAi represent a new class of promising anti-adenoviral therapeutics. However, as yet, their efficiency to treat hAd5 infections has only been investigated in vitro. In this study, we analyzed artificial microRNAs (amiRs) delivered by self-complementary adeno-associated virus (scAAV) vectors for treatment of hAd5 infections in immunosuppressed Syrian hamsters. In vitro evaluation of amiRs targeting the E1A, pTP, IVa2, and hexon genes of hAd5 revealed that two scAAV vectors containing three copies of amiR-pTP and three copies of amiR-E1A, or six copies of amiR-pTP, efficiently inhibited hAd5 replication and improved the viability of hAd5-infected cells. Prophylactic application of amiR-pTP/amiR-E1A- and amiR-pTP-expressing scAAV9 vectors, respectively, to immunosuppressed Syrian hamsters resulted in the reduction of hAd5 levels in the liver of up to two orders of magnitude and in reduction of liver damage. Concomitant application of the vectors also resulted in a decrease of hepatic hAd5 infection. No side effects were observed. These data demonstrate anti-adenoviral RNAi as a promising new approach to combat hAd5 infection. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. [Construction of recombinant adenovirus vector of calcitonin gene-related peptide gene and transfection to neonatal rat cardiomyocytes].

    PubMed

    Sun, Zhi-hui; Han, Jie; Shao, Lei; Wang, Li-hong; Song, Jun-xian; Wei, Zhong-hai; Zheng, Liang-rong

    2010-05-01

    To construct a recombinant adenovirus vector of calcitonin gene-related peptide (CGRP) by AdEasy system and to validate its expression in myocardial cells. The full-length of CGRP gene cDNA was acquired by RT-PCR and cloned into pShuttle-CMV. After linearization with Pme I, the recombinant plasmid (pShuttle-CMV-CGRP) was transformed into E.coli BJ5183 by electroporation to construct the recombinant adenovirus plasmid AdEasy-pShuttle-CGRP. The recombinant adenovirus plasmids were transformed into E.coli XL10-Gold cells to be amplified. Then the recombinant plasmid was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. PCR technique was used to detect target gene. The recombinant adenovirus particles were purified by CsC1 density gradient. The purified recombinant adenovirus was transfected to neonatal rat cardiomyocytes,and the recombinant adenovirus production was observed by fluorescent microscope. Expression of CGRP in hearts 7 days after intravenous delivery of adenoviral vectors AV-CGRP was determined by radioimmunoassay. The RT-PCR products confirmed a full-length cDNA of CGRP gene in PUC(57) by sequencing. The corresponding double endonuclease and PCR analysis certified the successful cloning of the gene into the pShuttle-CMV. The recombinant adenovirus plasmid AdEasy-pShuttle-CGRP was digested by Pac I endonuclease to form the typical DNA segments, whose length was about 3 kb and 30 kb. PCR analysis and fluorescent microscope observation confirmed that the CGRP gene was inserted into the adenovirus vector with very strong power of transfection. The recombinant adenovirus particles infected neonatal rat cardiomyocytes successfully. Radioimmunoassay showed that delivery of AV-CGRP significantly increased the expression of CGRP in mice hearts. The recombinant adenovirus vector of CGRP gene has been constructed,and it can infect neonatal rat cardiomyocytes successfully. Somatic delivery of CGRP gene can significantly

  4. Development of a replication defective adenovirus 5 vector expressing porcine interleukin-18 and a mutated analog

    USDA-ARS?s Scientific Manuscript database

    Cell-mediated immune responses against swine pathogens are sometimes necessary to elicit durable protective immunity. Cell mediated or Th1 immunity is dependent on the coordinated expression of several cytokines, including interferon-gamma to assist in the production of antigen-specific cytotoxic T...

  5. Combination of adenoviruses expressing melanoma differentiation-associated gene-7 and chemotherapeutic agents produces enhanced cytotoxicity on esophageal carcinoma.

    PubMed

    Ma, G; Kawamura, K; Shan, Y; Okamoto, S; Li, Q; Namba, M; Shingyoji, M; Tada, Y; Tatsumi, K; Hiroshima, K; Shimada, H; Tagawa, M

    2014-01-01

    We examined the combinatory antitumor effects of adenoviruses expressing human mda-7/IL-24 gene (Ad-mda-7) and chemotherapeutic agents on nine kinds of human esophageal carcinoma cells. All the carcinoma cells expressed the melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) receptor complexes, IL-20R2 and either IL-20R1 or IL-22R1, and were susceptible to Ad-mda-7, whereas fibroblasts were positive only for IL-20R2 gene and resistant to Ad-mda-7-mediated cytotoxicity. Sensitivity of these esophageal carcinoma cells to Ad-mda-7 was however lower than that to Ad expressing the wild-type p53 gene. We thereby investigated a possible combination of Ad-mda-7 and anticancer agents and found that Ad-mda-7 with 5-fluorouracil (5-FU), cisplatin, mitomycin C or etoposide produced greater cytotoxic effects than those by Ad-mda-7 or the agent alone. Half-maximal inhibitory concentration values of the agents in respective cells were decreased by the combination with Ad-mda-7. Cell cycle analyses showed that Ad-mda-7 and 5-FU increased G2/M-phase and S-phase populations, respectively, and the combination augmented sub-G1 populations. Ad-mda-7-treated cells showed cleavages of caspase-8, -9 and -3 and poly (ADP-ribose) polymerase, but the cleavage levels were not different from those of the combination-treated cells. Ad-mda-7 treatments upregulated Akt phosphorylation but suppressed IκB-α levels, whereas 5-FU treatments induced phosphorylation of p53 and extracellular signal-regulated protein kinases 1 and 2. Molecular changes caused by the combination were similar to those by Ad-mda-7 treatments, but the Ad-mda-7-mediated upregulation of Akt phosphorylation decreased with the combination. These data collectively suggest that Ad-mda-7 induced apoptosis despite Akt activation and that the combinatory antitumor effects with 5-FU were produced partly by downregulating the Ad-mda-7-induced Akt activation.

  6. Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection.

    PubMed

    Krzywkowski, Tomasz; Ciftci, Sibel; Assadian, Farzaneh; Nilsson, Mats; Punga, Tanel

    2017-06-01

    An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells.IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method

  7. Expression of coxsackievirus and adenovirus receptor (CAR)-Fc fusion protein in Pichia pastoris and characterization of its anti-coxsackievirus activity.

    PubMed

    Zhang, Kebin; Yu, Hua; Xie, Wei; Xu, Zihui; Zhou, Shiwen; Huang, Chunji; Sheng, Halei; He, Xiaomei; Xiong, Junzhi; Qian, Guisheng

    2013-04-15

    Coxsackievirus and adenovirus receptors (CARs) are the common cellular receptors which mediate coxsackievirus or adenovirus infection. Receptor trap therapy, which uses soluble viral receptors to block the attachment and internalization of virus, has been developed for the inhibition of virus infection. In this study, we have constructed a pPIC3.5K/CAR-Fc expression plasmid for the economical and scale-up production of CAR-Fc fusion protein in Pichia pastoris. The coding sequence of the fusion protein was optimized according to the host codon usage bias. The amount of the CAR-Fc protein to total cell protein was up to 10% by 1% methanol induction for 96h and the purity was up to 96% after protein purification. Next, the virus pull-down assay demonstrated the binding activity of the CAR-Fc to coxsackievirus. The analyses of MTT assay, immunofluorescence staining and quantitative real-time PCR after virus neutralization assay revealed that CAR-Fc could significantly block coxsackievirus B3 infection in vitro. In coxsackievirus B3 infected mouse models, CAR-Fc treatment reduced mortality, myocardial edema, viral loads and inflammation, suggesting the significant virus blocking effect in vivo. Our results indicated that the P. pastoris expression system could be used to produce large quantities of bioactive CAR-Fc for further clinical purpose.

  8. Long-term gene therapy in the CNS: reversal of hypothalamic diabetes insipidus in the Brattleboro rat by using an adenovirus expressing arginine vasopressin.

    PubMed

    Geddes, B J; Harding, T C; Lightman, S L; Uney, J B

    1997-12-01

    The ability of adenovirus (Ad) to transfect most cell types efficiently has already resulted in human gene therapy trials involving the systemic administration of adenoviral constructs. However, because of the complexity of brain function and the difficulty in noninvasively monitoring alterations in neuronal gene expression, the potential of Ad gene therapy strategies for treating disorders of the CNS has been difficult to assess. In the present study, we have used an Ad encoding the arginine vasopressin cDNA (AdAVP) in an AVP-deficient animal model of diabetes insipidus (the Brattleboro rat), which allowed us to monitor chronically the success of the gene therapy treatment by noninvasive assays. Injection of AdAVP into the supraoptic nuclei (SON) of the hypothalamus resulted in expression of AVP in magnocellular neurons. This was accompanied by reduced daily water intake and urine volume, as well as increased urine osmolality lasting 4 months. These data show that a single gene defect leading to a neurological disorder can be corrected with an adenovirus-based strategy. This study highlights the potential of using Ad gene therapy for the long-term treatment of disorders of the CNS.

  9. Adenovirus replication-competent vectors (KD1, KD3) complement the cytotoxicity and transgene expression from replication-defective vectors (Ad-GFP, Ad-Luc).

    PubMed

    Habib, Nagy A; Mitry, Ragai; Seth, Prem; Kuppuswamy, Mohan; Doronin, Konstantin; Toth, Karoly; Krajcsi, Peter; Tollefson, Ann E; Wold, William S M

    2002-08-01

    The successful clinical application of adenovirus (Ad) in cancer control has been of limited success because of the current inability to infect the majority of cancer cells with a large amount of vector. In this study, we show that when human lung tumors growing in immunodeficient nude mice were coinfected with a replication-defective (RD) Ad vector expressing green fluorescent protein and a replication-competent (RC) Ad vector named KD3, KD3 enhanced the expression of green fluorescent protein throughout the tumor. Also, KD3 and another RC vector named KD1 complemented the expression of luciferase from a RD vector in a human liver tumor xenotransplant in nude mice. Altogether, these results suggest that the combination of a RD vector with a RC vector might be a more effective treatment for cancer than either vector alone due to more widespread dissemination of the virus.

  10. [Construction of replication-deficient recombinant adenovirus vector with hTFPI-2 gene by AdMax system and expression in U937 monocytes in vitro].

    PubMed

    Pan, Junjie; Shi, Haiming; Luo, Xinping; Ma, Duan; Liang, Wang; Zhang, Jin; Zhu, Jun; Li, Jian

    2011-04-01

    We tried to construct and identify the recombinant replication-deficient adenovirus vector coding for human tissue factor pathway inhibitor 2 (hTFPI-2) gene by AdMax system in HEK293 cells. Firstly, we obtained hTFPI-2 gene from the recombinant plasmid pIRES2-EGFP-TFPI-2 by PCR using primers with restriction endonuclease site of EcoRI or SacI. After digesting the hTFPI-2 gene and plasmid PDC316-IRES-EGFP shuttle vector, we ligated them with T4 ligase and formed the recombinant shuttle vector PDC316-IRES-EGFP-hTFPI-2. It was confirmed that the ligation product was inserted the gene of hTFPI-2 correctly by sequencing. Then we took cotransfection of HEK293 cells with the recombinant shuttle vector and genomic plasmid pBHGloxdeltaE1,3Cre by liposome lipofectamine2000, and finished the package of recombinant adenovirus Ad-hTFPI-2. The results of the PCR test and restriction endonuclease digestion confirmed the successful construction of the recombinants Ad-hTFPI-2. Furthermore, we measured the titre of Ad-hTFPI-2 with the aid of green fluorescence protein expression after multiplication and purification. The titre was 0.931 x 10(12) pfu/ml. Finally, we infected U937 monocytes by purified Ad-hTFPI-2, and determined the infection efficiency and the TFPI-2's level and activity. The efficiency of Ad-hTFPI-2 infection in U937 cells was 89.33%. After infected by Ad-hTFPI-2, the TFPI-2's level in supernatant increased about 7 fold. Also the TFPI-2 in supernatant had activities of inhibiting trypsin and plasmin. The recombinant adenovirus with the hTFPI-2 gene was constructed successfully. It will be helpful for the further investigation of its potentiality to be applied in antiatherosclerosis.

  11. Effect of Exposure to UV-C Irradiation and Monochloramine on Adenovirus Serotype 2 Early Protein Expression and DNA Replication▿

    PubMed Central

    Sirikanchana, Kwanrawee; Shisler, Joanna L.; Mariñas, Benito J.

    2008-01-01

    The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20°C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants. PMID:18424543

  12. Effect of exposure to UV-C irradiation and monochloramine on adenovirus serotype 2 early protein expression and DNA replication.

    PubMed

    Sirikanchana, Kwanrawee; Shisler, Joanna L; Mariñas, Benito J

    2008-06-01

    The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20 degrees C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants.

  13. Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model

    PubMed Central

    Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

    2016-01-01

    Foxp3 is a master regulator of CD4+CD25+ regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4+ or CD8+ cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3DTR mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma. PMID:27633092

  14. Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model.

    PubMed

    Park, Soojin; Chung, Hwan-Suck; Shin, Dasom; Jung, Kyung-Hwa; Lee, Hyunil; Moon, Junghee; Bae, Hyunsu

    2016-09-16

    Foxp3 is a master regulator of CD4(+)CD25(+) regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4(+) or CD8(+) cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3(DTR) mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma.

  15. Core labeling of adenovirus with EGFP

    SciTech Connect

    Le, Long P.; Le, Helen N.; Nelson, Amy R.; Matthews, David A.; Yamamoto, Masato; Curiel, David T. . E-mail: curiel@uab.edu

    2006-08-01

    The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.

  16. [Effects of adenovirus-delivered angiopoietin-1 siRNA on expression of matrix metalloproteinases in rats with acute lung injury induced by phosgene].

    PubMed

    He, Daikun; Shao, Yiru; Shen, Jie; Zhang, Lin; Wang, Jing

    2014-09-01

    To investigate the effects of adenovirus-delivered angiopoietin-1 siRNA (Ad. Ang-1siRNA) on the expression of matrix metalloproteinase-2, 9 (MMP-2, 9) and tissue inhibitor of metallopro-teinase-1 (TIMP-1) in rats with acute lung injury (ALI) induced by phosgene (Psg). We first established a rat model of Psg-induced acute lung injury (ALI). The rats were randomly divided into 6 groups: air control group with exposure to air, air+adenovirus (air+Ad) group with caudal vein injection of 1×10(8) pfu/ml adenovirus 1 h after air exposure, air+Ad/Ang1 group with caudal vein injection of 1×10(8) pfu/ml Ad.Ang-1siRNA 1 h after air exposure, Psg group with exposure to 8.33 mg/L Psg (purity 100%, of the same volume as the inhaled air in the air control group) for 5 min, Psg+Ad group with caudal vein injection of 1×10(8) pfu/ml adenovirus 1 h after exposure to the same dose of Psg, and Psg+Ad/Ang1 group with caudal vein injection of 1×10(8) pfu/ml Ad.Ang-1siRNA 1 h after exposure to the same dose of Psg. Serum, bronchoalveolar lavage fluid (BALF), and lung tissue were collected 36 h after exposure. The protein expression of Ang-1, MMP-2, 9, and TIMP-1 in serum and BALF was determined by double-antibody sandwich ELISA. RT-PCR was used to determine the mRNA levels of Ang-1, MMP-2, 9, and TIMP-1 in lung tissue. The protein expression of MMP-2, 9 and TIMP-1 in lung tissue was determined by Western blot. A rat model of Psg-induced ALI was successfully established. The levels of MMP-2, 9 in serum, BALF, and lung tissue were significantly increased in the Psg group and Psg+Ad/Ang1 group as compared with the control group (P<0.01); no significant change was observed in serum TIMP-1 protein expression (P>0.05); interestingly, TIMP-1 protein expression in BALF and lung tissue was significantly increased (P<0.01). Compared with the Psg group, the Psg+Ad/Ang1 group showed a significant decrease in MMP-2, 9 expression in BALF, serum, and lung tissue (P<0.05), but no significant change

  17. Coxsackievirus and adenovirus receptor (CAR) is expressed in male germ cells and forms a complex with the differentiation factor JAM-C in mouse testis.

    PubMed

    Mirza, Momina; Hreinsson, Julius; Strand, Mona-Lisa; Hovatta, Outi; Söder, Olle; Philipson, Lennart; Pettersson, Ralf F; Sollerbrant, Kerstin

    2006-04-01

    The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein important for viral binding to target cells. Using RT-PCR, Western analysis, GST pull-down assay and indirect immunofluorescence, it was shown that CAR is expressed in male germ cells from mice, rats, and humans. CAR was detected in round spermatids in the testis as well as in purified, mature spermatozoa. The two membrane-bound isoforms of CAR occupied different subcellular sites in the acrosomal region of the spermatozoa. CAR was exposed on the surface of acrosome-reacted, but not acrosome-intact cells. Two CAR-binding proteins belonging to the ligand-of-numb protein-X (LNX) family also occupied distinct regions in spermatozoa. Finally, co-immunoprecipitation experiments demonstrated an interaction between CAR and JAM-C, a protein required for spermatid differentiation. Together, these findings imply a function for CAR in male fertility. The results also suggest that CAR in spermatozoa is inaccessible to adenovirus-based gene therapy vectors, and that the risk of germ line infection therefore is low.

  18. Constitutive episomal expression of polypeptide IX (pIX) in a 293-based cell line complements the deficiency of pIX mutant adenovirus type 5.

    PubMed Central

    Caravokyri, C; Leppard, K N

    1995-01-01

    The human adenovirus type 5 capsid is composed of a number of distinct polypeptides. It has been shown previously that one of these, polypeptide IX (pIX), is not absolutely required for the production of viable virus. However, viruses lacking this polypeptide have a significantly reduced packaging limit and, in the one case studied, also show a thermolabile virion phenotype. This report describes the use of eukaryotic episomal vectors based on the Epstein-Barr virus replicon to generate cells which stably express pIX. These cells provide pIX that is efficiently incorporated into virions that are genetically pIX-; such enhanced thermostability. These cells have also been used to isolate a genetically pIX- virus having a genome of length some 2.3 kbp in excess of the previously defined packaging limit for pIX- virus; the resulting virions have wild-type thermostability. These cells expand the theoretical capacity of adenovirus vectors for foreign DNA to around 9.2 kbp and may therefore be useful in gene therapy applications in which vector capacity is limiting. PMID:7474071

  19. Improvement of BCG protective efficacy with a novel chimpanzee adenovirus and a modified vaccinia Ankara virus both expressing Ag85A.

    PubMed

    Stylianou, E; Griffiths, K L; Poyntz, H C; Harrington-Kandt, R; Dicks, M D; Stockdale, L; Betts, G; McShane, H

    2015-11-27

    A replication-deficient chimpanzee adenovirus expressing Ag85A (ChAdOx1.85A) was assessed, both alone and in combination with modified vaccinia Ankara also expressing Ag85A (MVA85A), for its immunogenicity and protective efficacy against a Mycobacterium tuberculosis (M.tb) challenge in mice. Naïve and BCG-primed mice were vaccinated or boosted with ChAdOx1.85A and MVA85A in different combinations. Although intranasally administered ChAdOx1.85A induced strong immune responses in the lungs, it failed to consistently protect against aerosol M.tb challenge. In contrast, ChAdOx1.85A followed by MVA85A administered either mucosally or systemically, induced strong immune responses and was able to improve the protective efficacy of BCG. This vaccination regime has consistently shown superior protection over BCG alone and should be evaluated further.

  20. Improvement of BCG protective efficacy with a novel chimpanzee adenovirus and a modified vaccinia Ankara virus both expressing Ag85A

    PubMed Central

    Stylianou, E.; Griffiths, K.L.; Poyntz, H.C.; Harrington-Kandt, R.; Dicks, M.D.; Stockdale, L.; Betts, G.; McShane, H.

    2015-01-01

    A replication-deficient chimpanzee adenovirus expressing Ag85A (ChAdOx1.85A) was assessed, both alone and in combination with modified vaccinia Ankara also expressing Ag85A (MVA85A), for its immunogenicity and protective efficacy against a Mycobacterium tuberculosis (M.tb) challenge in mice. Naïve and BCG-primed mice were vaccinated or boosted with ChAdOx1.85A and MVA85A in different combinations. Although intranasally administered ChAdOx1.85A induced strong immune responses in the lungs, it failed to consistently protect against aerosol M.tb challenge. In contrast, ChAdOx1.85A followed by MVA85A administered either mucosally or systemically, induced strong immune responses and was able to improve the protective efficacy of BCG. This vaccination regime has consistently shown superior protection over BCG alone and should be evaluated further. PMID:26478198

  1. Down-regulation of collagen synthesis and matrix metalloproteinase expression in myofibroblasts from Dupuytren nodule using adenovirus-mediated relaxin gene therapy.

    PubMed

    Kang, Young-Mi; Choi, Yun-Rak; Yun, Chae-Ok; Park, Jin-Oh; Suk, Kyung-Soo; Kim, Hak-Sun; Park, Moon-Soo; Lee, Byung-Ho; Lee, Hwan-Mo; Moon, Seong-Hwan

    2014-04-01

    Dupuytren's disease is a fibroproliferative connective tissue disorder characterized by contracture of the palmer fascia of the hand. Relaxin (RLN) is a multifunctional factor which contributes to the remodeling of the pelvic ligament by inhibiting fibrosis and inflammatory activities. The aim of this study was to investigate the effect of the RLN gene on the inhibition of fibrosis in myofibroblastic cells. Myofibroblast cells with adenovirus LacZ (Ad-LacZ) as a marker gene or adenovirus relaxin (Ad-RLN) as therapeutic gene showed transgene expressions in beta-galactosidase assay and Western blot analysis. Myofibroblastic cells with Ad-RLN demonstrated a 22% and 48% reduction in collagen I and III mRNA expressions respectively, a 50% decrease in MMP-1, 70% decrease in MMP-2, 80% decrease in MMP-9, and a 15% reduction in MMP-13 protein expression compared with cultures with viral control and saline control. In addition, myofibroblastic cells with Ad-RLN showed a 40% decrease in TIMP 1 and a 15% increase in TIMP 3 protein expression at 48 h compared to cultures with viral control and saline control. Also, myofibroblastic cell with Ad-RLN demonstrated a 74% inhibition of fibronectin and a 52% decrease in total collagen synthesis at 48 h compared with cultures with viral control and saline control. In conclusion, the RLN gene render antifibrogenic effect on myofibroblastic cells from Dupuytren's nodule via direct inhibition of collagen synthesis not through collagenolytic pathway such as MMP-1, -13, TIMP 1, and 3. Therefore relaxin can be an alternative therapeutic strategy in initial stage of Dupuytren's disease by its antifibrogenic effect. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  2. The Adenovirus L4-22K Protein Has Distinct Functions in the Posttranscriptional Regulation of Gene Expression and Encapsidation of the Viral Genome

    PubMed Central

    Guimet, Diana

    2013-01-01

    The adenovirus L4-22K protein is multifunctional and critical for different aspects of viral infection. Packaging of the viral genome into an empty capsid absolutely requires the L4-22K protein to bind to packaging sequences in cooperation with other viral proteins. Additionally, the L4-22K protein is important for the temporal switch from the early to late phase of infection by regulating both early and late gene expression. To better understand the molecular mechanisms of these key functions of the L4-22K protein, we focused our studies on the role of conserved pairs of cysteine and histidine residues in the C-terminal region of L4-22K. We found that mutation of the cysteine residues affected the production of infectious progeny virus but did not interfere with the ability of the L4-22K protein to regulate viral gene expression. These results demonstrate that these two functions of L4-22K may be uncoupled. Mutation of the histidine residues resulted in a mutant with a similar phenotype as a virus deficient in the L4-22K protein, where both viral genome packaging and viral gene expression patterns were disrupted. Interestingly, both mutant L4-22K proteins bound to adenovirus packaging sequences, indicating that the paired cysteine and histidine residues do not function as a zinc finger DNA binding motif. Our results reveal that the L4-22K protein controls viral gene expression at the posttranscriptional level and regulates the accumulation of the L4-33K protein, another critical viral regulator, at the level of alternative pre-mRNA splicing. PMID:23637408

  3. Functional characterization of a PEI-CyD-FA-coated adenovirus as delivery vector for gene therapy.

    PubMed

    Yao, Hong; Chen, Shih-Chi; Shen, Zan; Huang, Yun-Chao; Zhu, Xiao; Wang, Xiao-mei; Jiang, Wenqi; Wang, Zi-Feng; Bian, Xiu-Wu; Ling, Eng-Ang; Kung, Hsiang-fu; Lin, Marie C

    2013-01-01

    The recombinant adenovirus is evolving as a promising gene delivery vector for gene therapy due to its efficiency in transducing different genes into most types of cells. However, the host-immune response elicited by primary inoculation of an adenovirus can cause rapid clearance of the vector, impairing the efficacy of the adenovirus and hence obstructing its clinical application. We have previously synthesized a biodegradable co-polymer consisting of a low molecular weight PEI (MW 600 Da), cross-linked with β-cyclodextrin, and conjugated with folic acid (PEI-CyD-FA, named H1). Here we report that coating the adenovirus vector (Adv) with H1 (H1/rAdv) could significantly improve both the efficacy and biosafety of Adv. Enhanced transfection efficiency as well as prolonged duration of gene expression were clearly demonstrated either by intratumoral or systemic injection of a single dose of H1/rAdv in immunocompetent mice. Importantly, repeated injections of H1/rAdv did not reduce the transfection efficiency in immunocompetent mice. Furthermore, H1 transformed the surface charge of the adenovirus capsomers from negative to positive in physiological solution, suggesting that H1 coated the capsid protein of the adenovirus. This could shelter the epitopes of capsid proteins of the adenovirus, resulting in a reduced host-immune response and enhanced transfection efficiency. Taken together, these findings suggest that H1/rAdv is an effective gene delivery system superior to the adenovirus alone and that it could be considered as a preferred vehicle for gene therapy.

  4. Widespread and efficient marker gene expression in the airway epithelia of fetal sheep after minimally invasive tracheal application of recombinant adenovirus in utero.

    PubMed

    Peebles, D; Gregory, L G; David, A; Themis, M; Waddington, S N; Knapton, H J; Miah, M; Cook, T; Lawrence, L; Nivsarkar, M; Rodeck, C; Coutelle, C

    2004-01-01

    Cystic fibrosis is a common lethal genetic disease caused by functional absence of the cystic fibrosis transmembrane conductance regulator (CFTR). Although a candidate disease for in utero gene therapy, demonstration of potentially therapeutic levels of transgene expression in the fetal airways after minimally invasive gene delivery is a mandatory prerequisite before application of this approach in humans can be considered. We report here on the delivery of a beta-galactosidase expressing adenovirus directly to the airways of fetal sheep in utero using ultrasound-guided percutaneous injection of the trachea in the fetal chest. Injection of adenoviral particles to the fetal airways was not associated with mortality and resulted in low-level expression in the peripheral airways. However, complexation of the virus with DEAE dextran, which confers a positive charge to the virus, and pretreatment of the airways with Na-caprate, which opens tight junctions, increased transgene expression, and a combination of these two enhancers resulted in widespread and efficient gene transfer of the fetal trachea and bronchial tree. Using a percutaneous ultrasound-guided injection technique, we have clearly demonstrated proof of principle for substantial transgene delivery to the fetal airways providing levels of gene expression that could be relevant for a therapeutic application of CFTR expressing vectors.

  5. Vaccine-Induced Immunity in Baboons by Using DNA and Replication-Incompetent Adenovirus Type 5 Vectors Expressing a Human Immunodeficiency Virus Type 1 gag Gene

    PubMed Central

    Casimiro, Danilo R.; Tang, Aimin; Chen, Ling; Fu, Tong-Ming; Evans, Robert K.; Davies, Mary-Ellen; Freed, Daniel C.; Hurni, William; Aste-Amezaga, Jose M.; Guan, Liming; Long, Romnie; Huang, Lingyi; Harris, Virginia; Nawrocki, Denise K.; Mach, Henryk; Troutman, Robert D.; Isopi, Lynne A.; Murthy, Krishna K.; Rice, Karen; Wilson, Keith A.; Volkin, David B.; Emini, Emilio A.; Shiver, John W.

    2003-01-01

    The cellular immunogenicity of formulated plasmid DNA and replication-defective human adenovirus serotype 5 (Ad5) vaccine vectors expressing a codon-optimized human immunodeficiency virus type 1 gag gene was examined in baboons. The Ad5 vaccine was capable of inducing consistently strong, long-lived CD8+-biased T-cell responses and in vitro cytotoxic activities. The DNA vaccine-elicited immune responses were weaker than those elicited by the Ad5 vaccine and highly variable; formulation with chemical adjuvants led to moderate increases in the levels of Gag-specific T cells. Increasing the DNA-primed responses with booster doses of either Ad5 or modified vaccinia virus Ankara vaccines suggests a difference in the relative levels of cytotoxic and helper responses. The implications of these results are discussed. PMID:12805466

  6. Effects of adenovirus-mediated expression of p27Kip1, p21Waf1 and p16INK4A in cell lines derived from t(2;5) anaplastic large cell lymphoma and Hodgkin's disease.

    PubMed

    Turturro, Franceso; Arnold, Marilyn D; Frist, Audrey Y; Seth, Prem

    2002-06-01

    We investigated the response of SUDHL-1 and L428 cells, derived from t(2;5)-anaplastic large cell lymphoma (ALCL) and Hodgkin's disease (HD), respectively, to recombinant adenoviruses expressing cyclin-dependent kinase inhibitors (CDKIs) p27Kip1 (Adp27), p21Waf1 (Adp21) and p16INK4A (Adp16). Cell cycle analysis of SUDHL-1 cells after 24 h of infection with 200 multiplicity of infection (MOI) of Adp27, Adp21, and Adp16, showed very high levels of cell debris in the subG1 area. The magnitude of cell debris-events was Adp27/Adp21 > Adp16. Cell cycle analysis of L428 cells revealed absence of cell debris and increased G2 phase in all the groups of cells tested as compared to the controls (mock and AdNull). A minimal increase in G1 phase was also evident in cells infected with Adp27 (52%) compared to uninfected cells (43%), AdNull (45%) and to cells infected with Adp21 (37%) and Adp16 (31%). The presence of significant levels of Coxsackie-adenovirus receptor (CAR) on the cell surface of L428 cells excluded the cell membrane-barrier as responsible for the differences in cell observed in response to the recombinant adenovirus-mediated CDKIs expression as compared to SUDHL-1. We also showed that the recombinant adenovirus-mediated cytotoxicity measured as apoptosis was MOI- and vector-dependent in SUDHL-1 cells at lower MOI (100). In conclusion, the therapeutic effect induced by recombinant adenoviruses expressing p27Kip1, p21Waf1 and p16INK4A is cell-dependent in cells derived from selected lymphoid malignancies. Biochemical cellular differences more than cell surface barriers seem to be responsible for differences in response to recombinant adenovirus-mediated expression of cytotoxic genes. Moreover, the cytotoxicity of recombinant adenoviruses expressing p27Kip1, p21Waf1 and p16INK4A may be further explored as a tool for gene therapy of t(2;5)-derived ALCL.

  7. Vasculature-Specific Adenovirus Vectors for Gene Therapy of Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    translation and, thus, never become available to the disulfide isomerases, the ER-localized enzymes that facilitate the formation of the disulfide bonds...duplexes and cloned into Bael-cut expression vectors. Upon transformation of Ecoli , colonies containing recombinant plasmids were identified by PCR...R. L. Crowell, and R. W. Finberg. 1997. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320-3. 6

  8. Canine Recombinant Adenovirus Vector Induces an Immunogenicity-Related Gene Expression Profile in Skin-Migrated CD11b+ -Type DCs

    PubMed Central

    Jouneau, Luc; Bourge, Mickael; Bouet-Cararo, Coraline; Bonneau, Michel; Zientara, Stephan; Klonjkowski, Bernard; Schwartz-Cornil, Isabelle

    2012-01-01

    Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs) could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2) as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b+ -type and CD103+ -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b+ -type DCs was far higher and broader than in the CD103+ -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b+ -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b+ DC type is more responsive to CAV2 than the CD103+ DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness. PMID:23300693

  9. Opa Expression Correlates with Elevated Transformation Rates in Neisseria gonorrhoeae

    PubMed Central

    Hill, Stuart A.

    2000-01-01

    Neisseria gonorrhoeae is naturally competent for DNA transformation. Under most conditions encountered in vivo, gonococci express one or more opacity (Opa) proteins on their surfaces. Recently, it was shown that DNA preferentially binds to the surfaces of Opa-expressing organisms compared to those of isogenic Opa-negative strains, presumably due to the numerous cationic residues in the predicted surface-exposed loops of the Opa protein. This study examined whether Opa-DNA interactions actually influence DNA transformation of the gonococcus. The data show that Opa-expressing gonococci are more efficient recipients of DNA for transformation and are more susceptible to exogenous DNase I treatment at early stages during the DNA transformation process than non-Opa expressors. Furthermore, inhibition of the transformation process was demonstrable for Opa+ populations when either nonspecific DNA or the polyanion heparin was used. Overall, the data suggest that Opa expression, with its presumptive positive surface charge contribution, promotes DNA transformation by causing a more prolonged sequestration of donor DNA at the cell surface, which translates into more efficient transformation over time. PMID:10613877

  10. Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

    SciTech Connect

    Vigl, Benjamin; Zgraggen, Claudia; Rehman, Nadia; Banziger-Tobler, Nadia E.; Detmar, Michael; Halin, Cornelia

    2009-01-15

    Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.

  11. A bidirectional Tet-dependent promotor construct regulating the expression of E1A for tight control of oncolytic adenovirus replication.

    PubMed

    Fechner, Henry; Wang, Xiaomin; Picó, Almudena Hurtado; Wildner, Judith; Suckau, Lennart; Pinkert, Sandra; Sipo, Isaac; Weger, Stefan; Poller, Wolfgang

    2007-01-20

    Tight regulation of oncolytic adenoviruses (oAdV) represents an important requirement for their safe application. Here we describe a new doxycycline (Dox)-dependent oAdV with a bidirectional expression cassette, which drives the expression of the reverse tetracycline-controlled transactivator (rtTA(s)-M2) from a lung tumor-specific promoter and, in the opposite direction, the expression of the adenoviral E1A gene from a second generation TetO(7) sequence linked to an isolated TATA box. In H441 lung cancer cells, this oAdV showed a strictly Dox-dependent E1A expression, adenoviral replication, cell killing activity and a 450-fold induction of progeny virus production. The virus could be shut off again by withdrawal of Dox and, in contrast to a control oAdV expressing E1A directly from the SP-B promoter, did not replicate in non-target cells. However, the absolute values of virus production and the cell killing activity in the presence of the inducer were still reduced as compared to the control oAdV. The results demonstrate, for the first time, Dox-dependent oAdV replication from a single adenoviral vector genome. Future improvement of the Dox-dependent E1A regulation cassette should lead to the generation of an oAdV well suited to meet the demands for a highly regulated and efficient oncolytic virus for in vivo applications.

  12. Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro.

    PubMed

    Vigl, Benjamin; Zgraggen, Claudia; Rehman, Nadia; Banziger-Tobler, Nadia E; Detmar, Michael; Halin, Cornelia

    2009-01-15

    Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.

  13. Transcriptome Sequencing and Development of an Expression Microarray Platform for Liver Infection in Adenovirus Type 5-Infected Syrian Golden Hamsters

    PubMed Central

    Ying, Baoling; Toth, Karoly; Spencer, Jacqueline F.; Aurora, Rajeev; Wold, William S.M.

    2015-01-01

    The Syrian golden hamster is an attractive animal for research on infectious diseases and other diseases. We report here the sequencing, assembly, and annotation of the Syrian hamster transcriptome. We include transcripts from ten pooled tissues from a naïve hamster and one stimulated with lipopolysaccharide. Our data set identified 42,707 non-redundant transcripts, representing 34,191 unique genes. Based on the transcriptome data, we generated a custom microarray and used this new platform to investigate the transcriptional response in the Syrian hamster liver following intravenous adenovirus type 5 (Ad5) infection. We found that Ad5 infection caused a massive change in regulation of liver transcripts, with robust up-regulation of genes involved in the antiviral response, indicating that the innate immune response functions in the host defense against Ad5 infection of the liver. The data and novel platforms developed in this study will facilitate further development of this important animal model. PMID:26319212

  14. Presence of the Coxsackievirus and Adenovirus Receptor (CAR) in human neoplasms: a multitumour array analysis

    PubMed Central

    Reeh, M; Bockhorn, M; Görgens, D; Vieth, M; Hoffmann, T; Simon, R; Izbicki, J R; Sauter, G; Schumacher, U; Anders, M

    2013-01-01

    Background: The Coxsackie- and Adenovirus Receptor (CAR) has been assigned two crucial attributes in carcinomas: (a) involvement in the regulation of growth and dissemination and (b) binding for potentially therapeutic adenoviruses. However, data on CAR expression in cancer types are conflicting and several entities have not been analysed to date. Methods: The expression of CAR was assessed by immunohistochemical staining of tissue microarrays (TMA) containing 3714 specimens derived from 100 malignancies and from 273 normal control tissues. Results: The expression of CAR was detected in all normal organs, except in the brain. Expression levels, however, displayed a broad range from being barely detectable (for example, in the thymus) to high abundance expression (for example, in the liver and gastric mucosa). In malignancies, a high degree of variability was notable also, ranging from significantly elevated CAR expression (for example, in early stages of malignant transformation and several tumours of the female reproductive system) to decreased CAR expression (for example, in colon and prostate cancer types). Conclusion: Our results provide a comprehensive insight into CAR expression in neoplasms and indicate that CAR may offer a valuable target for adenovirus-based therapy in a subset of carcinomas. Furthermore, these data suggest that CAR may contribute to carcinogenesis in an entity-dependent manner. PMID:24022195

  15. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy.

    PubMed

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-09-14

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

  16. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    NASA Astrophysics Data System (ADS)

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-09-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.

  17. A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

    PubMed Central

    Oka, Tetsuo; Kurozumi, Kazuhiko; Shimazu, Yosuke; Ichikawa, Tomotsugu; Ishida, Joji; Otani, Yoshihiro; Shimizu, Toshihiko; Tomita, Yusuke; Sakaguchi, Masakiyo; Watanabe, Masami; Nasu, Yasutomo; Kumon, Hiromi; Date, Isao

    2016-01-01

    Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma. PMID:27625116

  18. Umbilical cord blood-derived dendritic cells infected by adenovirus for SP17 expression induce antigen-specific cytotoxic T cells against NSCLC cells.

    PubMed

    Liu, Yang; Tian, Xin; Jiang, Shenyi; Ren, Xuemei; Liu, Fengjie; Yang, Jichun; Chen, Yanling; Jiang, Youhong

    2015-01-01

    Sperm protein 17 (SP17), a cancer/testis antigen, is expressed by non-small cell lung cancer (NSCLC). This study examined whether dendritic cells (DC) from human umbilical cord blood (UCB) could be induced for SP17 expression and induce antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) against NSCLC in vitro. We generated recombinant adenovirus of Ad-SP17 and control Ad-null. Infection with Ad-SP17, but not control, induced higher levels of SP17 expression in UCB-derived DC-Ad-SP17. Infection with Ad-SP17 significantly increased the frequency of CD80(+), CD83(+), CD86(+), and HLA-DR(+) DC that produced higher levels of IL-12, but lower IL-10. Co-culture of DC-Ad-SP17 with autologous UCB lymphocytes induced high frequency of IFNγ(+) CD8(+) CTLs, which had selective cytotoxicity against SP17(+) lung cancer CRL-5922 cells in a HLA-I restrictive manner. Thus, UCB-derived DC modulated for SP17 expression induced antigen-specific anti-tumor immunity against SP17(+) NSCLC, and SP17 may be a valuable target for development of immunotherapy against SP17(+) NSCLC.

  19. Expression of the fusogenic p14 FAST protein from a replication-defective adenovirus vector does not provide a therapeutic benefit in an immunocompetent mouse model of cancer

    PubMed Central

    Wong, C M; Nash, L A; Del Papa, J; Poulin, K L; Falls, T; Bell, J C; Parks, R J

    2016-01-01

    When injected directly into a tumor mass, adenovirus (Ad) vectors only transduce cells immediately along the injection tract. Expression of fusogenic proteins from the Ad vector can lead to syncytium formation, which efficiently spreads the therapeutic effect. Fusogenic proteins can also cause cancer cell death directly, and enhance the release of exosome-like particles containing tumor-associated antigens, which boosts the anti-tumor immune response. In this study, we have examined whether delivery of an early region 1 (E1)-deleted, replication-defective Ad vector encoding the reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein can provide therapeutic efficacy in an immunocompetent mouse tumor model. A high multiplicity of infection of AdFAST is required to induce cell fusion in mouse mammary carcinoma 4T1 cells in vitro, and FAST protein expression caused a modest reduction in cell membrane integrity and metabolic activity compared with cells infected with a control vector. Cells expressing FAST protein released significantly higher quantities of exosomes. In immunocompetent Balb/C mice harboring subcutaneous 4T1 tumors, AdFAST did not induce detectable cancer cell fusion, promote tumor regression or prolong mouse survival compared with untreated mice. This study suggests that in the context of the 4T1 model, Ad-mediated FAST protein expression did not elicit a therapeutic effect. PMID:27740615

  20. Genetic transformation and gene expression in white pine (pinus strobus)

    SciTech Connect

    Minocha, R.

    1987-10-01

    The objectives of the study were: (1) to develop protocols for transformation of white pine (Pinus strobus) embryonic tissue; and (2) to analyze the regulation of foreign gene expression in Pinus strobus. A number of Agrobacterium tumefaciens strains containing chimeric genes for neomycin phosphotransferase (NPTII for kanamycin resistance) and chloramphenicol acetyl transferase (CAT) under the control of either a constitutive promoter (NOS-nopaline synthase) or light-inducible promoters (RuBisCO small subunit and chlorophyll a/b binding protein) were used. A variety of tissues from white pine seedlings and mature trees was used. The techniques for transformation were modified from those used for tobacco transformation. The results show that white pine tissue from young seedlings is high suitable for transformation by A. tumefaciens. Whereas the normal tissues are very sensitive to kanamycin, transformed callus was quite resistant to this antibiotic.

  1. Nuclear actin and myosins in adenovirus infection.

    PubMed

    Fuchsova, Beata; Serebryannyy, Leonid A; de Lanerolle, Primal

    2015-11-01

    Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host's transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

    SciTech Connect

    Li Jianwei; Faber, Milosz; Papaneri, Amy; Faber, Marie-Luise; McGettigan, James P.; Schnell, Matthias J.; Dietzschold, Bernhard . E-mail: bernhard.dietzschold@jefferson.edu

    2006-12-20

    Rabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus.

  3. Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability.

    PubMed Central

    Theodorakis, N G; Morimoto, R I

    1987-01-01

    We have examined the posttranscriptional regulation of hsp70 gene expression in two human cell lines, HeLa and 293 cells, which constitutively express high levels of HSP70. HSP70 mRNA translates with high efficiency in both control and heat-shocked cells. Therefore, heat shock is not required for the efficient translation of HSP70 mRNA. Rather, the main effect of heat shock on translation is to suppress the translatability of non-heat shock mRNAs. Heat shock, however, has a marked effect on the stability of HSP70 mRNA; in non-heat-shocked cells the half-life of HSP70 mRNA is approximately 50 min, and its stability increases at least 10-fold upon heat shock. Moreover, HSP70 mRNA is more stable in cells treated with protein synthesis inhibitors, suggesting that a heat shock-sensitive labile protein regulates its turnover. An additional effect on posttranscriptional regulation of hsp70 expression can be found in adenovirus-infected cells, in which HSP70 mRNA levels decline precipititously late during infection although hsp70 transcription continues unabated. Images PMID:3437893

  4. Adenovirus-mediated ectopic expression of Msx2 in even-numbered rhombomeres induces apoptotic elimination of cranial neural crest cells in ovo.

    PubMed

    Takahashi, K; Nuckolls, G H; Tanaka, O; Semba, I; Takahashi, I; Dashner, R; Shum, L; Slavkin, H C

    1998-05-01

    Distinct cranial neural crest-derived cell types (a number of neuronal as well as non-neuronal cell lineages) are generated at characteristic times and positions in the rhombomeres of the hindbrain in developing vertebrate embryos. To examine this developmental process, we developed a novel strategy designed to test the efficacy of gain-of-function Msx2 expression within rhombomeres in ovo prior to the emigration of cranial neural crest cells (CNCC). Previous studies indicate that CNCC from odd-numbered rhombomeres (r3 and r5) undergo apoptosis in response to exogenous BMP4. We provide evidence that targeted infection in ovo using adenovirus containing Msx2 and a reporter molecule indicative of translation can induce apoptosis in either even- or odd-numbered rhombomeres. Furthermore, infected lacZ-control explants indicated that CNCC emigrated, and that 20% of these cells were double positive for crest cell markers HNK-1 and beta-gal. In contrast, there were no HNK-1 and Msx2 double positive cells emigrating from Msx2 infected explants. These results support the hypothesis that apoptotic elimination of CNCC can be induced by 'gain-of-function' Msx2 expression in even-numbered rhombomeres. These inductive interactions involve qualitative, quantitative, positional and temporal differences in TGF-beta-related signals, Msx2 expression and other transcriptional control.

  5. In Vitro Methylation of the BsuRI (5′-GGCC-3′) Sites in the E2a Region of Adenovirus Type 2 DNA Does Not Affect Expression in Xenopus laevis Oocytes

    PubMed Central

    Vardimon, Lily; Günthert, Ursula; Doerfler, Walter

    1982-01-01

    The early region 2a (E2a) of adenovirus type 2 (Ad2) DNA codes for a 72,000-dalton DNA-binding protein and is expressed in the Ad2-transformed hamster cell line HE1 but not in cell lines HE2 and HE3 (H. Esche, J. Virol. 41:1076-1082, 1982; K. Johansson et al., J. Virol. 27:628-639, 1978). An inverse correlation between DNA methylation at the 5′-CCGG-3′ sites of the E2a region and of gene expression in these cell lines has been observed (L. Vardimon et al., Nucleic Acids Res. 8:2461-2473, 1980). When the cloned E2a region of Ad2 DNA is methylated in vitro at the 5′-CCGG-3′ sites, the gene is not transcribed after being injected into the nuclei of Xenopus laevis oocytes, whereas unmethylated DNA is expressed (L. Vardimon et al., Eur. J. Cell Biol. 25:13-15, 1981; L. Vardimon et al., Proc. Natl. Acad. Sci. U.S.A. 79:1073-1077, 1982). These data demonstrate that DNA methylation is directly involved in the shut-off of transcription. In the present communication we investigated in detail the control region of the gene for the DNA-binding protein in Ad2-transformed cell lines and showed that the first late control region (map coordinate 72 on the viral DNA) of the E2a region is present in its entirety in cell lines HE1, HE2, and HE3. The HaeIII sites (5′-GGCC-3′) in the E2a region in all three cell lines were not methylated. When the DNA methyltransferase BsuRI was used, all 5′-GGCC-3′ sites in the cloned E2a region of Ad2 DNA were methylated in vitro. It was shown that methylation of these sites did not inhibit the expression of this viral gene in X. laevis oocytes. Thus, for methylation to affect gene expression in the E2a region it has to occur at specific sites (e.g., 5′-CCGG-3′) which may be different for other genes. Images PMID:14582198

  6. Therapy of Head and Neck Squamous Cell Carcinoma with Replicative Adenovirus Expressing Tissue Inhibitor of Metalloproteinase-2 and Chemoradiation

    PubMed Central

    McNally, Lacey R.; Rosenthal, Eben L.; Zhang, Wenyue; Buchsbaum, Donald J.

    2009-01-01

    Objective Recent studies have demonstrated efficacy of targeted therapy combined with radiotherapy in HNSCC. We hypothesized that a combination treatment including a replicating adenovirus armed with tissue inhibitor metalloproteinase-2 (TIMP-2), radiation, and Cisplatin will augment treatment response and reduce tumor growth in vivo of HNSCC xenografts. Design Both single-agent (TIMP-2 virus, radiation, and Cisplatin) and the combination therapies were evaluated in vitro and in vivo. The efficacy of both single agent and combination therapies in vivo was determined by monitoring of tumor growth and immunohistochemistry. Results Treatment with replicative Ad-TIMP-2 virus and radiation decreased cell viability in vitro and resulted in an additional anti-angiogenic response in vivo. Tumor response rates to treatment with replicative Ad-TIMP-2, radiation, Cisplatin, or combination therapies ranged from limited inhibition of tumor growth of the single-agent therapy to a statistically significant additive anti-tumor response with the combination therapies. Replicative Ad-TIMP-2 + radiation + Cisplatin in the SCC1 nude mice demonstrated the greatest response rates in tumor growth and angiogenesis. Conclusions Combination Ad-TIMP-2 gene therapy with radiation and the triple treatment group resulted in an augmented therapeutic response. This is the first report of the potential benefits of combining radiation and MMP inhibitor treatment. PMID:18846112

  7. Innate Immunity to Adenovirus

    PubMed Central

    Hendrickx, Rodinde; Stichling, Nicole; Koelen, Jorien; Kuryk, Lukasz; Lipiec, Agnieszka

    2014-01-01

    Abstract Human adenoviruses are the most widely used vectors in gene medicine, with applications ranging from oncolytic therapies to vaccinations, but adenovirus vectors are not without side effects. In addition, natural adenoviruses pose severe risks for immunocompromised people, yet infections are usually mild and self-limiting in immunocompetent individuals. Here we describe how adenoviruses are recognized by the host innate defense system during entry and replication in immune and nonimmune cells. Innate defense protects the host and represents a major barrier to using adenoviruses as therapeutic interventions in humans. Innate response against adenoviruses involves intrinsic factors present at constant levels, and innate factors mounted by the host cell upon viral challenge. These factors exert antiviral effects by directly binding to viruses or viral components, or shield the virus, for example, soluble factors, such as blood clotting components, the complement system, preexisting immunoglobulins, or defensins. In addition, Toll-like receptors and lectins in the plasma membrane and endosomes are intrinsic factors against adenoviruses. Important innate factors restricting adenovirus in the cytosol are tripartite motif-containing proteins, nucleotide-binding oligomerization domain-like inflammatory receptors, and DNA sensors triggering interferon, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 and cyclic guanosine monophosphate–adenosine monophosphate synthase. Adenovirus tunes the function of antiviral autophagy, and counters innate defense by virtue of its early proteins E1A, E1B, E3, and E4 and two virus-associated noncoding RNAs VA-I and VA-II. We conclude by discussing strategies to engineer adenovirus vectors with attenuated innate responses and enhanced delivery features. PMID:24512150

  8. Innate immunity to adenovirus.

    PubMed

    Hendrickx, Rodinde; Stichling, Nicole; Koelen, Jorien; Kuryk, Lukasz; Lipiec, Agnieszka; Greber, Urs F

    2014-04-01

    Human adenoviruses are the most widely used vectors in gene medicine, with applications ranging from oncolytic therapies to vaccinations, but adenovirus vectors are not without side effects. In addition, natural adenoviruses pose severe risks for immunocompromised people, yet infections are usually mild and self-limiting in immunocompetent individuals. Here we describe how adenoviruses are recognized by the host innate defense system during entry and replication in immune and nonimmune cells. Innate defense protects the host and represents a major barrier to using adenoviruses as therapeutic interventions in humans. Innate response against adenoviruses involves intrinsic factors present at constant levels, and innate factors mounted by the host cell upon viral challenge. These factors exert antiviral effects by directly binding to viruses or viral components, or shield the virus, for example, soluble factors, such as blood clotting components, the complement system, preexisting immunoglobulins, or defensins. In addition, Toll-like receptors and lectins in the plasma membrane and endosomes are intrinsic factors against adenoviruses. Important innate factors restricting adenovirus in the cytosol are tripartite motif-containing proteins, nucleotide-binding oligomerization domain-like inflammatory receptors, and DNA sensors triggering interferon, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 and cyclic guanosine monophosphate-adenosine monophosphate synthase. Adenovirus tunes the function of antiviral autophagy, and counters innate defense by virtue of its early proteins E1A, E1B, E3, and E4 and two virus-associated noncoding RNAs VA-I and VA-II. We conclude by discussing strategies to engineer adenovirus vectors with attenuated innate responses and enhanced delivery features.

  9. The HDAC inhibitor FK228 enhances adenoviral transgene expression by a transduction-independent mechanism but does not increase adenovirus replication.

    PubMed

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

    2011-02-17

    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.

  10. 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

  11. Recombinant adenovirus expressing the haemagglutinin of peste des petits ruminants virus (PPRV) protects goats against challenge with pathogenic virus; a DIVA vaccine for PPR

    PubMed Central

    2014-01-01

    Peste des petits ruminants virus (PPRV) is a morbillivirus that can cause severe disease in sheep and goats, characterised by pyrexia, pneumo-enteritis, and gastritis. The socio-economic burden of the disease is increasing in underdeveloped countries, with poor livestock keepers being affected the most. Current vaccines consist of cell-culture attenuated strains of PPRV, which induce a similar antibody profile to that induced by natural infection. Generation of a vaccine that enables differentiation of infected from vaccinated animals (DIVA) would benefit PPR control and eradication programmes, particularly in the later stages of an eradication campaign and for countries where the disease is not endemic. In order to create a vaccine that would enable infected animals to be distinguished from vaccinated ones (DIVA vaccine), we have evaluated the immunogenicity of recombinant fowlpox (FP) and replication-defective recombinant human adenovirus 5 (Ad), expressing PPRV F and H proteins, in goats. The Ad constructs induced higher levels of virus-specific and neutralising antibodies, and primed greater numbers of CD8+ T cells than the FP-vectored vaccines. Importantly, a single dose of Ad-H, with or without the addition of Ad expressing ovine granulocyte macrophage colony-stimulating factor and/or ovine interleukin-2, not only induced strong antibody and cell-mediated immunity but also completely protected goats against challenge with virulent PPRV, 4 months after vaccination. Replication-defective Ad-H therefore offers the possibility of an effective DIVA vaccine. PMID:24568545

  12. Pregnancy loss following coxsackievirus b3 infection in mice during early gestation due to high expression of coxsackievirus-adenovirus receptor (CAR) in uterus and embryo.

    PubMed

    Hwang, Ji Young; Lee, Kyung Min; Kim, Yun Hwa; Shim, Hye Min; Bae, Young Kyung; Hwang, Jung Hye; Park, Hosun

    2014-01-01

    Coxsackieviruses are important pathogens in children and the outcomes of neonatal infection can be serious or fatal. However, the outcomes of coxsackievirus infection during early gestation are not well defined. In this study, we examined the possibility of vertical transmission of coxsackievirus B3 (CVB3) and the effects of CVB3 infection on early pregnancy of ICR mice. We found that the coxsackievirus and adenovirus receptor (CAR) was highly expressed not only in embryos but also in the uterus of ICR mice. CVB3 replicated in the uterus 1 to 7 days post-infection (dpi), with the highest titer at 3 dpi. The pregnancy loss rate in mice infected with CVB3 during early gestation was 38.3%, compared to 4.7% and 2.7% in mock-infected and UV-inactivated-CVB3 infected pregnant mice, respectively. These data suggest that the uterus and embryo, which express abundant CAR, are important targets of CVB3 and that the vertical transmission of CVB3 during early gestation induces pregnancy loss.

  13. Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases

    NASA Astrophysics Data System (ADS)

    Sauter, Bernhard V.; Martinet, Olivier; Zhang, Wei-Jian; Mandeli, John; Woo, Savio L. C.

    2000-04-01

    Inhibition of angiogenesis has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in the large-scale production of the antiangiogenic proteins. This limitation may be resolved by in vivo delivery and expression of the antiangiogenic genes. We have constructed a recombinant adenovirus that expresses murine endostatin that is biologically active both in vitro, as determined in endothelial cell proliferation assays, and in vivo, by suppression of angiogenesis induced by vascular endothelial growth factor 165. Persistent high serum levels of endostatin (605-1740 ng/ml; mean, 936 ng/ml) were achieved after systemic administration of the vector to nude mice, which resulted in significant reduction of the growth rates and the volumes of JC breast carcinoma and Lewis lung carcinoma (P < 0.001 and P < 0.05, respectively). In addition, the endostatin vector treatment completely prevented the formation of pulmonary micrometastases in Lewis lung carcinoma (P = 0.0001). Immunohistochemical staining of the tumors demonstrated a decreased number of blood vessels in the treatment group versus the controls. In conclusion, the present study clearly demonstrates the potential of vector-mediated antiangiogenic gene therapy as a component in cancer therapy.

  14. Adenovirus-mediated expression of orphan nuclear receptor NR4A2 targeting hepatic stellate cell attenuates liver fibrosis in rats

    PubMed Central

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Yang, Quanjun; Huang, Jinlu; Gan, Run; Guo, Cheng

    2016-01-01

    Liver fibrosis is a wound-healing response characterized with the accumulation of extracellular matrix (ECM). And hepatic stellate cells (HSCs) are the principal cell source of ECM. NR4A2 (Nurr1) is a member of orphan nuclear receptor NR4A family and acts as transcription factor. It participates in regulating cell differentiation, proliferation and apoptosis. We previously demonstrated that NR4A2 expression in fibrotic liver reduced significantly compared with normal liver and NR4A2 knockout in HSCs promoted ECM production. In the present study we explored the role of NR4A2 on liver fibrosis. Studies in cultured HSCs demonstrated that NR4A2 over-expression suppressed the activation of HSCs, such as ECM production and invasion ability. Moreover cell cycle was arrested, cell apoptosis was promoted and cell signaling pathway was influenced. Adenovirus-mediated delivery of NR4A2 in rats ameliorated significantly dimethylnitrosamine (DMN) induced liver fibrosis. The In vivo experiments produced results consistent with in vitro experiments. Taken together these results demonstrate NR4A2 enhancement attenuates liver fibrosis via suppressing the activation of HSCs and NR4A2 may be an ideal target for anti-fibrotic therapy. PMID:27646469

  15. Non-replicating adenovirus vectors expressing avian influenza virus hemagglutinin and nucleocapsid proteins induce chicken specific effector, memory and effector memory CD8+ T lymphocytes

    PubMed Central

    Singh, Shailbala; Toro, Haroldo; Tang, De-Chu; Briles, Worthie E.; Yates, Linda M.; Kopulos, Renee T.; Collisson, Ellen W.

    2010-01-01

    Avian influenza virus (AIV) specific CD8+ T lymphocyte responses stimulated by intramuscular administration of an adenovirus (Ad) vector expressing either HA or NP were evaluated in chickens following ex vivo stimulation by non-professional antigen presenting cells. The CD8+ T lymphocyte responses were AIV specific, MHC-I restricted, and cross-reacted with heterologousH7N2 AIV strain. Specific effector responses, at 10 days post-inoculation (p.i.), were undetectable at 2 weeks p.i., and memory responses were detected from 3 to 8 weeks p.i. Effector memory responses, detected 1 week following a booster inoculation, were significantly greater than the primary responses and, within 7 days, declined to undetectable levels. Inoculation of an Ad-vector expressing human NP resulted in significantly greater MHC restricted, activation of CD8+ T cell responses specific for AIV. Decreases in all responses with time were most dramatic with maximum activation of T cells as observed following effector and effector memory responses. PMID:20557918

  16. Pregnancy Loss Following Coxsackievirus B3 Infection in Mice during Early Gestation Due toHigh Expression of Coxsackievirus-Adenovirus Receptor (CAR) in Uterus and Embryo

    PubMed Central

    Hwang, Ji Young; Lee, Kyung Min; Kim, Yun Hwa; Shim, Hye Min; Bae, Young Kyung; Hwang, Jung Hye; Park, Hosun

    2014-01-01

    Coxsackieviruses are important pathogens in children and the outcomes of neonatal infection can be serious or fatal. However, the outcomes of coxsackievirus infection during early gestation are not well defined. In this study, we examined the possibility of vertical transmission of coxsackievirus B3 (CVB3) and the effects of CVB3 infection on early pregnancy of ICR mice. We found that the coxsackievirus and adenovirus receptor (CAR) was highly expressed not only in embryos but also in the uterus of ICR mice. CVB3 replicated in the uterus 1 to 7 days post-infection (dpi), with the highest titer at 3 dpi. The pregnancy loss rate in mice infected with CVB3 during early gestation was 38.3%, compared to 4.7% and 2.7% in mock-infected and UV-inactivated-CVB3 infected pregnant mice, respectively. These data suggest that the uterus and embryo, which express abundant CAR, are important targets of CVB3 and that the vertical transmission of CVB3 during early gestation induces pregnancy loss. PMID:24521864

  17. Recombinant adenovirus expressing the haemagglutinin of Peste des petits ruminants virus (PPRV) protects goats against challenge with pathogenic virus; a DIVA vaccine for PPR.

    PubMed

    Herbert, Rebecca; Baron, Jana; Batten, Carrie; Baron, Michael; Taylor, Geraldine

    2014-02-26

    Peste des petits ruminants virus (PPRV) is a morbillivirus that can cause severe disease in sheep and goats, characterised by pyrexia, pneumo-enteritis, and gastritis. The socio-economic burden of the disease is increasing in underdeveloped countries, with poor livestock keepers being affected the most. Current vaccines consist of cell-culture attenuated strains of PPRV, which induce a similar antibody profile to that induced by natural infection. Generation of a vaccine that enables differentiation of infected from vaccinated animals (DIVA) would benefit PPR control and eradication programmes, particularly in the later stages of an eradication campaign and for countries where the disease is not endemic. In order to create a vaccine that would enable infected animals to be distinguished from vaccinated ones (DIVA vaccine), we have evaluated the immunogenicity of recombinant fowlpox (FP) and replication-defective recombinant human adenovirus 5 (Ad), expressing PPRV F and H proteins, in goats. The Ad constructs induced higher levels of virus-specific and neutralising antibodies, and primed greater numbers of CD8+ T cells than the FP-vectored vaccines. Importantly, a single dose of Ad-H, with or without the addition of Ad expressing ovine granulocyte macrophage colony-stimulating factor and/or ovine interleukin-2, not only induced strong antibody and cell-mediated immunity but also completely protected goats against challenge with virulent PPRV, 4 months after vaccination. Replication-defective Ad-H therefore offers the possibility of an effective DIVA vaccine.

  18. Protoplast Transformation as a Plant-Transferable Transient Expression System.

    PubMed

    Duarte, Patrícia; Ribeiro, Diana; Carqueijeiro, Inês; Bettencourt, Sara; Sottomayor, Mariana

    2016-01-01

    The direct uptake of DNA by naked plant cells (protoplasts) provides an expression system of exception for the quickly growing research in non-model plants, fuelled by the power of next-generation sequencing to identify novel candidate genes. Here, we describe a simple and effective method for isolation and transformation of protoplasts, and illustrate its application to several plant materials.

  19. Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    PubMed Central

    Rainbow, Andrew J.

    2013-01-01

    Previously, we have reported the use of a recombinant adenovirus (Ad)-based host cell reactivation (HCR) assay to examine nucleotide excision repair (NER) of UVC-induced DNA lesions in several mammalian cell types. The recombinant non-replicating Ad expresses the Escherichia coli β-galactosidase (β-gal) reporter gene under control of the cytomegalovirus immediate-early enhancer region. We have also used methylene blue plus visible light (MB + VL) to induce the major oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in the recombinant Ad-encoded reporter gene in order to study base excision repair (BER). The reported variability regarding 8-oxoG’s potential to block transcription by RNA polymerase II and data demonstrating that a number of factors play a role in transcriptional bypass of the lesion led us to examine the repair of 8-oxoG in the Ad reporter and its relationship to HCR for expression of the reporter gene. We have used Southern blotting to examine removal of UVC- and MB + VL-induced DNA damage by loss of endonuclease-sensitive sites from the Ad-encoded β-gal reporter gene in human and rodent cells. We show that repair of MB + VL-induced 8-oxoG via BER and UVC-induced cyclobutane pyrimidine dimers (CPDs) via NER is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. We also show that HCR for expression of the MB + VL-damaged and the UVC-damaged reporter gene is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. The difference between the human and rodent cells in the removal of both 8-oxoG and CPDs from the damaged reporter gene was comparable to the difference in HCR for expression of the damaged reporter gene. These results suggest that the major factor for HCR of the MB + VL-treated reporter gene in mammalian cells is DNA repair in the Ad rather than lesion bypass. PMID:23793457

  20. Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine.

    PubMed

    Leach, Derrik M; Zacal, Natalie J; Rainbow, Andrew J

    2013-09-01

    Previously, we have reported the use of a recombinant adenovirus (Ad)-based host cell reactivation (HCR) assay to examine nucleotide excision repair (NER) of UVC-induced DNA lesions in several mammalian cell types. The recombinant non-replicating Ad expresses the Escherichia coli β-galactosidase (β-gal) reporter gene under control of the cytomegalovirus immediate-early enhancer region. We have also used methylene blue plus visible light (MB + VL) to induce the major oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in the recombinant Ad-encoded reporter gene in order to study base excision repair (BER). The reported variability regarding 8-oxoG's potential to block transcription by RNA polymerase II and data demonstrating that a number of factors play a role in transcriptional bypass of the lesion led us to examine the repair of 8-oxoG in the Ad reporter and its relationship to HCR for expression of the reporter gene. We have used Southern blotting to examine removal of UVC- and MB + VL-induced DNA damage by loss of endonuclease-sensitive sites from the Ad-encoded β-gal reporter gene in human and rodent cells. We show that repair of MB + VL-induced 8-oxoG via BER and UVC-induced cyclobutane pyrimidine dimers (CPDs) via NER is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. We also show that HCR for expression of the MB + VL-damaged and the UVC-damaged reporter gene is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. The difference between the human and rodent cells in the removal of both 8-oxoG and CPDs from the damaged reporter gene was comparable to the difference in HCR for expression of the damaged reporter gene. These results suggest that the major factor for HCR of the MB + VL-treated reporter gene in mammalian cells is DNA repair in the Ad rather than lesion bypass.

  1. DNA damage and biological expression of adenovirus: A comparison of liquid versus frozen conditions of exposure to gamma rays

    SciTech Connect

    Bennett, C.B.; Rainbow, A.J. )

    1989-10-01

    Human adenovirus type 2 (Ad 2) was irradiated with 137Cs gamma rays in the liquid state at 0 degree C. DNA breaks were correlated with the inactivation of several viral functions and compared to results obtained previously for irradiation of Ad 2 under frozen conditions at -75 degrees C. Irradiation at 0 degree C induced 170 +/- 20 single-strand breaks and 2.6 +/- 0.4 double-strand breaks/Gy/10(12) Da in the viral DNA. Viral adsorption to human KB cells was inactivated with a D0 of 9.72 +/- 1.18 kGy, whereas the inactivation of Ad 2 plaque formation had a D0 of 0.99 +/- 0.14 or 1.1 +/- 0.29 kGy when corrected for the effect of radiation on virus adsorption. For the adsorbed virus, an average of 4.3 +/- 1.7 single-strand and 0.065 +/- 0.02 double-strand breaks were induced in the viral DNA per lethal hit. In contrast, irradiation of Ad 2 at -75 degrees C results in 2.6- to 3.4-fold less DNA breakage per Gy and a 5.6-fold increase in D0 for plaque formation of the adsorbed virus. Furthermore, although host cell reactivation (HCR) of Ad 2 viral structural antigen production for irradiated virus was substantially reduced in the xeroderma pigmentosum fibroblast strain (XP25RO) compared to normal strains for irradiation at -75 degrees C (57% HCR), it was only slightly reduced compared to normal for irradiation at 0 degree C (88% HCR). These results indicate that the spectrum of DNA damage is both quantitatively and qualitatively different for the two conditions of irradiation.

  2. A novel bicistronic high-capacity gutless adenovirus vector that drives constitutive expression of herpes simplex virus type 1 thymidine kinase and tet-inducible expression of Flt3L for glioma therapeutics.

    PubMed

    Puntel, Mariana; Muhammad, A K M G; Candolfi, Marianela; Salem, Alireza; Yagiz, Kader; Farrokhi, Catherine; Kroeger, Kurt M; Xiong, Weidong; Curtin, James F; Liu, Chunyan; Bondale, Niyati S; Lerner, Jonathan; Pechnick, Robert N; Palmer, Donna; Ng, Philip; Lowenstein, Pedro R; Castro, Maria G

    2010-06-01

    Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.

  3. Adenovirus vector E4 gene regulates connexin 40 and 43 expression in endothelial cells via PKA and PI3K signal pathways.

    PubMed

    Zhang, Fan; Cheng, Joseph; Lam, George; Jin, David K; Vincent, Loïc; Hackett, Neil R; Wang, Shiyang; Young, Lauren M; Hempstead, Barbara; Crystal, Ronald G; Rafii, Shahin

    2005-05-13

    Connexins (Cxs) provide a means for intercellular communication and play important roles in the pathophysiology of vascular cardiac diseases. Infection of endothelial cells (ECs) with first-generation E1/E3-deleted E4+ adenovirus (AdE4+) selectively modulates the survival and angiogenic potential of ECs by as of yet unrecognized mechanisms. We show here that AdE4+ vectors potentiate Cx expression in ECs in vitro and in mouse heart tissue. Infection of ECs with AdE4+, but not AdE4-, resulted in a time- and dose-dependent induction of junctional Cx40 expression and suppression of Cx43 protein and mRNA expression. Treatment of ECs with PKA inhibitor H89 or PI3K inhibitor LY294002 prevented the AdE4+-mediated regulation of Cx40 and Cx43 that was associated with diminished AdE4+-mediated survival of ECs. Moreover, both PKA activity and cAMP-response element (CRE)-binding activity were enhanced by treatment of ECs with AdE4+. However, there is no causal evidence of a cross-talk between the 2 modulatory pathways, PKA and PI3K. Remarkably, Cx40 immunostaining was markedly increased and Cx43 was decreased in the heart tissue of mice treated with intra-tracheal AdE4+. Taken together, these results suggest that AdE4+ may play an important role in the regulation of Cx expression in ECs, and that these effects are mediated by both the PKA/CREB and PI3K signaling pathways.

  4. Active targeting of RGD-conjugated bioreducible polymer for delivery of oncolytic adenovirus expressing shRNA against IL-8 mRNA

    PubMed Central

    Kim, Jaesung; Nam, Hye Yeong; Kim, Tae-il; Kim, Pyung-Hwan; Ryu, Jihoon; Yun, Chae-Ok; Kim, Sung Wan

    2011-01-01

    Even though oncolytic adenovirus (Ad) has been highlighted in the field of cancer gene therapy, transductional targeting and immune privilege still remain difficult challenges. The recent reports have noted the increasing tendency of adenoviral surface shielding with polymer to overcome the limits of its practical application. We previously reported the potential of the biodegradable polymer, poly(CBA-DAH) (CD) as a promising candidate for efficient gene delivery. To endow the selective-targeting moiety of tumor vasculature to CD, cRGDfC well-known as a ligand for cell-surface integrins on tumor endothelium was conjugated to CD using hetero-bifunctional cross-linker SM(PEG)n. The cytopathic effects of oncolytic Ad coated with the polymers were much more enhanced dose-dependently when compared with that of naked Ad in cancer cells selectively. Above all, the most potent oncolytic effect was assessed with the treatment of Ad/CD-PEG500-RGD in all cancer cells. The enhanced cytopathic effect of Ad/RGD-conjugated polymer was specifically inhibited by blocking antibodies to integrins, but not by blocking antibody to CAR. HT1080 cells treated with Ad/CD-PEG500-RGD showed strong induction of apoptosis and suppression of IL-8 and VEGF expression as well. These results suggest that RGD-conjugated bioreducible polymer might be used to deliver oncolytic Ad safely and efficiently for tumor therapy. PMID:21531456

  5. Immunogenicity and efficacy of a recombinant adenovirus expressing hemagglutinin from the H5N1 subtype of swine influenza virus in mice

    PubMed Central

    Wu, Yunpu; Qiao, Chuanling; Yang, Huanliang; Chen, Yan; Xin, Xiaoguang; Chen, Hualan

    2014-01-01

    The H5N1 influenza viruses infect a range of avian species and have recently been isolated from humans and pigs. In this study we generated a replication-defective recombinant adenovirus (rAd-H5HA-EGFP) expressing the hemagglutinin (HA) gene of H5N1 A/Swine/Fujian/1/2001 (SW/FJ/1/01) and evaluated its immunogenicity and protective efficacy in BALB/c mice. The recombinant virus induced high levels of hemagglutination inhibition (HI) antibody at a median tissue culture infective dose of 108 or 107. Compared with mice in the control groups, the mice vaccinated with rAd-H5HA-EGFP did not show apparent weight loss after challenge with either the homologous SW/FJ/1/01 or the heterologous H5N1 A/Chicken/Hunan/77/2005 (CK/HuN/77/05). Replication of the challenge virus was partially or completely inhibited, and viruses were detected at significantly lower numbers in the organs of the vaccinated mice, all of which survived the challenge with CK/HuN/77/05, whereas most of the control mice did not. These results indicate that rAd-H5HA-EGFP can provide effective immune protection from highly pathogenic H5N1 viruses in mice and is therefore a promising new candidate vaccine against H5N1 influenza in animals. PMID:24688173

  6. Leucine residues in conserved region of 33K protein of bovine adenovirus - 3 are important for binding to major late promoter and activation of late gene expression.

    PubMed

    Kulshreshtha, Vikas; Islam, Azharul; Ayalew, Lisanework E; Tikoo, Suresh K

    2015-09-01

    The L6 region of bovine adenovirus 3 (BAdV-3) encode 33K (spliced) and 22K (unspliced) proteins. Earlier, anti-33K serum detected five major and three minor proteins in BAdV-3 infected cells. Here, we demonstrate that anti-sera raised against L6-22K protein detected two proteins of 42 and 37 kDa in BAdV-3 infected cells and one protein of 42 kDa in transfected cells expressing splice-site variant 22K protein (pC.22K containing substituted splice acceptor/donor sequence). Unlike 22K, 33K stimulated the transcription from the major late promoter (MLP) by binding to the downstream sequence elements (DE). Analysis of the variant proteins demonstrated that amino acids 201-240 of the conserved C-terminus of 33K containing the potential leucine zipper and RS repeat are required for the activation of MLP. Furthermore, amino acid substitution analysis demonstrated that unlike arginine residues of RS repeat, the leucine residues (217, 224, 232 and 240) of the conserved leucine zipper appear required for the binding of 33K to the MLP.

  7. Protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing TgAMA1 as vaccines against Toxoplasma gondii infection in mice.

    PubMed

    Yu, Longzheng; Yamagishi, Junya; Zhang, Shoufa; Jin, Chunmei; Aboge, Gabriel Oluga; Zhang, Houshuang; Zhang, Guohong; Tanaka, Tetsuya; Fujisaki, Kozo; Nishikawa, Yoshifumi; Xuan, Xuenan

    2012-09-01

    A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection.

  8. Combination therapy with conditionally replicating adenovirus and replication defective adenovirus.

    PubMed

    Lee, Choon-Taek; Park, Kyung-Ho; Yanagisawa, Kiyoshi; Adachi, Yasushi; Ohm, Joyce E; Nadaf, Sorena; Dikov, Mikhail M; Curiel, David T; Carbone, David P

    2004-09-15

    Low gene transfer rate is the most substantial hurdle in the practical application of gene therapy. One strategy to improve transfer efficiency is the use of a conditionally replicating adenovirus (CRAD) that can selectively replicate in tumor cells. We hypothesized that conventional E1-deleted adenoviruses (ad) can become replication-competent when cotransduced with a CRAD to selectively supply E1 in trans in tumors. The resulting selective production of large numbers of the E1-deleted ad within the tumor mass will increase the transduction efficiency. We used a CRAD (Delta24RGD) that produces a mutant E1 without the ability to bind retinoblastoma but retaining viral replication competence in cancer cells with a defective pRb/p16. Ad-lacZ, adenovirus-luciferase (ad-luc), and adenovirus insulin-like growth factor-1R/dominant-negative (ad-IGF-1R/dn; 482, 950) are E1-deleted replication-defective adenoviruses. The combination of CRAD and ad-lacZ increased the transduction efficiency of lacZ to 100% from 15% observed with ad-lacZ alone. Transfer of media of CRAD and ad-lacZ cotransduced cells induced the transfer of lacZ (media transferable bystander effect). Combination of CRAD and ad-IGF-1R/dn increased the production of truncated IGF-1R or soluble IGF-1R > 10 times compared with transduction with ad-IGF-1R/dn alone. Combined intratumoral injection of CRAD and ad-luc increased the luciferase expression about 70 times compared with ad-luc alone without substantial systemic spread. Combined intratumoral injection of CRAD and ad-IGF-1R/482 induced stronger growth suppression of established lung cancer xenografts than single injections. The combination of CRAD and E1-deleted ad induced tumor-specific replication of CRAD and E1-deleted ad and increased the transduction rate and therapeutic efficacy of these viruses in model tumors.

  9. Adenovirus (For Parents)

    MedlinePlus

    ... the virus by holding hands or sharing a toy with an infected person. Adenovirus can survive on ... by sharing contaminated objects (such as towels or toys), or by touch. Once a child is exposed ...

  10. Genetic stability of a recombinant adenovirus vaccine vector seed library expressing human papillomavirus type 16 E6 and E7 proteins.

    PubMed

    Wu, Jie; Chen, Ke-DA; Gao, Meng; Chen, Gang; Jin, Su-Feng; Zhuang, Fang-Cheng; Wu, Xiao-Hong; Jiang, Yun-Shui; Li, Jian-Bo

    2015-04-01

    The aim of the present study was to understand the genetic stability of a master seed bank (MSB) and a working seed bank (WSB) of an adenovirus vector vaccine expressing the human papillomavirus (HPV) type 16 E6 and E7 fusion proteins (Ad-HPV16E6E7). Microscopic examination and viral infectious efficacy were used to measure the infectious titers of the Ad-HPV16E6E7 MSB and WSB. Polymerase chain reaction was used to analyze the stability of the Ad-HPV16E6E7 target gene insertion, while western blot analysis and immunofluorescence were used to assess the expression levels of the Ad-HPV16E6E7 target protein. A C57BL/6 mouse TC-1 tumor cell growth inhibition model was used to evaluate the biological effect of Ad-HPV16E6E7 administration. The infectious titers of the Ad-HPV16E6E7 MSB and WSB were 6.31×10(9) IU/ml and 3.0×10(9) IU/ml, respectively. In addition, the expression levels of the inserted target genes and target proteins were found to be stable. In the mouse TC-1 tumor inhibition analysis, when the virus titers of the Ad-HPV16E6E7 MSB and WSB were 10(9) IU/ml, the tumor inhibition rate was 100%, which was significantly different when compared with the control group (χ(2)MSB=20.00 and χ(2)WSB=20.00; P<0.01). Therefore, the Ad-HPV16E6E7 vaccine seed bank is genetically stable and meets the requirements for vaccine development.

  11. Essential Role for the Lectin Pathway in Collagen Antibody-Induced Arthritis Revealed Through Use of Adenovirus Programming Complement Inhibitor MAp44 Expression

    PubMed Central

    Banda, Nirmal K.; Mehta, Gaurav; Kjaer, Troels R.; Takahashi, Minoru; Schaack, Jerome; Morrison, Thomas E.; Thiel, Steffen; Arend, William P.; Holers, V. Michael

    2014-01-01

    Previous studies using mannose-binding lectin (MBL) and complement C4 deficient mice have suggested that the lectin pathway (LP) is not required for the development of inflammatory arthritis in the collagen antibody-induced arthritis (CAIA) model. MBL, ficolins and collectin-11 are key LP pattern recognition molecules that associate with three serine proteases, MASP-1, MASP-2 and MASP-3, and also with two MBL-associated proteins designated sMAP and MAp44. Recent studies have shown that MAp44, an alternatively spliced product of the MASP-1/3 gene, is a competitive inhibitor of the binding of the recognition molecules to all three MASPs. In these studies we examined the effect of treatment of mice with adenovirus (Ad) programmed to express human MAp44 (AdhMAp44) on the development of CAIA. AdhMAp44 and Ad programming Green fluorescent protein (AdGFP) expression were injected intraperitoneally in C57BL/6 wild-type mice prior to the induction of CAIA. AdhMAp44 significantly reduced the clinical disease activity score (CDA) by 81% compared to mice injected with AdGFP. Similarly, histopathologic injury scores for inflammation, pannus, cartilage and bone damage, as well as C3 deposition in the cartilage and synovium, were significantly reduced by AdhMAp44 pretreatment. Mice treated with AdmMAp44, programming expression of mouse MAp44, also showed significantly decreased CDA and histopathologic injury scores. Additionally, administration of AdhMAp44 significantly diminished the severity of Ross River Virus-induced arthritis, a LP-dependent model. Our study provides conclusive evidence that an intact complement LP is essential to initiate CAIA, and that MAp44 may be an appropriate treatment for inflammatory arthritis. PMID:25070856

  12. Nickel-induced heritable alterations in retroviral transforming gene expression.

    PubMed Central

    Biggart, N W; Gallick, G E; Murphy, E C

    1987-01-01

    Determination of the mutagenic effects of carcinogenic nickel compounds has been difficult because, like many metals, nickel is poorly or nonmutagenic in procaryotic mutagenicity assays. We attempted to characterize nickel-induced genetic lesions by assessing the effect of nickel chloride on the conditionally defective expression of the v-mos transforming gene in normal rat kidney cells infected with the Murine sarcoma virus mutant ts110 (MuSVts110) retrovirus. MuSVts110 contains an out-of-frame gag gene-mos gene junction that prevents the expression of the v-mos gene at the nonpermissive temperature (39 degrees C). In MuSVts110-infected cells (6m2 cells) grown at 33 degrees C, however, this defect can be suppressed by a splicing event that restores the mos reading frame, allowing the expression of a gag-mos fusion protein which induces the transformed phenotype. The capacity to splice the viral transcript at 33 degrees C, but not at 39 degrees C, is an intrinsic property of the viral RNA. This property allowed us to target the MuSVts110 genome using a positive selection scheme whereby nickel was used to induce genetic changes which resulted in expression of the transformed phenotype at 39 degrees C. We treated 6m2 cells with NiCl2 and isolated foci consisting of cells which had reverted to the transformed phenotype at 39 degrees C. We found that brief nickel treatment increased the reversion frequency of 6m2 cells grown at 39 degrees C sevenfold over the spontaneous reversion frequency. The nickel-induced revertants displayed the following heritable characteristics: They stably maintained the transformed phenotype at 39 degrees C; unlike the MuSVts110 RNA in 6m2 cells, the nickel-induced revertant viral RNA could be spliced efficiently at 39 degrees C; as a consequence of the enhanced accumulation of spliced viral RNA, the nickel-induced revertants produced substantial amounts of the transforming v-mos protein P85gag-mos at 39 degrees C; the nickel

  13. Adenovirus co-expressing CD40 ligand and interleukin (IL)-2 contributes to maturation of dendritic cells and production of IL-12

    PubMed Central

    Guo, Zhi; Gao, Hong-Yan; Zhang, Tian-Yang; Lou, Jin-Xing; Yang, Kai; Liu, Xiao-Dong; He, Xue-Peng; Chen, Hui-Ren

    2016-01-01

    The aim of the present study was to construct a chimeric adenovirus (Ad)5/F35 co-expressing human CD4O ligand (CD4OL) and interleukin (IL)-2 (Ad5/F35 CD40L-IL-2). The infection efficiency to human monocyte-derived dendritic cells (Mo-DCs), expression of genes, phenotype changes and IL-12 production of Mo-DC by Ad5/F35 CD40L-IL-2 were investigated. CD40L and IL-2 from total RNA extracted from human peripheral blood mononuclear cells (PBMCs) were cloned by reverse transcription-polymerase chain reaction and used to construct Ad5/F35 CD40L-IL-2. The infection efficiency, expression of CD40L, and phenotype changes of Mo-DC infected with Ad5/F35 CD40L-IL-2 were analyzed using flow cytometry. The quantities of IL-2 and IL-12 in the supernatants of Mo-DC following infection of Ad5/F35 CD40L-IL-2 were measured by enzyme-linked immunosorbent assay. The CD40L and IL-2 genes were successfully cloned and the Ad5/F35 CD40L-IL-2 was constructed. Ad5/F35 CD40L-IL-2 efficiently infected Mo-DCs with an infection efficiency of >75%, and the infected Mo-DCs expressed CD40L and secreted IL-2. The expression levels of cluster of differentiation (CD)80, CD86, CD40, and human leukocyte antigen-antigen D related on Mo-DC were moderate; however, CD83 was low prior to infection of Ad5/F35 CD40L-IL-2. Those molecules, particularly CD83, were markedly upregulated 24 h after the infection. Increasing quantities of IL-12 in the supernatants were detected subsequent to infection at different time points in a time-dependent manner. Thus, Ad5/F35 CD40L-IL-2 efficiently infected human Mo-DCs and its products, CD40L and IL-2, were subsequently expressed. In addition, infection with Ad5/F35 CD40L-IL-2 stimulated the maturation of Mo-DC and high levels of IL-12 production. PMID:27882218

  14. Armed Oncolytic Adenovirus-Expressing PD-L1 Mini-Body Enhances Antitumor Effects of Chimeric Antigen Receptor T Cells in Solid Tumors.

    PubMed

    Tanoue, Kiyonori; Rosewell Shaw, Amanda; Watanabe, Norihiro; Porter, Caroline; Rana, Bhakti; Gottschalk, Stephen; Brenner, Malcolm; Suzuki, Masataka

    2017-04-15

    Chimeric antigen receptor-modified T cells (CAR T cells) produce proinflammatory cytokines that increase expression of T-cell checkpoint signals such as PD-L1, which may inhibit their functionality against solid tumors. In this study, we evaluated in human tumor xenograft models the proinflammatory properties of an oncolytic adenovirus (Onc.Ad) with a helper-dependent Ad (HDAd) that expresses a PD-L1 blocking mini-antibody (mini-body; HDPDL1) as a strategy to enhance CAR T-cell killing. Coadministration of these agents (CAd-VECPDL1) exhibited oncolytic effects with production of PD-L1 mini-body locally at the tumor site. On their own, HDPDL1 exhibited no antitumor effect and CAd-VECPDL1 alone reduced tumors only to volumes comparable to Onc.Ad treatment. However, combining CAd-VECPDL1 with HER2.CAR T cells enhanced antitumor activity compared with treatment with either HER2.CAR T cells alone or HER2.CAR T cells plus Onc.Ad. The benefits of locally produced PD-L1 mini-body by CAd-VECPDL1 could not be replicated by infusion of anti-PD-L1 IgG plus HER2.CAR T cells and coadministration of Onc.Ad in an HER2(+) prostate cancer xenograft model. Overall, our data document the superiority of local production of PD-L1 mini-body by CAd-VECPDL1 combined with administration of tumor-directed CAR T cells to control the growth of solid tumors. Cancer Res; 77(8); 2040-51. ©2017 AACR. ©2017 American Association for Cancer Research.

  15. Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle.

    PubMed

    Sreenivasa, B P; Mohapatra, J K; Pauszek, S J; Koster, M; Dhanya, V C; Tamil Selvan, R P; Hosamani, M; Saravanan, P; Basagoudanavar, Suresh H; de Los Santos, T; Venkataramanan, R; Rodriguez, L L; Grubman, M J

    2017-05-01

    Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O, A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutralizing antibody response in indigenous cattle (Bos indicus). Purified Ad5-FMD viruses were inoculated in cattle as monovalent (5×10(9) pfu/animal) or trivalent (5×10(9) pfu/animal per serotype) vaccines. Animals vaccinated with monovalent Ad5-FMD vaccines were boosted 63days later with the same dose. After primary immunization, virus neutralization tests (VNT) showed seroconversion in 83, 67 and 33% of animals vaccinated with Ad5-FMD O, A and Asia 1, respectively. Booster immunization elicited seroconversion in all of the animals (100%) in the monovalent groups. When used in a trivalent form, the Ad5-FMD vaccine induced neutralizing antibodies in only 33, 50 and 16% of animals against serotypes O, A and Asia 1, respectively on primo-vaccination, and titers were significantly lower than when the same vectors were used in monovalent form. Neutralizing antibody titers differed by serotype for both Ad5-FMD monovalent and trivalent vaccines, with Asia 1 serotype inducing the lowest titers. Antibody response to Ad5 vector in immunized cattle was also assessed by VNT. It appeared that the vector immunity did not impact the recall responses to expressed FMDV antigens on booster immunization. In summary, the study suggested that the recombinant Ad5-FMD vaccine has a potential use in monovalent form, while its application in multivalent form is not currently encouraging. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Synergistic effect of interferon-gamma and tumor necrosis factor-alpha on coxsackievirus and adenovirus receptor expression: an explanation of cell sloughing during testicular inflammation in mice.

    PubMed

    Gao, Ying; Lui, Wing-Yee

    2014-03-01

    Coxsackievirus and adenovirus receptor (CAR) is a junction molecule that expresses on Sertoli and germ cells. It mediates Sertoli-germ cell adhesion and facilitates migration of preleptotene/leptotene spermatocytes across the blood-testis barrier, suggesting that CAR-based cell adhesion and migration are crucial for spermatogenesis. Interferon-gamma (IFNG) and tumor necrosis factor alpha (TNF) are two major cytokines that are elevated during testicular inflammation and cause reduced fertility. We investigated the mechanism by which IFNG and TNF exert their disruptive effects on testicular cell adhesion. We have demonstrated that combined treatment with IFNG and TNF (IFNG+TNF) exerts a synergistic effect by downregulating CAR mRNA and protein levels. Immunofluorescence staining revealed that IFNG+TNF treatment effectively removes CAR from the site of cell-cell contact. Using inhibitor and co-immunoprecipitation, we confirmed that IFNG+TNF mediates CAR protein degradation via ubiquitin-proteasome and NFKB pathways. Blockage of ubiquitin-proteasome pathway significantly inhibits CAR degradation, as indicated by the reappearance of CAR at the site of cell-cell contact. Additionally, IFNG+TNF reduces CAR mRNA via transcriptional regulation. Mutational studies have shown that IFNG+TNF-induced CAR repression is achieved by suppression of the basal transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays further confirmed that IFNG+TNF treament not only inhibits binding of the basal transcription factors but also promotes binding of NFKB subunits and Sp1 (negative regulators) to the CAR promoter region. Taken together, IFNG+TNF treatment significantly downregulates CAR expression, which provides an explanation of how cell sloughing in the epithelium mediates, by loss of CAR-based cell adhesion, during testicular inflammation.

  17. Pre-Clinical Development of a Recombinant, Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

    PubMed Central

    Alexander, Jeff; Mendy, Jason; Vang, Lo; Avanzini, Jenny B.; Garduno, Fermin; Manayani, Darly J.; Ishioka, Glenn; Farness, Peggy; Ping, Li-Hua; Swanstrom, Ronald; Parks, Robert; Liao, Hua-Xin; Haynes, Barton F.; Montefiori, David C.; LaBranche, Celia; Smith, Jonathan; Gurwith, Marc; Mayall, Tim

    2013-01-01

    Background There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated. Methods The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets. Results Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization. Conclusions The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical

  18. Regulation of the Target Protein (Transgene) Expression in the Adenovirus Vector Using Agonists of Toll-Like Receptors

    PubMed Central

    Bagaev, A. V.; Pichugin, A. V.; Lebedeva, E. S.; Lysenko, A. A.; Shmarov, M. M.; Logunov, D. Yu.; Naroditsky, B. S.; Ataullakhanov, R. I.; Khaitov, R. M.; Gintsburg, A. L.

    2014-01-01

    Replication-defective adenoviral vectors are effective molecular tools for both gene therapy and gene vaccination. Using such vectors one can deliver and express target genes in different epithelial, liver, hematopoietic and immune system cells of animal and human origin. The success of gene therapy and gene vaccination depends on the production intensity of the target protein encoded by the transgene. In this work, we studied influence of Toll-like receptors (TLR) agonists on transduction and expression efficacy of adenoviral vectors in animal and human antigen-presenting cells. We found that agonists of TLR2, 4, 5, 7, 8 and 9 significantly enhance a production of the target protein in cells transduced with adenoviral vector having the target gene insert. The enhancement was observed in dendritic cells and macrophages expressing cytoplasmic (GFP), membrane (HA) or secretory (SEAP) proteins encoded by the respective rAd-vectors. Experiments in mice showed that enhancement of the transgene expression can be achieved in the organism of animals using a pharmaceutical-grade TLR4-agonist. In contrast to other TLR-agonists, the agonist of TLR3 substantially suppressed the expression of transgene in cells transduced with adenoviral vectors having insert of GFP or SEAP target genes. We propose that the enhancement of transgene expression is linked to the activation of MyD88→ NF-kB, while the inhibition of transgene expression depends on TRIF→ IRF signaling pathways. Both of these pathways jointly exploited by TLR4-agonists lead to the enhancement of transgene expression due to the dominant role of the MyD88→ NF-kB signaling. PMID:25558392

  19. Prognostic value of transformerexpression in prostate cancer.

    PubMed

    Diao, Yan; Wu, Dong; Dai, Zhijun; Kang, Huafeng; Wang, Ziming; Wang, Xijing

    2015-01-01

    Deregulation of transformer 2β (Tra2β) has been implicated in several cancers. However, the role of Tra2β expression in prostate cancer (PCa) is unclear. Therefore, this study was to investigate the expression of Tra2β in PCa and evaluated its association with clinicopathological variables and prognosis. Thirty paired fresh PCa samples were analyzed for Tra2β expression by Western blot analysis. Immunohistochemistry (IHC) assay was performed in 160 PCa samples after radical prostatectomy and adjacent non-cancerous tissues. Tra2β protein expression was divided into high expression group and low expression group by IHC. We also investigated the association of Tra2β expression with clinical and pathologic parameters. Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the association between Tra2β protein expression and prognosis of PCa patients. Our results showed that Tra2β was significantly upregulated in PCa tissues by western blot and IHC. Our data indicated that high expression of Tra2β was significantly associated with lymph node metastasis (P=0.002), clinical stage (P=0.015), preoperative prostate-specific antigen (P=0.003), Gleason score (P=0.001), and biochemical recurrence (P=0.021). High Tra2β expression was a significant predictor of poor biochemical recurrence free survival and overall survival both in univariate and multivariate analysis. We show that Tra2β was significantly upregulated in PCa patients after radical prostatectomy, and multivariate analysis confirmed Tra2β as an independent prognostic factor.

  20. Immune responses induced by replication-defective adenovirus expressing the C-terminal portion of the Mycoplasma hyopneumoniae P97 adhesin.

    PubMed

    Okamba, F R; Moreau, E; Cheikh Saad Bouh, K; Gagnon, C A; Massie, B; Arella, M

    2007-06-01

    Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine, causing significant economic losses to swine production worldwide. Vaccination is the most cost-effective strategy for the control and prevention of this disease. The goal of this study was to design and evaluate a replication-defective recombinant adenovirus, rAdP97c, expressing the C-terminal portion of P97 adhesin (P97c), an important pathogenesis-associated protein of M. hyopneumoniae, as a new vaccine candidate against M. hyopneumoniae infection. P97c-specific immune responses were evaluated in BALB/c mice following intranasal and intramuscular inoculation with rAdP97c. Mice inoculated by both routes of immunization produced significant levels of specific immunoglobulin G (IgG) antibodies in the serum and in bronchoalveolar lavage fluids (BALs). Animals immunized intranasally also produced a significant level of P97c-specific IgA in BALs. Intramuscular inoculation of rAdP97c induced a systemic and mucosal Th1-type biased response, evidenced by the predominance of IgG2a in the serum and BALs, whereas intranasal inoculation resulted in a mixed Th1/Th2-type response (balanced levels of IgG1 and IgG2a) in both sytemic and mucosal compartments. P97c-specific antibodies were able to inhibit the growth of M. hyopneumoniae cells in vitro. These data suggest that rAdP97c vaccine may represent a new strategy for controlling infection by M. hyopneumoniae.

  1. Hemagglutinin-targeting Artificial MicroRNAs Expressed by Adenovirus Protect Mice From Different Clades of H5N1 Infection

    PubMed Central

    Tang, Xinying; Zhang, Hongbo; Song, Yufeng; Zhou, Dongming; Wang, Jieru

    2016-01-01

    Influenza virus (IV) is a continuously evolving virus that widely spreads in humans and contributes to substantial morbidity and mortality. Re-emergence of human infection with avian influenza virus H5N1 poses extra challenge to IV control. Artificial microRNA (amiRNA)-mediated RNA interference has become a powerful antiviral approach due to its high specificity and rapid effect. Here, we designed several amiRNAs targeting the hemagglutinin gene of H5N1, a major determinant of pathogenicity. Expression and delivery efficiency were enhanced by presenting functional amiRNA with chimpanzee adenovirus serotype 68 (AdC68). One amiRNA, HA-1405, significantly limited H5N1 replication in vitro and inhibited 96.7% of clade 2.3.2 replication. AdC68-conjugated HA-1405 treatment remarkably decreased different clades of H5N1 plaque formation in Madin–Darby canine kidney cells. Moreover, prophylactic administration with rAd(HA-1405) markedly alleviated clinical symptoms and reduced ~3- to 40-folds of lung viral RNA copies against four clades of H5N1 in Institute of Cancer Research (ICR) mice. Our results further showed that rAd(HA-1405) conferred 70 and 40% immediate protection against lethal clade 2.3.2 and clade 2.3.4 H5N1 challenge, respectively. In conclusion, these data provided information that HA-targeting amiRNA delivered by AdC68 could be pursued as a potential agent for highly pathogenic avian influenza viruses prevention. PMID:27093169

  2. A recombinant adenovirus expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice.

    PubMed

    Li, Wu; Deng, Guangcun; Li, Min; Zeng, Jin; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2014-11-01

    Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide, which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report, the generation of Ad5-CEAB, a recombinant adenovirus expressing Mtb antigens of CFP10, ESAT6, Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB, mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response, particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines, IFN-γ, TNF-α, IL-2 and IL-12. In addition, the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore, an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice, suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo, which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.

  3. Adrenal Gland Infection by Serotype 5 Adenovirus Requires Coagulation Factors

    PubMed Central

    Franken, Philippe R.; Darcourt, Jacques; Cornilleau, Gaétan; Benihoud, Karim; Vassaux, Georges

    2013-01-01

    Recombinant, replication-deficient serotype 5 adenovirus infects the liver upon in vivo, systemic injection in rodents. This infection requires the binding of factor X to the capsid of this adenovirus. Another organ, the adrenal gland is also infected upon systemic administration of Ad, however, whether this infection is dependent on the cocksackie adenovirus receptor (CAR) or depends on the binding of factor X to the viral capsid remained to be determined. In the present work, we have used a pharmacological agent (warfarin) as well as recombinant adenoviruses lacking the binding site of Factor X to elucidate this mechanism in mice. We demonstrate that, as observed in the liver, adenovirus infection of the adrenal glands in vivo requires Factor X. Considering that the level of transduction of the adrenal glands is well-below that of the liver and that capsid-modified adenoviruses are unlikely to selectively infect the adrenal glands, we have used single-photon emission computed tomography (SPECT) imaging of gene expression to determine whether local virus administration (direct injection in the kidney) could increase gene transfer to the adrenal glands. We demonstrate that direct injection of the virus in the kidney increases gene transfer in the adrenal gland but liver transduction remains important. These observations strongly suggest that serotype 5 adenovirus uses a similar mechanism to infect liver and adrenal gland and that selective transgene expression in the latter is more likely to be achieved through transcriptional targeting. PMID:23638001

  4. Regulation of Human Adenovirus Replication by RNA Interference

    PubMed Central

    Nikitenko, N. A.; Speiseder, T.; Lam, E.; Rubtsov, P. M.; Tonaeva, Kh. D.; Borzenok, S. A.; Dobner, T.; Prassolov, V. S.

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy. PMID:26483965

  5. Regulation of Human Adenovirus Replication by RNA Interference.

    PubMed

    Nikitenko, N A; Speiseder, T; Lam, E; Rubtsov, P M; Tonaeva, Kh D; Borzenok, S A; Dobner, T; Prassolov, V S

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

  6. Affinity Maturation of an Anti-V Antigen IgG Expressed In Situ Via Adenovirus Gene Delivery Confers Enhanced Protection Against Yersinia pestis Challenge

    PubMed Central

    Van Blarcom, Thomas J.; Sofer-Podesta, Carolina; Ang, John; Boyer, Julie L.; Crystal, Ronald G.; Georgiou, George

    2013-01-01

    Genetic transfer of neutralizing antibodies has been shown to confer strong and persistent protection against bacterial and viral infectious agents. While it is well established that for many exogenous neutralizing antibodies increased antigen affinity correlates with protection, the effect of antigen affinity on antibodies produced in situ following adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal antibody 2C12.4 recognizes the Yersinia pestis Type III secretion apparatus protein LcrV (V antigen) and confers protection in mice when administered as an IgG intraperitoneally or, following genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad) 1. 2C12.4 was expressed as a scFv fragment in E. coli and was shown to display a KD=3.5 nM by surface plasmon resonance (SPR) analysis. The 2C12.4 scFv was subjected to random mutagenesis and variants with increased affinity were isolated by flow cytometry using the Anchored Periplasmic Expression (APEx) bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower KD values (H8, KD=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen antibodies 3 days post-immunization with 109, 1010 or 1011 particle units. Following intranasal challenge with 363 LD50Y. pestis CO92, 54% of the mice immunized with 1010 pu of AdαV.H8 survived at the 14 day end point compared to only 15% survivors for the group immunized with AdαV expressing the lower affinity 2C12.4 (P<0.04, AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing antibody delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but possibly for other pathogens. PMID:20393511

  7. An oncolytic adenovirus that expresses the HAb18 and interleukin 24 genes exhibits enhanced antitumor activity in hepatocellular carcinoma cells

    PubMed Central

    Yuan, Sujing; Fang, Xianlong; Xu, Yanni; Ni, Aimin; Liu, Xin-Yuan; Chu, Liang

    2016-01-01

    Hepatocellular carcinoma (HCC) is characterized by alterations in multiple genes. High expression of CD147 on the surface of HCC cells promotes proliferation. The monoclonal antibody HAb18 recognizes CD147. We constructed an oncolytic adenoviral vector to express HAb18 (ZD55-HAb18) in HCC cells. Interleukin 24 (IL24) was co-expressed through the use of an F2A linker. ZD55-HAb18-IL24 decreased HCC cell viability to a greater degree than either ZD55-HAb18 or ZD55-IL24 alone. ZD55-HAb18-IL24 also induced apoptosis and autophagy in PLC/PRF/5 HCC cells. Intratumoral injection of ZD55-HAb18-IL24 repressed tumor growth in a PLC/PRF/5 xenograft model. Our results suggest that antibody-antitumor gene conjugation elicited a stronger antitumor effect than the antibody alone, and that this strategy could broaden the applications of antibody-based therapies in HCC. PMID:27528029

  8. Expression of the pRb-Binding Regions of E1A Enables Efficient Transformation of Primary Epithelial Cells by v-src

    PubMed Central

    Fischer, Robert S.; Quinlan, Margaret P.

    1998-01-01

    Primary cultures of rat embryo fibroblasts have been shown to be resistant to transformation by dominant oncogenes such as v-src. We sought to determine if similar resistance is displayed by primary epithelial cells, and, if so, whether an immortalizing oncogene such as E1A could enhance transformation of primary epithelial cells by v-src. Transformation of primary rat epithelial cells by v-src was synergistically enhanced when E1A expression plasmids were cotransfected with a v-src expression plasmid. Foci were more numerous and observed earlier (9 to 14 days) with E1A plus v-src than with v-src alone (18 to 28 days). This cotransformation ability was abrogated by deletions in CR1 or CR2 of E1A, which encode the binding regions for the pRb family and are responsible for E1A-mediated cell cycle activation. Mutations in the p300 binding site or the second exon, which abolish immortalization, did not affect v-src cooperation, in contrast to ras and adenovirus E1B. While kinase activation was required for growth in soft agar, differential activation of Src kinase did not correlate with transformation efficiency. Cell morphology and actin structures were not dramatically impacted by E1A expression; thus, hypertransformation, as previously described for ras cotransformation, was not observed with v-src and second-exon mutants of E1A. However, growth rates for cells expressing both E1A and v-Src were higher than those for cells expressing only v-Src. These results suggest that functions involved in cell cycle activation encoded by E1A first exon can enhance v-src transformation of primary epithelial cells. PMID:9525601

  9. Suppression of Adenovirus Replication by Cardiotonic Steroids.

    PubMed

    Grosso, Filomena; Stoilov, Peter; Lingwood, Clifford; Brown, Martha; Cochrane, Alan

    2017-02-01

    The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies.

  10. Survivin expression in oral lichen planus: Role in malignant transformation

    PubMed Central

    Suganya, G; Bavle, Radhika M; Paremala, K; Makarla, Soumya; Sudhakar, M; Reshma, V

    2016-01-01

    Context: Oral lichen planus (OLP) is a potentially malignant disease with a prevalence rate of 0.5–2.2%. It is a T-cell-mediated autoimmune disease, in which cytotoxic CD8+ T-cells trigger apoptosis of the basal cells of oral epithelium. The reported progression of OLP to oral squamous cell carcinoma (OSCC) ranges from 0.4% to 6.5%. Apoptosis plays a major role in the maintenance of tissue homeostasis. The evasion of apoptosis in the form of dysregulation of inhibitors of apoptosis proteins (IAPs) may lead to malignant transformation. Survivin belongs to the second gene family of IAPs, which is overexpressed in many tumors such as OSCC and gastric carcinomas, and its expression is widely involved in apoptosis as well as in tumor metastasis. Materials and Methods: Sections were obtained from the paraffin-embedded archival blocks of patients diagnosed histologically as OLP, and cases with normal epithelium were used for comparison whereas cases with OSCC were used as positive control. Results: We analyzed the expression of survivin in OLP and normal epithelium. Survivin expression with moderate intensity was seen in the cells of basal layer with nuclear positivity in cases of OLP, whereas mild to nil expression was seen in normal epithelium with nuclear and cytoplasmic positivity in different layers. Conclusions: Survivin positivity was seen predominantly in the basal cells of OLP suggesting increased longevity of these cells which in turn might acquire dysplastic changes leading to increased risk of malignant transformation of this premalignant condition. Although the conversion rate may be low, the potential exists in the indolent course of the disease. PMID:27601815

  11. ACTH adenomas transforming their clinical expression: report of 5 cases.

    PubMed

    Zoli, Matteo; Faustini-Fustini, Marco; Mazzatenta, Diego; Marucci, Gianluca; De Carlo, Eugenio; Bacci, Antonella; Pasquini, Ernesto; Lanzino, Giuseppe; Frank, Giorgio

    2015-02-01

    OBJECT Adrenocorticotropic hormone (ACTH) adenomas have been recognized as a more aggressive and invasive subtype of pituitary adenomas. An additional and clinically relevant peculiarity of these tumors is their ability to modify their clinical expression from a silent form to Cushing disease or vice versa. The aim of this study was to review a series of patients with pituitary adenomas and analyze the clinical implications of the transformation of clinical expression in 5 cases that showed this phenomenon. METHODS The authors retrospectively reviewed a series of patients with pituitary adenoma and collected clinical, biohumoral, and neuroradiological data of those who presented with a transformation from silent ACTH adenomas to functioning tumors or vice versa. In all the cases, preoperative assessment consisted of brain MRI, ophthalmological examination, and complete baseline endocrinological investigation. In patients with clinical and/or biochemical findings suspicious for Cushing syndrome, a low-dose dexamethasone suppression test was performed to rule in or out this diagnosis. Endocrinological evaluations were repeated 1 month after surgery, 3 months after surgery, and every 6 months or annually thereafter. Ophthalmological evaluations and brain MRIs were repeated after 3 months and then every 6 or 12 months thereafter. RESULTS Five patients (2 men and 3 women) included in this series had corticotropic tumors that showed transformation from an endocrinologically silent form to manifest Cushing disease and vice versa. The mean age at presentation was 40 years (range 18-51 years). In 3 of these patients, a transformation from silent to functioning ACTH adenoma with manifest Cushing disease occurred. In 1 patient, the authors observed the transition from a functioning to a silent adenoma with spontaneous resolution of hypercortisolism. Another patient's silent adenoma "shifted" to a functioning adenoma and then regressed back to a silent form with spontaneous

  12. Adenovirus Replaces Mitotic Checkpoint Controls

    PubMed Central

    Turner, Roberta L.; Groitl, Peter; Dobner, Thomas

    2015-01-01

    ABSTRACT Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins, including the E1B-55K and E4orf3 proteins, inhibit signaling in response to DNA damage. A fraction of cells infected with an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this state at late times of infection. This arrested state, which displays hallmarks of mitotic catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However, E1B-55K mutant virus-infected cells became trapped in a mitotic-like state in the presence of the microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by possibly mislocalizing and functionally inactivating cyclin B1. When expressed in noninfected cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1 variant. IMPORTANCE Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis, these adenoviral proteins act to prevent the infected cell from arresting in a

  13. Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

    PubMed

    Komorek, Jessica; Kuppuswamy, Mohan; Subramanian, T; Vijayalingam, S; Lomonosova, Elena; Zhao, Ling-Jun; Mymryk, Joe S; Schmitt, Kimberly; Chinnadurai, G

    2010-03-01

    The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

  14. Adenovirus Type 5 E1A and E6 Proteins of Low-Risk Cutaneous Beta-Human Papillomaviruses Suppress Cell Transformation through Interaction with FOXK1/K2 Transcription Factors ▿

    PubMed Central

    Komorek, Jessica; Kuppuswamy, Mohan; Subramanian, T.; Vijayalingam, S.; Lomonosova, Elena; Zhao, Ling-jun; Mymryk, Joe S.; Schmitt, Kimberly; Chinnadurai, G.

    2010-01-01

    The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2. PMID:20053746

  15. Temporal regulation of adenovirus major late alternative RNA splicing.

    PubMed

    Akusjarvi, Goran

    2008-05-01

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced mRNAs during replication. The accumulation of viral mRNAs is subjected to a temporal regulation, a mechanism that ensures that proteins that are needed at certain stages of the virus life cycle are produced in a timely fashion. The complex interactions between the virus and the host cell RNA splicing machinery has been studied in detail during the last decade. These studies have resulted in the characterization of two viral proteins, E4-ORF4 and L4-33K, that adenovirus uses to remodel the host cell RNA splicing machinery. Here I will review the current knowledge of how mRNA expression from the adenovirus major late transcription unit is controlled with a particular emphasis on how cis-acting sequence element, trans-acting factors and mechanisms regulating adenovirus major late L1 alternative RNA splicing is controlled.

  16. Labeling of Adenovirus Particles with PARACEST Agents

    PubMed Central

    Vasalatiy, Olga; Gerard, Robert D; Zhao, Piyu; Sun, Xiankai; Sherry, A. Dean

    2009-01-01

    Recombinant adenovirus type 5 particles (AdCMVLuc) were labeled with two different bifunctional ligands capable of forming stable complexes with paramagnetic lanthanide ions. The number of covalently attached ligands varied between 630 and 1960 per adenovirus particle depending upon the chemical reactivity of the bifunctional ligand (NHS ester versus isothiocyanide), the amount of excess ligand added, and the reaction time. The bioactivity of each labeled adenovirus derivative, as measured by the ability of the virus to infect cells and express luciferase, was shown to be highly dependent upon the number of covalently attached ligands. This indicates that certain amino groups, likely on the surface of the adenovirus fiber protein where cell binding is known to occur, are critical for viral attachment and infection. Addition of 177Lu3+ to chemically modified versus control viruses demonstrated a significant amount of nonspecific binding of 177Lu3+ to the virus particles that could not be sequestered by addition of excess DTPA. Thus, it became necessary to implement a prelabeling strategy for conjugation of preformed lanthanide ligand chelates to adenovirus particles. Using preformed Tm3+-L2, a large number of chelates having chemical exchange saturation transfer (CEST) properties were attached to the surface residues of AdCMVLuc without nonspecific binding of metal ions elsewhere on the virus particle. The potential of such conjugates to act as PARACEST imaging agents was tested using an on-resonance WALTZ sequence for CEST activation. A 12% decrease in bulk water signal intensity was observed relative to controls. This demonstrates that viral particles labeled with PARACEST-type imaging agents can potentially serve as targeted agents for molecular imaging. PMID:18254605

  17. General Strategy for Broadening Adenovirus Tropism

    PubMed Central

    Fontana, Laura; Nuzzo, Maurizio; Urbanelli, Lorena; Monaci, Paolo

    2003-01-01

    In spite of its broad host range, adenovirus type 5 (Ad5) transduces a number of clinically relevant tissues and cell types inefficiently, mostly because of low expression of the coxsackievirus-adenovirus receptor (CAR). To improve gene transfer to such cells, we modified the Ad5 fiber knob to recognize novel receptors. We expressed a functional Ad5 fiber knob domain on the capsid of phage λ and employed this display system to construct a large collection of ligands in the HI loop of the Ad5 knob. Panning this library on the CAR-negative mouse fibroblast cell line NIH 3T3 resulted in the identification of three clones with increased binding to these cells. Adenoviruses incorporating these ligands in the fiber gene transduced NIH 3T3 cells 2 or 3 orders of magnitude better than the parent vector. The same nonnative tropism was revealed in other cell types, independently of CAR expression. These Ad5 derivatives proved capable of transducing mouse and human primary immature dendritic cells with up to 100-fold increased efficiency. PMID:14512557

  18. Oncolytic virotherapy for osteosarcoma using midkine promoter-regulated adenoviruses.

    PubMed

    Takagi-Kimura, M; Yamano, T; Tagawa, M; Kubo, S

    2014-03-01

    Oncolytic virotherapy using adenoviruses has potential therapeutic benefits for a variety of cancers. We recently developed MOA5, a tumor-specific midkine promoter-regulated oncolytic vector based on human adenovirus serotype 5 (Ad5). We modified the binding tropism of MOA5 by replacing the cell-binding domain of the Ad5 fiber knob with that from another adenovirus serotype 35 (Ad35); the resulting vector was designated MOA35. Here we evaluated the therapeutic efficacies of MOA5 and MOA35 for human osteosarcoma. Midkine mRNA expression and its promoter activity was significantly high in five human osteosarcoma cell lines, but was restricted in normal cells. Very low levels of adenovirus cellular receptor coxsackievirus/adenovirus receptor (CAR) (Ad5 receptor) expression were observed in MNNG-HOS and MG-63 cells, whereas high levels of CAR expression were seen in the other osteosarcoma cell lines. By contrast, CD46 (Ad35 receptor) was highly expressed in all osteosarcoma cell lines. Infectivity and in vitro cytocidal effect of MOA35 was significantly enhanced in MNNG-HOS and MG-63 cells compared with MOA5, although the cytocidal effects of MOA5 were sometimes higher in high CAR-expressing cell lines. In MG-63 xenograft models, MOA35 significantly enhanced antitumor effects compared with MOA5. Our findings indicate that MOA5 and MOA35 allow tailored virotherapy and facilitate more effective treatments for osteosarcoma.

  19. Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3′-side of the transgene

    PubMed Central

    Putranto, Endy Widya; Kinoshita, Rie; Watanabe, Masami; Sadahira, Takuya; Murata, Hitoshi; Yamamoto, Ken-Ichi; Futami, Junichiro; Kataoka, Ken; Inoue, Yusuke; Winarsa Ruma, I. Made; Sumardika, I. Wayan; Youyi, Chen; Kubo, Miyoko; Sakaguchi, Yoshihiko; Saito, Kenji; Nasu, Yasutomo; Kumon, Hiromi; Huh, Nam-Ho; Sakaguchi, Masakiyo

    2017-01-01

    Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5′ located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5′ located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy. PMID:28693271

  20. Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3'-side of the transgene.

    PubMed

    Putranto, Endy Widya; Kinoshita, Rie; Watanabe, Masami; Sadahira, Takuya; Murata, Hitoshi; Yamamoto, Ken-Ichi; Futami, Junichiro; Kataoka, Ken; Inoue, Yusuke; Winarsa Ruma, I Made; Sumardika, I Wayan; Youyi, Chen; Kubo, Miyoko; Sakaguchi, Yoshihiko; Saito, Kenji; Nasu, Yasutomo; Kumon, Hiromi; Huh, Nam-Ho; Sakaguchi, Masakiyo

    2017-07-01

    Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5' located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy.

  1. Antiviral antibodies target adenovirus to phagolysosomes and amplify the innate immune response.

    PubMed

    Zaiss, Anne K; Vilaysane, Akosua; Cotter, Matthew J; Clark, Sharon A; Meijndert, H Christopher; Colarusso, Pina; Yates, Robin M; Petrilli, Virginie; Tschopp, Jurg; Muruve, Daniel A

    2009-06-01

    Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-kappaB-dependent genes, type I IFNs, and caspase-dependent IL-1beta maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1beta maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

  2. Human papillomavirus E6E7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes.

    PubMed

    Balagué, C; Noya, F; Alemany, R; Chow, L T; Curiel, D T

    2001-08-01

    Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.

  3. Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

    PubMed Central

    Balagué, Cristina; Noya, Francisco; Alemany, Ramon; Chow, Louise T.; Curiel, David T.

    2001-01-01

    Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells. PMID:11462032

  4. Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response.

    PubMed

    Moraes, Mauro Pires; Segundo, Fayna Diaz-San; Dias, Camila C; Pena, Lindomar; Grubman, Marvin J

    2011-11-28

    We previously demonstrated that an adenovirus-based foot-and-mouth disease virus (FMDV) serotype A24 capsid subunit vaccine, Ad5-A24, expressed under the control of a cytomegalovirus promoter (CMV) can protect swine and bovines against homologous challenge, but in a similar approach using swine vaccinated with an Ad5-vectored FMDV O1 Campos vaccine, Ad5-O1C, the animals were only partially protected when challenged at 21 days post-vaccination (dpv). Recently, we demonstrated that inclusion of the complete coding region of nonstructural protein 2B in the Ad5-A24 vector resulted in improved immune responses in pigs. We also found that inclusion of a modified CMV promoter (pCI), Ad5-CI-A24-2B, enhanced the efficacy of the vector. To address the limited immunogenicity of Ad5-O1C, we have produced a new set of Ad5 vectors with the complete 2B coding region under the control of either the original or the modified version of the CMV promoter, Ad5-O1C-2B, or Ad5-CI-O1C-2B, respectively. To evaluate the potency and efficacy of the new vectors we performed 2 sets of experiments in cattle. In the first experiment we compared the original vector with vectors containing the pCI promoter and partial or full-length 2B. All groups were challenged, intradermally in the tongue, at 21 dpv with FMDV O1C. We found that in all vaccinated groups 2 of 4 animals were protected from clinical disease. In the second experiment we directly compared the efficacy of vectors with a partial or full-length 2B under the control of the original CMV promoter. While all animals in the control group developed clinical disease, 2 of 4 animals in the group receiving Ad5-O1C vaccine and 3 of 4 animals in the group receiving Ad5-O1C-2B vaccine were completely protected after challenge. We also observed a 100-fold reduction of virus shedding in Ad5-O1C vaccinated animals and the group receiving Ad5-O1C-2B had an additional 10-fold reduction compared with the Ad5-O1C vaccinated group. There was no difference

  5. Expression of adenovirus-2 early region 4: assignment of the early region 4 polypeptides to their respective mRNAs, using in vitro translation.

    PubMed

    Tigges, M A; Raskas, H J

    1982-12-01

    Adenovirus-2 early region 4 (E4; map positions 91.3 to 99.1) encodes six 5' and 3' coterminal, differently spliced mRNAs, which are 2.5, 2.1, 1.8, 1.5, 1.2, and 0.8 kilobases (kb) long. Hybridization selection with five cloned viral DNA fragments that hybridize with subsets of E4 mRNAs was used to purify these six mRNAs and a previously unreported 3.0-kb mRNA from virus-infected cells. E4 mRNAs which were purified by hybridization selection with cloned EcoRI fragment C (map positions 89.7 to 100) were also fractionated by size. The purified mRNAs were then translated in rabbit reticulocyte or wheat germ lysate systems. The full complement of E4 mRNAs specified as many as 16 different polypeptides, with molecular weights ranging from 24,000 (24K) to 10K. The most abundant E4 mRNA, which was 2.1 kb long, specified an 11K polypeptide. The 1.5-kb mRNA, which differed from the 2.1-kb mRNA only by deletion of a second intron from the 3' untranslated region, also specified an 11K polypeptide. The second most abundant mRNA, which was 1.8 kb long, and the 1.2-kb mRNA, which had an intron deleted from the 3' untranslated region, specified a 15K polypeptide. This polypeptide was labeled more intensely with [5,6-(3)H]leucine than with [35S]methionine. The 3.0- and 2.5-kb mRNAs specified four polypeptides (24K, 22K, 19K, and 17K). Translation of E4 mRNAs with a mean size of 0.8 kb, which accumulated preferentially in the presence of cycloheximide, yielded at least 10 polypeptides that migrated in polyacrylamide gels with apparent molecular weights ranging from 21,800 to 10,000. On the basis of translation in wheat germ lysates and the distribution of polypeptides encoded by size-fractionated mRNAs, we concluded that the 0.8-kb mRNA size class includes a heterogeneous mixture of mRNAs which are probably formed as the result of utilization of alternate splice acceptor and donor sites during removal of the second intron. Our polypeptide assignments for the 2.1-, 1.8-, 1.5-, and 1

  6. Oncolytic adenovirus co-expressing IL-12 and IL-18 improves tumor-specific immunity via differentiation of T cells expressing IL-12Rβ2 or IL-18Rα

    PubMed Central

    Choi, I-K; Lee, J-S; Zhang, S-N; Park, J; Lee, K-M; Sonn, C H; Yun, C-O

    2011-01-01

    The oncolytic adenovirus (Ad) is currently being advanced as a promising antitumor remedy as it selectively replicates in tumor cells and can transfer and amplify therapeutic genes. Interleukin (IL)-12 induces a potent antitumor effect by promoting natural killer (NK) cell and cytotoxic T cell activities. IL-18 also augments cytotoxicity of NK cells and proliferation of T cells. This effect further enhances the function of IL-12 in a synergistic manner. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18, RdB/IL-12/IL-18. Intratumoral administration of RdB/IL-12/IL-18 improved antitumor effects, as well as increased survival, in B16-F10 murine melanoma model. The ratio of T-helper type 1/2 cytokine as well as the levels of IL-12, IL-18, interferon-γ and granulocyte–macrophage colony-stimulating factor was markedly elevated in RdB/IL-12/IL-18-treated tumors. Mice injected with RdB/IL-12/IL-18 also showed enhanced cytotoxicity of tumor-specific immune cells. Consistent with these results, immense necrosis and infiltration of NK cells, as well as CD4+ and CD8+ T cells, were observed in RdB/IL-12/IL-18-treated tumor tissues. Importantly, tumors treated with RdB/IL-12/IL-18 showed an elevated number of T cells expressing IL-12Rβ2 or IL-18Rα. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-18 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity. PMID:21451575

  7. Avian influenza mucosal vaccination in chickens with replication-defective recombinant adenovirus vaccine

    USDA-ARS?s Scientific Manuscript database

    We evaluated protection conferred by mucosal vaccination with replication competent adenovirus (RCA)-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene (AdTW68.H5ck). Commercial layer-type chicken groups were singly vaccinated ocularly at 5 days of age, or singly v...

  8. Molecular evolution of human species D adenoviruses

    PubMed Central

    Robinson, Christopher M.; Seto, Donald; Jones, Morris S.; Dyer, David W.; Chodosh, James

    2011-01-01

    Adenoviruses are medium-sized double stranded DNA viruses that infect vertebrates. Human adenoviruses cause an array of diseases. Currently there are 56 human adenovirus types recognized and characterized within seven species (A-G). Of those types, a majority belongs to species D. In this review, the genomic conservation and diversity are examined amongst human adenoviruses within species D, particularly in contrast to other human adenovirus species. Specifically, homologous recombination is presented as a driving force for the molecular evolution of human adenoviruses and the emergence of new adenovirus pathogens. PMID:21570490

  9. Perforin and gamma interferon expression are required for CD4+ and CD8+ T-cell-dependent protective immunity against a human parasite, Trypanosoma cruzi, elicited by heterologous plasmid DNA prime-recombinant adenovirus 5 boost vaccination.

    PubMed

    de Alencar, Bruna C G; Persechini, Pedro M; Haolla, Filipe A; de Oliveira, Gabriel; Silverio, Jaline C; Lannes-Vieira, Joseli; Machado, Alexandre V; Gazzinelli, Ricardo T; Bruna-Romero, Oscar; Rodrigues, Mauricio M

    2009-10-01

    A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4(+) and CD8(+) T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4(+) and CD8(+) T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-gamma) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8(+) T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-gamma or IFN-gamma/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-gamma in the presence of highly cytotoxic T cells. Vaccinated IFN-gamma-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-gamma in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy.

  10. Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination▿

    PubMed Central

    de Alencar, Bruna C. G.; Persechini, Pedro M.; Haolla, Filipe A.; de Oliveira, Gabriel; Silverio, Jaline C.; Lannes-Vieira, Joseli; Machado, Alexandre V.; Gazzinelli, Ricardo T.; Bruna-Romero, Oscar; Rodrigues, Mauricio M.

    2009-01-01

    A heterologous prime-boost strategy using plasmid DNA, followed by replication-defective recombinant adenovirus 5, is being proposed as a powerful way to elicit CD4+ and CD8+ T-cell-mediated protective immunity against intracellular pathogens. We confirmed this concept and furthered existing research by providing evidence that the heterologous prime-boost regimen using the gene encoding amastigote surface protein 2 elicited CD4+ and CD8+ T-cell-mediated protective immunity (reduction of acute parasitemia and prolonged survival) against experimental infection with Trypanosoma cruzi. Protective immunity correlated with the presence of in vivo antigen-specific cytotoxic activity prior to challenge. Based on this, our second goal was to determine the outcome of infection after heterologous prime-boost immunization of perforin-deficient mice. These mice were highly susceptible to infection. A detailed analysis of the cell-mediated immune responses in immunized perforin-deficient mice showed an impaired gamma interferon (IFN-γ) secretion by immune spleen cells upon restimulation in vitro with soluble recombinant antigen. In spite of a normal numeric expansion, specific CD8+ T cells presented several functional defects detected in vivo (cytotoxicity) and in vitro (simultaneous expression of CD107a/IFN-γ or IFN-γ/tumor necrosis factor alpha) paralleled by a decreased expression of CD44 and KLRG-1. Our final goal was to determine the importance of IFN-γ in the presence of highly cytotoxic T cells. Vaccinated IFN-γ-deficient mice developed highly cytotoxic cells but failed to develop any protective immunity. Our study thus demonstrated a role for perforin and IFN-γ in a number of T-cell-mediated effector functions and in the antiparasitic immunity generated by a heterologous plasmid DNA prime-adenovirus boost vaccination strategy. PMID:19651871

  11. Adenovirus DNA polymerase is a phosphoprotein.

    PubMed

    Ramachandra, M; Nakano, R; Mohan, P M; Rawitch, A B; Padmanabhan, R

    1993-01-05

    Biological activities of many of the eukaryotic DNA replication proteins are modulated by protein phosphorylation. Investigations of the phosphorylation of adenovirus DNA polymerase (AdPol) have been difficult mainly because of its low level of synthesis in adenovirus-infected HeLa cells. However, when AdPol was overproduced using the recombinant vaccinia virus (RV-AdPol) and the baculovirus expression systems, or by a large scale metabolic labeling of adenovirus 2-infected HeLa cells (native AdPol), in vivo phosphorylation of AdPol could be demonstrated. Phosphoamino acid analysis of [32P]AdPol indicated the presence of phosphoserine independent of the source of AdPol. Comparison of tryptic peptide maps of native AdPol and RV-AdPol revealed that the majority of phosphopeptides were common. Fractionation by high performance liquid chromatography and sequencing of one of the major phosphopeptides revealed serine 67 as a site of phosphorylation. Interestingly, this site is located close to the nuclear localization signal of AdPol and has a consensus substrate recognition sequence for histone H1 (cdc2-related) kinases and mitogen-activated protein kinases. Dephosphorylation of AdPol with calf intestinal alkaline phosphatase resulted in significant decrease in its activity in the in vitro DNA replication initiation assay, suggesting that phosphorylation is important for its biological activity.

  12. Elasticity and Binding of Adenovirus

    NASA Astrophysics Data System (ADS)

    Matthews, Garrett; Negishi, Atsuko; Seeger, Adam; McCarty, Doug; Taylor, Russell; Samulshi, Jude; Superfine, Richard

    1999-11-01

    Adenovirus was the first human virus found to cause the transformation of cells and is one of the more common vectors being used for the development of gene therapy. As such, much is known about the viral structure and genome; however, the events of the early infection cycle, such as binding of the virus to the cell membrane and the release of genetic material from the capsid, for this and other nonenveloped viruses, are not fully understood. With the atomic force microscope (AFM) we are able to image the virus in both air and liquids, allowing us to change the surrounding environment, varying such physiologically relevant parameters as osmolality or pH. We additionally have the ability to do manipulations on single virus particles in these environments using the nanoManipulator. The nanoManipulator is an advanced interface for AFM that allows real time three dimensional rendering of the topographical data, allows the sample surface to be non-destructively felt using a hand held stylus that responds to the information being sensed at the tip, and allows controlled modification of the surface. Using this tool we have translated single virions over various surfaces, allowing us to measure the adhesion between the capsid and these surfaces. Additionally, we are able to place the tip directly atop individual viruses and measure their elasticity under a compressive load being supplied by that tip. We can explore how this property changes as a function of the properties of the surrounding liquid.

  13. Retargeted adenoviruses for radiation-guided gene delivery

    PubMed Central

    Kaliberov, S A; Kaliberova, L N; Yan, H; Kapoor, V; Hallahan, D E

    2016-01-01

    The combination of radiation with radiosensitizing gene delivery or oncolytic viruses promises to provide an advantage that could improve the therapeutic results for glioblastoma. X-rays can induce significant molecular changes in cancer cells. We isolated the GIRLRG peptide that binds to radiation-inducible 78 kDa glucose-regulated protein (GRP78), which is overexpressed on the plasma membranes of irradiated cancer cells and tumor-associated microvascular endothelial cells. The goal of our study was to improve tumor-specific adenovirus-mediated gene delivery by selectively targeting the adenovirus binding to this radiation-inducible protein. We employed an adenoviral fiber replacement approach to conduct a study of the targeting utility of GRP78-binding peptide. We have developed fiber-modified adenoviruses encoding the GRP78-binding peptide inserted into the fiber-fibritin. We have evaluated the reporter gene expression of fiber-modified adenoviruses in vitro using a panel of glioma cells and a human D54MG tumor xenograft model. The obtained results demonstrated that employment of the GRP78-binding peptide resulted in increased gene expression in irradiated tumors following infection with fiber-modified adenoviruses, compared with untreated tumor cells. These studies demonstrate the feasibility of adenoviral retargeting using the GRP78-binding peptide that selectively recognizes tumor cells responding to radiation treatment. PMID:27492853

  14. Exploiting features of adenovirus replication to support mammalian kinase production

    PubMed Central

    Cotten, Matt; Stegmueller, Kerstin; Eickhoff, Jan; Hanke, Miriam; Herzberger, Katrin; Herget, Thomas; Choidas, Axel; Daub, Henrik; Godl, Klaus

    2003-01-01

    Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated. PMID:14576328

  15. Exploiting features of adenovirus replication to support mammalian kinase production.

    PubMed

    Cotten, Matt; Stegmueller, Kerstin; Eickhoff, Jan; Hanke, Miriam; Herzberger, Katrin; Herget, Thomas; Choidas, Axel; Daub, Henrik; Godl, Klaus

    2003-11-01

    Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.

  16. Vaccination with recombinant adenoviruses expressing Ebola virus glycoprotein elicits protection in the interferon alpha/beta receptor knock-out mouse.

    PubMed

    O'Brien, Lyn M; Stokes, Margaret G; Lonsdale, Stephen G; Maslowski, David R; Smither, Sophie J; Lever, Mark S; Laws, Thomas R; Perkins, Stuart D

    2014-03-01

    The resistance of adult immunocompetent mice to infection with ebolaviruses has led to the development of alternative small animal models that utilise immunodeficient mice, for example the interferon α/β receptor knock-out mouse (IFNR(-/-)). IFNR(-/-) mice have been shown to be susceptible to infection with ebolaviruses by multiple routes but it is not known if this murine model is suitable for testing therapeutics that rely on the generation of an immune response for efficacy. We have tested recombinant adenovirus vectors for their ability to protect IFNR(-/-) mice from challenge with Ebola virus and have analysed the humoral response generated after immunisation. The recombinant vaccines elicited good levels of protection in the knock-out mouse and the antibody response in IFNR(-/-) mice was similar to that observed in vaccinated wild-type mice. These results indicate that the IFNR(-/-) mouse is a relevant small animal model for studying ebolavirus-specific therapeutics.

  17. Further reduction in adenovirus vector-mediated liver transduction without largely affecting transgene expression in target organ by exploiting microrna-mediated regulation and the Cre-loxP recombination system.

    PubMed

    Bennett, David; Sakurai, Fuminori; Shimizu, Kahori; Matsui, Hayato; Tomita, Kyoko; Suzuki, Takayuki; Katayama, Kazufumi; Kawabata, Kenji; Mizuguchi, Hiroyuki

    2012-12-03

    In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into the 3'-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3'-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen.

  18. Transforming growth factor-beta induces endothelin-1 expression through activation of the Smad signaling pathway.

    PubMed

    Rodríguez-Pascual, Fernando; Reimunde, Francisco Manuel; Redondo-Horcajo, Mariano; Lamas, Santiago

    2004-11-01

    Expression of the endothelin-1 gene is subject to complex regulation by different factors, among which transforming growth factor-beta is one of the most important. We have analyzed the mechanism by which transforming growth factor-beta increases endothelin-1 expression in vascular endothelial cells. Transcriptional activation of the endothelin-1 promoter accounted for the transforming growth factor-beta-induced increase in endothelin-1 mRNA levels. Two DNA elements within the promoter are responsible for this effect: a Smad binding element and a proximal activator protein-1 site. Mutation of both elements abolished transforming growth factor-beta responsiveness. Overexpression of the Smad3 isoform strongly potentiates transforming growth factor-beta- induced endothelin-1 promoter activity in a phosphorylation-dependent manner. These results demonstrate that transforming growth factor-beta induces endothelin-1 expression by a functional cooperation between Smads and activator protein-1 through activation of the Smad signaling pathway.

  19. Adenovirus-Mediated Human Paraoxonase 1 Gene Transfer to Provide Protection Against the Toxicity of the Organophosphorus Pesticide Toxicant Diazoxon

    DTIC Science & Technology

    2011-03-01

    tested whether PON1 gene transfer using adenovirus provides protection against the toxicity of the OP diazoxon. Using an adenovirus construct...expressed PON1 at 600-3480 U ml(-1) on day 5 post-treatment, which is 8-50 fold above endogenous. Six mice expressing hPON1 survived 2LD(50) doses of diazoxon

  20. Anti-tumor immunity elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1.

    PubMed

    Hasegawa, K; Tagawa, M; Takagi, K; Tsukamoto, H; Tomioka, Y; Suzuki, T; Nishioka, Y; Ohrui, T; Numasaki, M

    2016-08-01

    Interleukin (IL)-28A/interferon (IFN)-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect anti-tumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of an anti-proliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8(+) T cells have a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8(+) T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific anti-tumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ-deficient mice, anti-tumor activities of AdIL-28A were completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided a synergistic anti-tumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer immunogene therapy.

  1. Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

    USGS Publications Warehouse

    Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

    2001-01-01

    This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

  2. Mechanisms of pathogenesis of emerging adenoviruses

    PubMed Central

    Cook, James; Radke, Jay

    2017-01-01

    Periodic outbreaks of human adenovirus infections can cause severe illness in people with no known predisposing conditions. The reasons for this increased viral pathogenicity are uncertain. Adenoviruses are constantly undergoing mutation during circulation in the human population, but related phenotypic changes of the viruses are rarely detected because of the infrequency of such outbreaks and the limited biological studies of the emergent strains. Mutations and genetic recombinations have been identified in these new strains. However, the linkage between these genetic changes and increased pathogenicity is poorly understood. It has been observed recently that differences in virus-induced immunopathogenesis can be associated with altered expression of non-mutant viral genes associated with changes in viral modulation of the host innate immune response. Initial small animal studies indicate that these changes in viral gene expression can be associated with enhanced immunopathogenesis in vivo. Available evidence suggests the hypothesis that there is a critical threshold of expression of certain viral genes that determines both the sustainability of viral transmission in the human population and the enhancement of immunopathogenesis. Studies of this possibility will require extension of the analysis of outbreak viral strains from a sequencing-based focus to biological studies of relationships between viral gene expression and pathogenic responses. Advances in this area will require increased coordination among public health organizations, diagnostic microbiology laboratories, and research laboratories to identify, catalog, and systematically study differences between prototype and emergent viral strains that explain the increased pathogenicity that can occur during clinical outbreaks. PMID:28184296

  3. Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs

    PubMed Central

    Park, Jong-Hyeon; Lee, Kwang-Nyeong; Kim, Se-Kyung; You, Su-Hwa; Kim, Taeseong; Tark, Dongseob; Lee, Hyang-Sim; Seo, Min-Goo; Kim, Byounghan

    2015-01-01

    ABSTRACT Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-αγ and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-αγ and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD. IMPORTANCE The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against

  4. Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs.

    PubMed

    Kim, Su-Mi; Park, Jong-Hyeon; Lee, Kwang-Nyeong; Kim, Se-Kyung; You, Su-Hwa; Kim, Taeseong; Tark, Dongseob; Lee, Hyang-Sim; Seo, Min-Goo; Kim, Byounghan

    2015-08-01

    Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-αγ and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-αγ and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD. The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against FMDV, although the

  5. Bovine adenovirus-3 as a vaccine delivery vehicle.

    PubMed

    Ayalew, Lisanework E; Kumar, Pankaj; Gaba, Amit; Makadiya, Niraj; Tikoo, Suresh K

    2015-01-15

    The use of vaccines is an effective and relatively inexpensive means of controlling infectious diseases, which cause heavy economic losses to the livestock industry through animal loss, decreased productivity, treatment expenses and decreased carcass quality. However, some vaccines produced by conventional means are imperfect in many respects including virulence, safety and efficacy. Moreover, there are no vaccines for some animal diseases. Although genetic engineering has provided new ways of producing effective vaccines, the cost of production for veterinary use is a critical criterion for selecting the method of production and delivery of vaccines. The cost effective production and intrinsic ability to enter cells has made adenovirus vectors a highly efficient tool for delivery of vaccine antigens. Moreover, adenoviruses induce both humoral and cellular immune responses to expressed vaccine antigens. Since nonhuman adenoviruses are species specific, the development of animal specific adenoviruses as vaccine delivery vectors is being evaluated. This review summarizes the work related to the development of bovine adenovirus-3 as a vaccine delivery vehicle in animals, particularly cattle.

  6. Resident corneal c-fms+ macrophages and dendritic cells mediate early cellular infiltration in adenovirus keratitis

    PubMed Central

    Ramke, Mirja; Zhou, Xiaohong; Materne, Emma Caroline; Rajaiya, Jaya; Chodosh, James

    2016-01-01

    The cornea contains a heterogeneous population of antigen-presenting cells with the capacity to contribute to immune responses. Adenovirus keratitis is a severe corneal infection with acute and chronic phases. The role of resident corneal antigen-presenting cells in adenovirus keratitis has not been studied. We utilized transgenic MaFIA mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, in a mouse model of adenovirus keratitis. Clinical keratitis and recruitment of myeloperoxidase and CD45+ cells were diminished in c-fms depleted, adenovirus infected mice, as compared to controls, consistent with a role for myeloid-lineage cells in adenovirus keratitis. PMID:27185163

  7. Resident corneal c-fms(+) macrophages and dendritic cells mediate early cellular infiltration in adenovirus keratitis.

    PubMed

    Ramke, Mirja; Zhou, Xiaohong; Materne, Emma Caroline; Rajaiya, Jaya; Chodosh, James

    2016-06-01

    The cornea contains a heterogeneous population of antigen-presenting cells with the capacity to contribute to immune responses. Adenovirus keratitis is a severe corneal infection with acute and chronic phases. The role of resident corneal antigen-presenting cells in adenovirus keratitis has not been studied. We utilized transgenic MaFIA mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, in a mouse model of adenovirus keratitis. Clinical keratitis and recruitment of myeloperoxidase and CD45(+) cells were diminished in c-fms depleted, adenovirus infected mice, as compared to controls, consistent with a role for myeloid-lineage cells in adenovirus keratitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Effects of cold atmospheric plasmas on adenoviruses in solution

    NASA Astrophysics Data System (ADS)

    Zimmermann, J. L.; Dumler, K.; Shimizu, T.; Morfill, G. E.; Wolf, A.; Boxhammer, V.; Schlegel, J.; Gansbacher, B.; Anton, M.

    2011-12-01

    Experiments were performed with cold atmospheric plasma (CAP) to inactivate adenovirus, a non-enveloped double stranded DNA virus, in solution. The plasma source used was a surface micro-discharge technology operating in air. Various plasma diagnostic measurements and tests were performed in order to determine the efficacy of CAPs and to understand the inactivation mechanism(s). Different stages of the adenovirus ‘life cycle’ were investigated—infectivity and gene expression as well as viral replication and spread. Within 240 s of CAP treatment, inactivation of up to 6 decimal log levels can be achieved.

  9. Interferon Regulatory Factor 4 Contributes to Transformation of v-Rel-Expressing Fibroblasts

    PubMed Central

    Hrdličková, Radmila; Nehyba, Jiří; Bose, Henry R.

    2001-01-01

    The avian homologue of the interferon regulatory factor 4 (IRF-4) and a novel splice variant lacking exon 6, IRF-4ΔE6, were isolated and characterized. Chicken IRF-4 is expressed in lymphoid organs, less in small intestine, and lungs. IRF-4ΔE6 mRNA, though less abundant than full-length IRF-4, was detected in lymphoid tissues, with the highest levels observed in thymic cells. IRF-4 is highly expressed in v-Rel-transformed lymphocytes, and the expression of IRF-4 is increased in v-Rel- and c-Rel-transformed fibroblasts relative to control cells. The expression of IRF-4 from retrovirus vectors morphologically transformed primary fibroblasts, increased their saturation density, proliferation, and life span, and promoted their growth in soft agar. IRF-4 and v-Rel cooperated synergistically to transform fibroblasts. The expression of IRF-4 antisense RNA eliminated formation of soft agar colonies by v-Rel and reduced the proliferation of v-Rel-transformed cells. v-Rel-transformed fibroblasts produced interferon 1 (IFN1), which inhibits fibroblast proliferation. Infection of fibroblasts with retroviruses expressing v-Rel resulted in an increase in the mRNA levels of IFN1, the IFN receptor, STAT1, JAK1, and 2′,5′-oligo(A) synthetase. The exogenous expression of IRF-4 in v-Rel-transformed fibroblasts decreased the production of IFN1 and suppressed the expression of several genes in the IFN transduction pathway. These results suggest that induction of IRF-4 expression by v-Rel likely facilitates transformation of fibroblasts by decreasing the induction of this antiproliferative pathway. PMID:11533227

  10. Construction, production, and purification of recombinant adenovirus vectors.

    PubMed

    Miravet, Susana; Ontiveros, Maria; Piedra, Jose; Penalva, Cristina; Monfar, Mercè; Chillón, Miguel

    2014-01-01

    Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. In this chapter, a standard procedure for their generation and small-scale production is described. Homologous recombination in E. coli between shuttle plasmids and full-length adenovirus backbones (E1-deleted) is used for the generation of recombinant adenoviral vectors genomes. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized and transfected to generate a recombinant adenoviral vector in permissive human cells. These vectors are then purified by two sequential CsCl gradient centrifugations and subjected to a chromatography step in order to eliminate the CsCl and exchange buffers. Finally, the viral stock is characterized through the quantification of its viral particle content and its infectivity.

  11. Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus

    SciTech Connect

    Steel, Jason C.; Morrison, Brian J.; Mannan, Poonam; Abu-Asab, Mones S.; Wildner, Oliver; Miles, Brian K.; Yim, Kevin C.; Ramanan, Vijay; Prince, Gregory A.; Morris, John C.

    2007-12-05

    Oncolytic adenoviruses as a treatment for cancer have demonstrated limited clinical activity. Contributing to this may be the relevance of preclinical animal models used to study these agents. Syngeneic mouse tumor models are generally non-permissive for adenoviral replication, whereas human tumor xenograft models exhibit attenuated immune responses to the vector. The cotton rat (Sigmodon hispidus) is susceptible to human adenovirus infection, permissive for viral replication and exhibits similar inflammatory pathology to humans with adenovirus replicating in the lungs, respiratory passages and cornea. We evaluated three transplantable tumorigenic cotton rat cell lines, CCRT, LCRT and VCRT as models for the study of oncolytic adenoviruses. All three cells lines were readily infected with adenovirus type-5-based vectors and exhibited high levels of transgene expression. The cell lines supported viral replication demonstrated by the induction of cytopathogenic effect (CPE) in tissue culture, increase in virus particle numbers and assembly of virions seen on transmission electron microscopy. In vivo, LCRT and VCRT tumors demonstrated delayed growth after injection with replicating adenovirus. No in vivo antitumor activity was seen in CCRT tumors despite in vitro oncolysis. Adenovirus was also rapidly cleared from the CCRT tumors compared to LCRT and VCRT tumors. The effect observed with the different cotton rat tumor cell lines mimics the variable results of human clinical trials highlighting the potential relevance of this model for assessing the activity and toxicity of oncolytic adenoviruses.

  12. Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.

    PubMed Central

    Weitzman, M D; Fisher, K J; Wilson, J M

    1996-01-01

    Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication-defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus. PMID:8627709

  13. Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.

    PubMed

    Weitzman, M D; Fisher, K J; Wilson, J M

    1996-03-01

    Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication-defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus.

  14. Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

    PubMed

    Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

    2015-12-29

    Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene).

  15. Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

    USDA-ARS?s Scientific Manuscript database

    The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

  16. Overexpression of several Arabidopsis histone genes increases Agrobacterium-medicated transformation and transgene expression in plants

    USDA-ARS?s Scientific Manuscript database

    The Arabidopsis histone H2A-1 is important for Agrobacterium-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, in the rat5 mutant results in decreased T-(transferred) DNA integration into the plant genome, whereas over-expression of HTA1 increases transformation freq...

  17. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    USDA-ARS?s Scientific Manuscript database

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  18. Noninvasive visualization of adenovirus replication with a fluorescent reporter in the E3 region.

    PubMed

    Ono, Hidetaka A; Le, Long P; Davydova, Julia G; Gavrikova, Tatyana; Yamamoto, Masato

    2005-11-15

    To overcome the inefficacy and undesirable side effects of current cancer treatment strategies, conditionally replicative adenoviruses have been developed to exploit the unique mechanism of oncolysis afforded by tumor-specific viral replication. Despite rapid translation into clinical trials and the established safety of oncolytic adenoviruses, the in vivo function of these agents is not well understood due to lack of a noninvasive detection system for adenovirus replication. To address this issue, we propose the expression of a reporter from the adenovirus E3 region as a means to monitor replication. Adenovirus replication reporter vectors were constructed with the enhanced green fluorescent protein (EGFP) gene placed in the deleted E3 region under the control of the adenoviral major late promoter while retaining expression of the adenovirus death protein to conserve the native oncolytic capability of the virus. Strong EGFP fluorescence was detected from these vectors in a replication-dependent manner, which correlated with viral DNA replication. Fluorescence imaging in vivo confirmed the ability to noninvasively detect fluorescent signal during replication, which generally corresponded with the underlying level of viral DNA replication. EGFP representation of viral replication was further confirmed by Western blot comparison with the viral DNA content in the tumors. Imaging reporter expression controlled by the adenoviral major late promoter provides a viable approach to noninvasively monitor adenovirus replication in preclinical studies and has the potential for human application with clinically relevant imaging reporters.

  19. Behavioral analysis of Drosophila transformants expressing human taste receptor genes in the gustatory receptor neurons.

    PubMed

    Adachi, Ryota; Sasaki, Yuko; Morita, Hiromi; Komai, Michio; Shirakawa, Hitoshi; Goto, Tomoko; Furuyama, Akira; Isono, Kunio

    2012-06-01

    Transgenic Drosophila expressing human T2R4 and T2R38 bitter-taste receptors or PKD2L1 sour-taste receptor in the fly gustatory receptor neurons and other tissues were prepared using conventional Gal4/UAS binary system. Molecular analysis showed that the transgene mRNAs are expressed according to the tissue specificity of the Gal4 drivers. Transformants expressing the transgene taste receptors in the fly taste neurons were then studied by a behavioral assay to analyze whether transgene chemoreceptors are functional and coupled to the cell response. Since wild-type flies show strong aversion against the T2R ligands as in mammals, the authors analyzed the transformants where the transgenes are expressed in the fly sugar receptor neurons so that they promote feeding ligand-dependently if they are functional and activate the neurons. Although the feeding preference varied considerably among different strains and individuals, statistical analysis using large numbers of transformants indicated that transformants expressing T2R4 showed a small but significant increase in the preference for denatonium and quinine, the T2R4 ligands, as compared to the control flies, whereas transformants expressing T2R38 did not. Similarly, transformants expressing T2R38 and PKD2L1 also showed a similar preference increase for T2R38-specific ligand phenylthiocarbamide (PTC) and a sour-taste ligand, citric acid, respectively. Taken together, the transformants expressing mammalian taste receptors showed a small but significant increase in the feeding preference that is taste receptor and also ligand dependent. Although future improvements are required to attain performance comparable to the endogenous robust response, Drosophila taste neurons may serve as a potential in vivo heterologous expression system for analyzing chemoreceptor function.

  20. Cloning, biological characterization and high-level expression of rat interleukin-3 using recombinant adenovirus: description of a new splicing variant.

    PubMed

    del C Esandi, M; van Someren, G D; van der Velde, I; van Bekkum, D W; Valerio, D; Noteboom, J L; Bout, A

    1998-04-28

    In the present study, we describe the cloning and sequence analysis of rat IL-3. Two different mRNA isoforms were isolated after transfection of COS cells with the cytokine genomic sequences. One of the isoforms has been predicted before by Cohen et al. (1986), and the other one is identical except that it encodes a protein with an insertion of three amino acids at position 56. As names for the two isoforms, we propose IL-3alpha for the predicted and IL-3beta for the novel molecule. IL-3beta mRNA was detected as the predominant isoform in rat lymphocytes in vivo. High levels of the cytokine were obtained after infection of human cells (A549) with a recombinant adenovirus harboring rIL-3beta cDNA (IG.Ad.CMV.IL-3beta). The biological properties of the IL-3beta protein were tested in a FDC-P1 proliferation assay and in a hematopoietic progenitor colony forming assay. To assess in-vivo bioactivity, lysed 293 cells containing IG.Ad.CMV.rIL-3beta virus were injected subcutaneously into F344 rats. Stimulation of hematopoiesis and leucocytosis were observed during the treatment. After subcutaneous injections of the lysed adeno-producer cells in mice, the only effect observed was a cellular infiltration at the site of injection, confirming the poor cross-reactivity between the two species. The biological properties in vitro and in vivo demonstrate that the cDNA sequences of IL-3beta presented here encode active rat IL-3 protein.

  1. Immunogenicity of the recombinant adenovirus type-5 vector -based Ebola vaccine (Ad5-EBOV) expressing the glycoprotein of the 2014 epidemic strain in mice.

    PubMed

    Wang, Ling; Liu, Jing Jing; Kong, Yan; Hou, Li Hua; Li, Yu Hua

    2017-08-10

    The 2014 Ebola outbreak in West Africa has brought great threat to the public health worldwide. Development of Ebola vaccine is urgent. A novel recombinant adenovirus type-5 vector-based Ebola vaccine (Ad5-EBOV) based on the 2014 Zaire Guinea epidemic strain was developed in China. A good safety profile and robust immune response elicited by Ad5-EBOV were confirmed in Phase I and Phase II clinical trial. However, clinical studies of Ebola vaccine are still at an early stage and no solid efficacy data for human beings yet. For efficacy evaluation and quality control of Ad5-EBOV, the cellular and humoral immune responses were examined at different time points in BALB/c mice which were vaccinated with the Ad5-EBOV. EBOV GP-specific T-cell responses were detected early in the first week, increased overtime, reached the peak at week 4 and lasted to week 6 by ELISpot and Flow cytometric analysis. High titer of EBOV glycoprotein-specific antibody was found at week 1 at 1783(geometric mean, GM), peaked at week 10 at 26,214(GM), and lasted to six months at 1351(GM). The titer of neutralizing antibody based on pseudovirus also increased overtime, peaked at 1:16 in one mouse and 1:8 in nine mice at week 6, then decreased to zero at week 12. These results suggest that BALB/c mice can be used to evaluate the effectiveness of Ad5-EBOV, while cellular immune response and humoral immune response can be used as indicators for the evaluation of vaccine effectiveness. Determination of methods and indicators as soon as possible is critical and valuable for efficacy evaluation of Ebola vaccine, and is expected to provide an important guarantee for quality control of Ad5-EBOV.

  2. Screening promoters for Anthurium transformation using transient expression

    USDA-ARS?s Scientific Manuscript database

    Different promoters and tissue types were evaluated for transient '-glucoronidase (GUS) expression in Anthurium andreanum Hort. ‘Marian Seefurth’ following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35 S promoter fr...

  3. Caveolin-1 inhibits epidermal growth factor-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3). Transformation suppressor effects of adenovirus-mediated gene delivery of caveolin-1.

    PubMed

    Zhang, W; Razani, B; Altschuler, Y; Bouzahzah, B; Mostov, K E; Pestell, R G; Lisanti, M P

    2000-07-07

    Caveolin-1 is a principal component of caveolae membranes that may function as a transformation suppressor. For example, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (D7S522; 7q31.1) that is deleted in human cancers, including mammary carcinomas. However, little is known about the role of caveolins in regulating cell movement, a critical parameter in determining metastatic potential. Here, we examine the role of caveolin-1 in cell movement. For this purpose, we employed an established cellular model, MTLn3, a metastatic rat mammary adenocarcinoma cell line. In this system, epidermal growth factor (EGF) stimulation induces rapid lamellipod extension and cell migration. Interestingly, we find that MTLn3 cells fail to express detectable levels of endogenous caveolin-1. To restore caveolin-1 expression in MTLn3 cells efficiently, we employed an inducible adenoviral gene delivery system to achieve tightly controlled expression of caveolin-1. We show here that caveolin-1 expression in MTLn3 cells inhibits EGF-stimulated lamellipod extension and cell migration and blocks their anchorage-independent growth. Under these conditions, EGF-induced activation of the p42/44 mitogen-activated protein kinase cascade is also blunted. Our results suggest that caveolin-1 expression in motile MTLn3 cells induces a non-motile phenotype.

  4. Development of novel types of plastid transformation vectors and evaluation of factors controlling expression.

    PubMed

    Herz, Stefan; Füssl, Monika; Steiger, Sandra; Koop, Hans-Ulrich

    2005-12-01

    Two new vector types for plastid transformation were developed and uidA reporter gene expression was compared to standard transformation vectors. The first vector type does not contain any plastid promoter, instead it relies on extension of existing plastid operons and was therefore named "operon-extension" vector. When a strongly expressed plastid operon like psbA was extended by the reporter gene with this vector type, the expression level was superior to that of a standard vector under control of the 16S rRNA promoter. Different insertion sites, promoters and 5'-UTRs were analysed for their effect on reporter gene expression with standard and operon-extension vectors. The 5'-UTR of phage 7 gene 10 in combination with a modified N-terminus was found to yield the highest expression levels. Expression levels were also strongly dependent on external factors like plant or leaf age or light intensity. In the second vector type, named "split" plastid transformation vector, modules of the expression cassette were distributed on two separate vectors. Upon co-transformation of plastids with these vectors, the complete expression cassette became inserted into the plastome. This result can be explained by successive co-integration of the split vectors and final loop-out recombination of the duplicated sequences. The split vector concept was validated with different vector pairs.

  5. Adenovirus-Mediated Expression of the p14 Fusion-Associated Small Transmembrane Protein Promotes Cancer Cell Fusion and Apoptosis In Vitro but Does Not Provide Therapeutic Efficacy in a Xenograft Mouse Model of Cancer

    PubMed Central

    Wong, Carmen M.; Poulin, Kathy L.; Tong, Grace; Christou, Carin; Kennedy, Michael A.; Falls, Theresa; Bell, John C.; Parks, Robin J.

    2016-01-01

    Adenoviruses (Ads) are used in numerous preclinical and clinical studies for delivery of anti-cancer therapeutic genes. Unfortunately, Ad has a poor ability to distribute throughout a tumor mass after intratumoral injection, and infects cells primarily within the immediate area of the injection tract. Thus, Ad-encoded transgene expression is typically limited to only a small percentage of cells within the tumor. One method to increase the proportion of the tumor impacted by Ad is through expression of fusogenic proteins. Infection of a single cell with an Ad vector encoding a fusogenic protein should lead to syncytium formation with adjacent cells, effectively spreading the effect of Ad and Ad-encoded therapeutic transgenes to a greater percentage of the tumor mass. Moreover, syncytium formation can be cytotoxic, suggesting that such proteins may be effective sole therapeutics. We show that an early region 1 (E1)-deleted Ad expressing reptilian reovirus p14 fusion-associated small transmembrane (FAST) protein caused extensive cell fusion in the replication-permissive 293 cell line and at high multiplicity of infection in non-permissive human lung adenocarcinoma A549 cells in vitro. FAST protein expression in the A549 cancer cell line led to a loss of cellular metabolic activity and membrane integrity, which correlated with induction of apoptosis. However, in an A549 xenograft CD-1 nude mouse cancer model, Ad-mediated FAST gene delivery did not induce detectable cell fusion, reduce tumor burden nor enhance mouse survival compared to controls. Taken together, our results show that, although AdFAST can enhance cancer cell killing in vitro, it is not effective as a sole therapeutic in the A549 tumor model in vivo. PMID:26986751

  6. An Adenovirus Vector Incorporating Carbohydrate Binding Domains Utilizes Glycans for Gene Transfer

    PubMed Central

    Nakayama, Masaharu; Ak, Ferhat; Ugai, Hideyo; Curiel, David T.

    2013-01-01

    Background Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. Methodology/Principal Findings As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. Conclusions/Significance These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers. PMID:23383334

  7. Epigenetic mechanisms in human adenovirus type 12 oncogenesis.

    PubMed

    Doerfler, Walter

    2009-06-01

    For the past 30 years, my laboratory has concentrated its work on demonstrating that the epigenetic consequences of foreign DNA insertion into established mammalian genomes -de novo DNA methylation of the integrate and alterations of methylation patterns across the recipient genome - are essential elements in setting the stage towards oncogenic transformation. We have primarily studied human adenovirus type 12 (Ad12) which induces undifferentiated tumors in Syrian hamsters (Mesocricetus auratus) either at the site of subcutaneous Ad12 injection or intraperitoneally upon intramuscular injection. Up to 90% of the hamsters injected with Ad12 develop tumors within 3-6 weeks. Integration of foreign DNA, its de novo methylation, and the consequences of insertion on the cellular methylation and transcription profiles have been studied in detail. While viral infections are a frequent source of foreign genomes entering mammalian and other hosts and often their genomes, we have also pursued the fate of food-ingested foreign DNA in the mouse organism. The persistence of this DNA in the animals is transient and there is no evidence for the expression or germ line fixation of foreign DNA. Nevertheless, the occasional cell that carries integrated genomes from that foreign source deserves the oncologist's sustained interest.

  8. Adenoviruses in the immunocompromised host.

    PubMed Central

    Hierholzer, J C

    1992-01-01

    Adenoviruses are among the many pathogens and opportunistic agents that cause serious infection in the congenitally immunocompromised, in patients undergoing immunosuppressive treatment for organ and tissue transplants and for cancers, and in human immunodeficiency virus-infected patients. Adenovirus infections in these patients tend to become disseminated and severe, and the serotypes involved are clustered according to the age of the patient and the nature of the immunosuppression. Over 300 adenovirus infections in immunocompromised patients, with an overall case fatality rate of 48%, are reviewed in this paper. Children with severe combined immunodeficiency syndrome and other primary immunodeficiencies are exposed to the serotypes of subgroups B and C that commonly infect young children, and thus their infections are due to types 1 to 7 and 31 of subgenus A. Children with bone marrow and liver transplants often have lung and liver adenovirus infections that are due to an expanded set of subgenus A, B, C, and E serotypes. Adults with kidney transplants have viruses of subgenus B, mostly types 11, 34, and 35, which cause cystitis. This review indicates that 11% of transplant recipients become infected with adenoviruses, with case fatality rates from 60% for bone marrow transplant patients to 18% for renal transplant patients. Patients with AIDS become infected with a diversity of serotypes of all subgenera because their adult age and life-style expose them to many adenoviruses, possibly resulting in antigenically intermediate strains that are not found elsewhere. Interestingly, isolates from the urine of AIDS patients are generally of subgenus B and comprise types 11, 21, 34, 35, and intermediate strains of these types, whereas isolates from stool are of subgenus D and comprise many rare, new, and intermediate strains that are untypeable for practical purposes. It has been estimated that adenoviruses cause active infection in 12% of AIDS patients and that 45% of

  9. Leishmania pifanoi: kinetics of messenger RNA expression during amastigote to promastigote transformation in vitro.

    PubMed

    Campbell, S M; Rainey, P M

    1993-08-01

    Conditions have been developed which induce axenically grown Leishmania pifanoi amastigotes to transform into the promastigote stage in a highly reproducible fashion. Transformation was induced by a temperature shift from 31 to 22 degrees C, was inhibited by high cell concentration (> or = 40 x 10(6) cells/ml), and was unaffected by pH from 5.5-7.2. Morphologic transformation was first evident at 8 hr after induction, had occurred in > 50% of cells by 24 hr, and was > 90% complete by 48 hr. This system enabled study of the kinetics of mRNA expression during the transformation of Leishmania. The differentially expressed mRNAs for ATPase 1a and 1b, alpha- and beta-tubulin, P100/11E, Pro-1, and pLm 2, 7, 14, and 16 exhibited complex patterns of temporal expression, suggesting a highly regulated process. Differentiation on the biochemical level was evident within an hour and continued throughout the course of morphologic transformation. In addition, transformed L. pifanoi promastigotes in the plateau growth phase expressed genes characteristic of metacyclic promastigotes. Axenically cultured L. pifanoi should provide an excellent model for the study of differentiation in Leishmania.

  10. Expression of Foreign Genes Demonstrates the Effectiveness of Pollen-Mediated Transformation in Zea mays

    PubMed Central

    Yang, Liyan; Cui, Guimei; Wang, Yixue; Hao, Yaoshan; Du, Jianzhong; Zhang, Hongmei; Wang, Changbiao; Zhang, Huanhuan; Wu, Shu-Biao; Sun, Yi

    2017-01-01

    Plant genetic transformation has arguably been the core of plant improvement in recent decades. Efforts have been made to develop in planta transformation systems due to the limitations present in the tissue-culture-based methods. Herein, we report an improved in planta transformation system, and provide the evidence of reporter gene expression in pollen tube, embryos and stable transgenicity of the plants following pollen-mediated plant transformation with optimized sonication treatment of pollen. The results showed that the aeration at 4°C treatment of pollen grains in sucrose prior to sonication significantly improved the pollen viability leading to improved kernel set and transformation efficiency. Scanning electron microscopy observation revealed that the removal of operculum covering pollen pore by ultrasonication might be one of the reasons for the pollen grains to become competent for transformation. Evidences have shown that the eGfp gene was expressed in the pollen tube and embryos, and the Cry1Ac gene was detected in the subsequent T1 and T2 progenies, suggesting the successful transfer of the foreign genes to the recipient plants. The Southern blot analysis of Cry1Ac gene in T2 progenies and PCR-identified Apr gene segregation in T2 seedlings confirmed the stable inheritance of the transgene. The outcome illustrated that the pollen-mediated genetic transformation system can be widely applied in the plant improvement programs with apparent advantages over tissue-culture-based transformation methods. PMID:28377783

  11. Altered gene expression and miRNA expression associated with cancerous IEC-6 cell transformed by MNNG.

    PubMed

    Zhang, Bo; Wang, Xukai; Wang, Yan

    2009-04-28

    Tumorigenesis is thought to be the consequence of gene mutation and disordered gene expression. However, the detailed molecular mechanism underlying the development and progress of colon cancer have not been elucidate completely. This study aimed to find out the genes associated with cancer biological pathways involved in transformation and tumorigenesis. Normal intestinal cell line 6 (IEC-6) cells were transformed to cancer cells by treatment with cancerogenic agent of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and Phorbol 12-myristate 13 acetate (PMA). Then we investigated the altered gene expression of transformed IEC-6 cells by the microarray containing 113 genes associated with cancer pathway. Also the altered miRNAs of transformed IEC-6 cells were analyzed by array hybridization (miRCURY Array v9.2, Exiqon). The levels of acetylated histone H3 in transformed IEC-6 cells was evaluated by western blot. Cell proliferation was significantly increased as IEC-6 cells were transformed and tumor xenografts could be detected in animals as transformed IEC-6 cells were inoculated subcutaneously in nude mice. Result of microarray showed nine genes were increased and two decreased, as well as 13 miRNA were increased and 97 decreased. Verification by real-time PCR implies that the data obtained from microarray analysis were reliable. Western blot showed the levels of acetylated histone H3 were increased dramatically after MNNG/PMA treatment. Our results showed many important biological pathways and miRNAs were involved in transformation and tumorigenesis of IEC-6 cells, which suggested the transformation of normal cells was involved with large mount of genetic and epigenetic variation.

  12. Six-Month Assessment of a Phase I Trial of Angiogenic Gene Therapy for the Treatment of Coronary Artery Disease Using Direct Intramyocardial Administration of an Adenovirus Vector Expressing the VEGF121 cDNA

    PubMed Central

    Rosengart, Todd K.; Lee, Leonard Y.; Patel, Shailen R.; Kligfield, Paul D.; Okin, Peter M.; Hackett, Neil R.; Isom, O. Wayne; Crystal, Ronald G.

    1999-01-01

    Objective To summarize the 6-month follow-up of a cohort of patients with clinically significant coronary artery disease who received direct myocardial injection of an E1−E3− adenovirus (Ad) gene transfer vector (AdGVVEGF121.10) expressing the human vascular endothelial growth factor (VEGF) 121 cDNA to induce therapeutic angiogenesis. Background Therapeutic angiogenesis describes a novel approach to the treatment of vascular occlusive disease that uses the administration of growth factors known to induce neovascularization of ischemic tissues. Methods Direct myocardial injection of AdGVVEGF121.10 into an area of reversible ischemia was carried out in 21 patients as an adjunct to conventional coronary artery bypass grafting (group A, n = 15) or as sole therapy using a minithoracotomy (group B, n = 6). Results No evidence of systemic or cardiac-related adverse events related to vector administration was observed up to 6 months after therapy. Trends toward improvement in angina class and exercise treadmill testing at 6-month follow-up in the sole therapy group suggest the effects of this therapy are persistent for ≥6 months. Conclusions This study suggests that direct myocardial administration of AdGVVEGF121.10 appears to be well tolerated in patients with clinically significant coronary artery disease. Initiation of phase II evaluation of this therapy appears warranted. PMID:10522716

  13. The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection.

    PubMed Central

    Tollefson, A E; Scaria, A; Saha, S K; Wold, W S

    1992-01-01

    We have reported that an 11,600-MW (11.6K) protein is coded by region E3 of adenovirus. We have now prepared two new antipeptide antisera that have allowed us to characterize this protein further. The 11.6K protein migrates as multiple diffuse bands having apparent Mws of about 14,000, 21,000, and 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting as well as virus mutants with deletions in the 11.6K gene were used to show that the various gel bands represent forms of 11.6K. The 11.6K protein was synthesized in very low amounts during early stages of infection, from the scarce E3 mRNAs d and e which initiate from the E3 promoter. However, 11.6K was synthesized very abundantly at late stages of infection, approximately 400 times the rate at early stages, from new mRNAs termed d' and e'. Reverse transcriptase-polymerase chain reaction and RNA blot experiments indicated that mRNAs d' and e' had the same body (the coding portion) and the same middle exon (the y leader) as early E3 mRNAs d and e, but mRNAs d' and e' were spliced at their 5' termini to the major late tripartite leader which is found in all mRNAs in the major late transcription unit. mRNAs d' and e' and the 11.6K protein were the only E3 mRNAs and protein that were scarce early and were greatly amplified at late stages of infection. This suggests that specific cis- or trans-acting sequences may function to enhance the splicing of mRNAs d' and e' at late stages of infection and perhaps to suppress the splicing of mRNAs d and e at early stages of infection. We propose that the 11.6K gene be considered not only a member of region E3 but also a member of the major late transcription unit. Images PMID:1316473

  14. Therapy of Breast Cancers Using Conditionally Replicating Adenovirus

    DTIC Science & Technology

    2005-08-01

    virotherapy on breast cancer cells in vitro. We have developed a CRAd using the fit-I promoter element for specific EIA gene expression (CRAdflt), RGD...replicating adenoviruses (CRAd) and investigate effects of CRAd virotherapy on endothelial cells and breast cancer cells in vitro. Vascular targeting...determined the capacity of CRAdRGDflt-mda-7 virotherapy to induce breast cancer cell death. To verify the levels of MDA-7/IL-24 protein expression in vitro

  15. TRANSFORMATION

    SciTech Connect

    LACKS,S.A.

    2003-10-09

    Transformation, which alters the genetic makeup of an individual, is a concept that intrigues the human imagination. In Streptococcus pneumoniae such transformation was first demonstrated. Perhaps our fascination with genetics derived from our ancestors observing their own progeny, with its retention and assortment of parental traits, but such interest must have been accelerated after the dawn of agriculture. It was in pea plants that Gregor Mendel in the late 1800s examined inherited traits and found them to be determined by physical elements, or genes, passed from parents to progeny. In our day, the material basis of these genetic determinants was revealed to be DNA by the lowly bacteria, in particular, the pneumococcus. For this species, transformation by free DNA is a sexual process that enables cells to sport new combinations of genes and traits. Genetic transformation of the type found in S. pneumoniae occurs naturally in many species of bacteria (70), but, initially only a few other transformable species were found, namely, Haemophilus influenzae, Neisseria meningitides, Neisseria gonorrheae, and Bacillus subtilis (96). Natural transformation, which requires a set of genes evolved for the purpose, contrasts with artificial transformation, which is accomplished by shocking cells either electrically, as in electroporation, or by ionic and temperature shifts. Although such artificial treatments can introduce very small amounts of DNA into virtually any type of cell, the amounts introduced by natural transformation are a million-fold greater, and S. pneumoniae can take up as much as 10% of its cellular DNA content (40).

  16. ALDH1 and podoplanin expression patterns predict the risk of malignant transformation in oral leukoplakia

    PubMed Central

    Habiba, Umma; Hida, Kyoko; Kitamura, Tetsuya; Matsuda, Aya Yanagawa; Higashino, Fumihiro; Ito, Yoichi M.; Ohiro, Yoichi; Totsuka, Yasunori; Shindoh, Masanobu

    2017-01-01

    Oral leukoplakia (OL) is a clinically diagnosed preneoplastic lesion of the oral cavity with an increased oral cancer risk. However, the risk of malignant transformation is still difficult to assess. The objective of the present study was to examine the expression patterns of aldehyde dehydrogenase 1 (ALDH1) and podoplanin in OL, and to determine their roles in predicting oral cancer development. In the present study, the expression patterns of ALDH1 and podoplanin were determined in samples from 79 patients with OL. The association between protein expression and clinicopathological parameters, including oral cancer-free survival, was analyzed during a mean follow-up period of 3.4 years. Expression of ALDH1 and podoplanin was observed in 61 and 67% patients, respectively. Kaplan-Meier analysis demonstrated that the expression of the proteins was correlated with the risk of progression to oral cancer. Multivariate analysis revealed that expression of ALDH1 and podoplanin was associated with 3.02- and 2.62-fold increased risk of malignant transformation, respectively. The malignant transformation risk of OL was considerably higher in cases with expression of both proteins. Point-prevalence analysis revealed that 66% of patients with co-expression of ALDH1 and podoplanin developed oral cancer. Taken together, our data indicate that ALDH1 and podoplanin expression patterns in OL are associated with oral cancer development, suggesting that ALDH1 and podoplanin may be useful biomarkers to identify OL patients with a substantially high oral cancer risk. PMID:28123562

  17. ALDH1 and podoplanin expression patterns predict the risk of malignant transformation in oral leukoplakia.

    PubMed

    Habiba, Umma; Hida, Kyoko; Kitamura, Tetsuya; Matsuda, Aya Yanagawa; Higashino, Fumihiro; Ito, Yoichi M; Ohiro, Yoichi; Totsuka, Yasunori; Shindoh, Masanobu

    2017-01-01

    Oral leukoplakia (OL) is a clinically diagnosed preneoplastic lesion of the oral cavity with an increased oral cancer risk. However, the risk of malignant transformation is still difficult to assess. The objective of the present study was to examine the expression patterns of aldehyde dehydrogenase 1 (ALDH1) and podoplanin in OL, and to determine their roles in predicting oral cancer development. In the present study, the expression patterns of ALDH1 and podoplanin were determined in samples from 79 patients with OL. The association between protein expression and clinicopathological parameters, including oral cancer-free survival, was analyzed during a mean follow-up period of 3.4 years. Expression of ALDH1 and podoplanin was observed in 61 and 67% patients, respectively. Kaplan-Meier analysis demonstrated that the expression of the proteins was correlated with the risk of progression to oral cancer. Multivariate analysis revealed that expression of ALDH1 and podoplanin was associated with 3.02- and 2.62-fold increased risk of malignant transformation, respectively. The malignant transformation risk of OL was considerably higher in cases with expression of both proteins. Point-prevalence analysis revealed that 66% of patients with co-expression of ALDH1 and podoplanin developed oral cancer. Taken together, our data indicate that ALDH1 and podoplanin expression patterns in OL are associated with oral cancer development, suggesting that ALDH1 and podoplanin may be useful biomarkers to identify OL patients with a substantially high oral cancer risk.

  18. Translation of adenovirus 2 late mRNAs microinjected into cultured African green monkey kidney cells

    SciTech Connect

    Richardson, W.D.; Anderson, C.W.

    1984-08-01

    Adenovirus 2-infected monkey cells fail to synthesize fiber, a 62,000 M/sub r/ virion polypeptide expressed at late times in productively infected cells. Yet these cells contain fiber mRNA that, after isolation, can be translated in vitro. The reason for the failure of monkey cells to translate fiber mRNA has been approached by microinjecting adenovirus mRNA into the cytoplasm of cultured monkey cells. Late adenovirus 2 mRNA, isolated from infected HeLa cells, was efficiently expressed when microinjected into the African green monkey kidney cell line CV-C. Expressed viral proteins identified by immunoprecipitation included the adenovirus fiber polypeptide. This result demonstrates that the monkey cell translational apparatus is capable of recognizing and expressing functional adenovirus mRNA. Microinjection of late virus mRNA into cells previously infected with wild-type adenovirus 2 failed to increase significantly the yield of infectious virus. 26 references, 2 figures, 1 table.

  19. Adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids.

    PubMed

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H; Haydon, Rex C; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.

  20. CEACAM1: Expression and Role in Melanocyte Transformation

    PubMed Central

    Turcu, Gabriela; Ion, Daniela Adriana; Brînzea, Alice; Cioplea, Mirela Daniela; Jilaveanu, Lucia Beatrice

    2016-01-01

    Metastases represent the main cause of death in melanoma patients. Despite the current optimized targeted therapy or immune checkpoint inhibitors the treatment of metastatic melanoma is unsatisfactory. Because of the poor prognosis of advanced melanoma there is an urgent need to identify new biomarkers to differentiate melanoma cells from normal melanocytes, to stratify patients according to their risk, and to identify subgroups of patients that require close follow-up or more aggressive therapy. Furthermore, melanoma progression has been associated with the dysregulation of cell adhesion molecules. We have reviewed the literature and have discussed the important role of the expression of the carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) in the development of melanoma. Thus, novel insights into CEACAM1 may lead to promising strategies in melanoma treatment, in monitoring melanoma patients, in assessing the response to immunotherapy, and in completing the standard immunohistochemical panel used in melanoma examination. PMID:27642217

  1. Temporal Control of Transforming Growth Factor (TGF) - Betal Expression on Mammary Cell Multistep Transformation

    DTIC Science & Technology

    2000-10-01

    phosphorylation of Smad2 tumors, EMT appears to be initiated by TGF-P produced and Smad3 at specific Erk consensus sites in the linker by peritumoral host...1243-1252. linker region of Smad2 and Smad3 , which, in turn, inhibit Smad accumula- Inhibition of autocrine TGF-j signaling, by expression of dominant...mediated mostly by TGF-P1 and TGF-j2 are potent immunosuppressants the receptor specific Smad2 and Smad3 proteins [47,48], [73]. Thus, elevated levels

  2. Cadophora finlandia and Phialocephala fortinii: Agrobacterium-mediated transformation and functional GFP expression.

    PubMed

    Gorfer, Markus; Klaubauf, Sylvia; Bandian, Dragana; Strauss, Joseph

    2007-07-01

    Hygromycin B resistance was transferred to the sterile mycelia of Cadophora finlandia and Phialocephala fortinii by co-cultivation with Agrobacterium tumefaciens. Constitutively expressed green fluorescent protein (GFP) was also introduced using the same vector. Confocal laser scanning microscopy (CLSM) revealed strong fluorescence of transformants. Both traits were mitotically stable during one year of subculturing on non-selective growth medium. Southern blot analysis showed that the majority of the transformants contained single-copy integrations at random sites in the genome.

  3. RNAi suppressor P19 can be broadly exploited for enhanced adenovirus replication and microRNA knockdown experiments.

    PubMed

    Rauschhuber, Christina; Mueck-Haeusl, Martin; Zhang, Wenli; Nettelbeck, Dirk M; Ehrhardt, Anja

    2013-01-01

    RNA interference (RNAi) is a key regulator of various biological systems including viral infection. Within a virus life cycle gene products can be modulated by the RNA interference (RNAi) pathway which can crucially impact productive virus replication. Herein we explored the RNA interference suppressor protein P19 derived from a plant virus and we found that P19 enhanced adenovirus replication up to 100-fold. Critical factors responsible for this observation were overexpression of adenovirus encoded genes on mRNA and protein levels. To investigate the impact of this phenomenon on recombinant viruses, we exploited its feasibility for therapeutic and genomic applications. We found that P19 significantly increased recombinant adenovirus yields enabling up-scaling for preclinical and clinical studies. Moreover, adenoviruses possessed significantly higher oncolytic activity by expression of P19. Finally, we show that introducing a p19 expression cassette into high-capacity adenovirus provides a strategy to analyze RNAi knockdown in a tissue-specific manner.

  4. Adenovirus-receptor interaction with human lymphocytes.

    PubMed

    Mentel, R; Döpping, G; Wegner, U; Seidel, W; Liebermann, H; Döhner, L

    1997-03-01

    Lymphocytes play a key role in cell-mediated immunity and are host cells for several viral and bacterial pathogens. Their importance in adenovirus (Ad) infections is not yet fully understood. The initial event, the attachment of Ad to lymphocytes and their subsets, was examined using flow cytometry. The study included analysis of stimulated T cells in binding assays with FITC-labeled Ad fiber. The results confirm that native peripheral lymphocytes express very small amounts of Ad receptors. Stimulation with PHA and interleukin 2 induced the expression. The presence of Ad DNA as a sign of internalization in stimulated cells was demonstrated using the polymerase chain reaction. The findings suggest that lymphocytes after stimulation can turn into target cells for Ad. This is particularly important if there are indications for persistence of Ad, and in the case of immunocompromised patients severe, life-threatening diseases can develop.

  5. Vaccine Design: Replication-Defective Adenovirus Vectors.

    PubMed

    Zhou, Xiangyang; Xiang, Zhiquan; Ertl, Hildegund C J

    2016-01-01

    Replication-defective adenovirus (Ad) vectors were initially developed for gene transfer for correction of genetic diseases. Although Ad vectors achieved high levels of transgene product expression in a variety of target cells, expression of therapeutic proteins was found to be transient as vigorous T cell responses directed to components of the vector as well as the transgene product rapidly eliminate Ad vector-transduced cells. This opened the use of Ad vectors as vaccine carriers and by now a multitude of preclinical as well as clinical studies has shown that Ad vectors induce very potent and sustained transgene product-specific T and B cell responses. This chapter provides guidance on developing E1-deleted Ad vectors based on available viral molecular clones. Specifically, it describes methods for cloning, viral rescue and purification as well as quality control studies.

  6. E1A RNA transcripts amplify adenovirus-mediated tumor reduction.

    PubMed

    Dion, L D; Goldsmith, K T; Strong, T V; Bilbao, G; Curiel, D T; Garver, R I

    1996-11-01

    Previous work by this group has established that E1-defective, recombinant adenoviruses can be replication-enabled by the codelivery of a plasmid encoding the deleted E1 functions, a strategy now designated conditional replication-enablement system for adenovirus (CRESA). In the studies reported here, the original replication-enabling plasmid was replaced by two separate plasmids that encoded the necessary E1A and E1B functions, respectively. An RNA transcript encoding the requisite E1A functions was shown to substitute functionally for the E1A plasmid without significant loss of new adenovirus production in in vitro experiments. No replication competent adenovirus was detectable in the cells treated with the plasmids, or the RNA and plasmid combinations. Subcutaneous human tumor nodules containing a fraction of cells cotransduced with the replication-enabling RNA + DNA and an adenovirus containing a herpes simplex virus thymidine kinase (HSVtk) expression cassette were reduced to a greater extent than control nodules containing the same fraction of cells cotransduced with the virus and an irrelevant plasmid. These experiments show that an E1-defective adenovirus can be conditionally replication-enabled by an RNA transcript encoding the required E1 functions, and that the replication-enablement is sufficient to produce an augmentation of an adenovirus-mediated therapeutic effect in vivo.

  7. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.

  8. Biobased monoliths for adenovirus purification.

    PubMed

    Fernandes, Cláudia S M; Gonçalves, Bianca; Sousa, Margarida; Martins, Duarte L; Barroso, Telma; Pina, Ana Sofia; Peixoto, Cristina; Aguiar-Ricardo, Ana; Roque, A Cecília A

    2015-04-01

    Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (-20 °C and -80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at -80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

  9. Differential in vitro phenotype pattern, transforming growth factor-beta(1) activity and mRNA expression of transforming growth factor-beta(1) in Apert osteoblasts.

    PubMed

    Locci, P; Baroni, T; Pezzetti, F; Lilli, C; Marinucci, L; Martinese, D; Becchetti, E; Calvitti, M; Carinci, F

    1999-09-01

    The phenotype of Apert osteoblasts differs from that of normal osteoblasts in the accumulation of macromolecules in the extracellular matrix. Apert osteoblasts increase type I collagen, fibronectin and glycosaminoglycans secretion compared with normal osteoblasts. Because the extracellular matrix macromolecule accumulation is greatly modulated by transforming growth factor-beta(1), we examined the ability of normal and Apert osteoblasts to secrete transforming growth factor-beta(1) by CCL-64 assay and to produce transforming growth factor-beta(1 )by analysis of the mRNA expression of transforming growth factor-beta(1). Northern blot analysis revealed an increased amount of transforming growth factor-beta(1) mRNA expression in Apert osteoblasts compared with normal ones. Moreover, the level of the active transforming growth factor-beta(1) isoform was higher in Apert than in normal media. In pathologic cells, the increase in transforming growth factor-beta(1) gene expression was associated with a parallel increase in the factor secreted into the medium. The level of transforming growth factor-beta(1) was decreased by the addition of basic fibroblast growth factor. Transforming growth factor-beta(1) is controlled temporally and spatially during skeletal tissue development and produces complex stimulatory and inhibitory changes in osteoblast functions. We hypothesise that in vitro differences between normal and Apert osteoblasts may be correlated to different transforming growth factor-beta(1) cascade patterns, probably due to an altered balance between transforming growth factor-beta(1) and basic fibroblast growth factor.

  10. Transient GUS Gene Expression in Date Palm Fruit Using Agroinjection Transformation Technique.

    PubMed

    Solliman, Mohei El-Din M; Mohasseb, Hebaallah A; Al-Khateeb, Abdullatif A; Al-Khayri, Jameel M; Al-Khateeb, Suliman A

    2017-01-01

    Transient expression of foreign genes in plant tissue is a valuable tool for testing the efficacy of transformation methods. In this work, we present, for the first time, the utilization of agroinjection as an efficient transformation system for gene delivery in date palm fruit. The research utilized Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pRI201-AN-GUS carrying the beta-glucuronidase (GUS) gene, under the control of a CaMV 35S and kanamycin (NPTII) as an antibiotic gene under the control of a NOS promoter. Based on histochemical assay of agroinjected fruit for the GUS gene expressions, this protocol has proved to be an efficient and reliable tool for transgene expression in date palm. PCR for plasmid DNA, extracted from the transformed Agrobacterium, demonstrated the generation of the expected amplicon, corresponding to the GUS gene using GUS primers.

  11. TRANSFORMER

    DOEpatents

    Baker, W.R.

    1959-08-25

    Transformers of a type adapted for use with extreme high power vacuum tubes where current requirements may be of the order of 2,000 to 200,000 amperes are described. The transformer casing has the form of a re-entrant section being extended through an opening in one end of the cylinder to form a coaxial terminal arrangement. A toroidal multi-turn primary winding is disposed within the casing in coaxial relationship therein. In a second embodiment, means are provided for forming the casing as a multi-turn secondary. The transformer is characterized by minimized resistance heating, minimized external magnetic flux, and an economical construction.

  12. Methods for transforming and expression screening of filamentous fungal cells with a DNA library

    DOEpatents

    Teter, Sarah; Lamsa, Michael; Cherry, Joel; Ward, Connie

    2015-06-02

    The present invention relates to methods for expression screening of filamentous fungal transformants, comprising: (a) isolating single colony transformants of a DNA library introduced into E. coli; (b) preparing DNA from each of the single colony E. coli transformants; (c) introducing a sample of each of the DNA preparations of step (b) into separate suspensions of protoplasts of a filamentous fungus to obtain transformants thereof, wherein each transformant contains one or more copies of an individual polynucleotide from the DNA library; (d) growing the individual filamentous fungal transformants of step (c) on selective growth medium, thereby permitting growth of the filamentous fungal transformants, while suppressing growth of untransformed filamentous fungi; and (e) measuring activity or a property of each polypeptide encoded by the individual polynucleotides. The present invention also relates to isolated polynucleotides encoding polypeptides of interest obtained by such methods, to nucleic acid constructs, expression vectors, and recombinant host cells comprising the isolated polynucleotides, and to methods of producing the polypeptides encoded by the isolated polynucleotides.

  13. Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.

    PubMed

    Cheng, Yun-Qing; Yang, Jun; Xu, Feng-Ping; An, Li-Jia; Liu, Jian-Feng; Chen, Zhi-Wen

    2009-12-01

    A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research.

  14. Concordant activity of transgene expression cassettes inserted into E1, E3 and E4 cloning sites in the adenovirus genome

    PubMed Central

    Pham, Linh; Nakamura, Takafumi; Rosales, A. Gabriela; Carlson, Stephanie K.; Bailey, Kent R.; Peng, Kah-Whye; Russell, Stephen J.

    2009-01-01

    Background Expression cassettes can be inserted at several positions into recombinant adenoviral genomes but the implications of this choice for transgene expression level have not been determined. Knowledge of the relative expression levels of transgenes inserted at different sites in the adenoviral genome is of particular significance for transgene expression monitoring approaches that rely on the concordant expression of a marker transgene inserted elsewhere in the viral genome. Methods Three expression cassettes, each comprising a cytomegalovirus promoter driving one of three marker peptides [serum carcinoembryonic antigen (sCEA), beta subunit of human chorionic gonadotropin (βhCG) or human sodium iodide symporter (hNIS)], were inserted into E1, E3 or E4 cloning sites in a recombinant adenoviral vector backbone. High titer stocks of bicistronic adenoviral vectors coding for combinations of marker peptides were prepared. A panel of human cells of various lineages was infected with the vectors and expression ratios of the transgene-encoded proteins were analysed. Serum levels of the soluble proteins and hepatic uptake of radioactive iodine were also compared in vivo in nude rats after intravenous vector infusion. Results High concordance of expression between the inserted transgenes was observed in all of the bicistronic vectors irrespective of whether the expression cassettes were placed in the E1, E3 or E4 regions. Concordance was maintained across multiple cell lineages. In vivo, in athymic rats, blood and urine levels of βhCG were highly concordant with serum levels of sCEA at all timepoints after intravenous infusion of the bicistronic vectors encoding both of these soluble markers. Hepatic radioiodine uptake was concordant with serum CEA concentration in mice infused with a bicistronic vector expressing CEA and NIS. Conclusions The expression level of a given transgene in an adenoviral vector genome can be accurately and quantitatively inferred from the

  15. Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts.

    PubMed Central

    Lin, C S; Aebersold, R H; Kent, S B; Varma, M; Leavitt, J

    1988-01-01

    The phosphoprotein plastin was originally identified as an abundant transformation-induced polypeptide of chemically transformed neoplastic human fibroblasts. This abundant protein is normally expressed only in leukocytes, suggesting that it may play a role in hemopoietic cell differentiation. Protein microsequencing of plastin purified from leukemic T lymphocytes by high-resolution two-dimensional gel electrophoresis produced eight internal oligopeptide sequences. An oligodeoxynucleotide probe corresponding to one of the oligopeptides was used to clone cDNAs from transformed human fibroblasts that encoded the seven other oligopeptides predicted for human plastin. Sequencing and characterization of two cloned cDNAs revealed the existence of two distinct, but closely related, isoforms of plastin--l-plastin, which is expressed in leukocytes and transformed fibroblasts, and t-plastin, which is expressed in normal cells of solid tissues and transformed fibroblasts. The leukocyte isoform l-plastin is expressed in a diverse variety of human tumor cell lines, suggesting that it may be involved in the neoplastic process of some solid human tumors. Images PMID:3211125

  16. Adenovirus-mediated inhibition of SPARC attenuates liver fibrosis in rats.

    PubMed

    Camino, Alejandra M; Atorrasagasti, Catalina; Maccio, Daniela; Prada, Federico; Salvatierra, Edgardo; Rizzo, Miguel; Alaniz, Laura; Aquino, Jorge B; Podhajcer, Osvaldo L; Silva, Marcelo; Mazzolini, Guillermo

    2008-09-01

    The interaction between fibrogenic cells and extracellular matrix plays a role in liver fibrosis, yet the mechanisms are largely unknown. Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that is expressed by hepatic stellate cells and is overexpressed in fibrotic livers. We investigated the in vivo role of SPARC in experimentally induced liver fibrosis in rats. A recombinant adenovirus carrying antisense SPARC was constructed (AdasSPARC). Advanced liver fibrosis was induced in Sprague-Dawley rats by prolonged intraperitoneal administration of thioacetamide. Animals received injections of AdasSPARC or Ad beta gal (control adenovirus) via the tail vein and directly into the liver 1 week after the first dose. The pathological changes in liver tissues and indices of fibrosis were assessed at eight weeks. Expression of SPARC, transforming growth factor (TGF)-beta and alpha-smooth muscle actin were evaluated by quantitative real-time polymerase chain reaction, western blotting, enzyme-linked immunosorbent assay and immunohistochemistry. Hepatic SPARC expression significantly increased during the development of liver fibrosis. AdasSPARC markedly attenuated the development of hepatic fibrosis in rats treated with thiocetamide, as assessed by decreased collagen deposition, lower hepatic content of hydroxyproline and less advanced morphometric stage of fibrosis. AdasSPARC treatment reduced inflammatory activity (Knodell score) and suppressed transdifferentiation of hepatic stellate cell to the myofibroblasts like phenotype in vivo. Furthermore, in vitro inhibition of SPARC on hepatic stellate cells decreases the production of TGF-beta. This is the first study to demonstrate that knockdown of hepatic SPARC expression ameliorates thioacetamide-induced liver fibrosis in rats with chronic liver injury. SPARC is a potential target for gene therapy in liver fibrosis. (c) 2008 John Wiley & Sons, Ltd.

  17. Acute respiratory infection with mouse adenovirus type 1

    PubMed Central

    Weinberg, Jason B.; Stempfle, Gregory S.; Wilkinson, John E.; Younger, John G.; Spindler, Katherine R.

    2005-01-01

    Studies of the pathogenesis of adenovirus respiratory disease are limited by the strict species-specificity of the adenoviruses. Following intranasal inoculation of adult C57BL/6 mice with mouse adenovirus type 1 (MAV-1), we detected MAV-1 early region 3 (E3) and hexon gene expression in the lungs at 7 days post-infection (dpi). We detected MAV-1 E3 protein in the respiratory epithelium 7 dpi. We did not detect viral mRNA or protein at 14 dpi, but MAV-1 DNA was detected by PCR at 21 dpi. Chemokine transcript levels increased between 7 and 14 dpi in the lungs of infected mice. MAV-1 infection induced a patchy cellular infiltrate in lungs at 7 and 14 dpi. This is the first report demonstrating the presence of MAV-1 in the respiratory epithelium of infected mice and describing chemokine responses in the lung induced by MAV-1 respiratory infection. MAV-1 infection of mice has the potential to serve as a model for inflammatory changes seen in human adenovirus respiratory disease. PMID:16054189

  18. Design and synthesis of a peptide-PEG transporter tool for carrying adenovirus vector into cells.

    PubMed

    Maeda, Mitsuko; Kida, Shinya; Hojo, Keiko; Eto, Yusuke; Gaob, Jian-Qing; Kurachi, Shinnosuke; Sekiguchi, Fumiko; Mizuguchi, Hiroyuki; Hayakawa, Takao; Mayumi, Tadanori; Nakagawa, Shinsaku; Kawasaki, Koichi

    2005-02-01

    The adenovirus vector is a promising carrier for the efficient transfer of genes into cells via the coxackie-adenovirus receptor (CAR) and integrins (alphavbeta3 and alphavbeta5). The clinical use of the adenovirus vector remains problematic however. Successful administration of this vector is associated with side effects because antibodies to this vector are commonly found throughout the human body. To make the adenovirus vector practicable for clinical use, it is necessary to design an auxiliary transporter. The present study describes the use of Arg-Gly-Asp(RGD)-related peptide, a peptide that binds to integrins, as an auxiliary transporter to aid efficient transport of adenovirus vector. Furthermore, poly(ethylene glycol) (PEG) was also used as a tool to modify the adenovirus such that the risk of side effects incurred during clinical application was reduced. The present study describes the design, preparation and use of (acetyl-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-(beta)Ala)(2)Lys-PEG-(beta)Ala-Cys-NH(2)[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC] as an efficient peptide-PEG transporter tool for carrying adenovirus vector into cells. (Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC was coupled with 6-maleimidohexanoic acid N-hydroxysuccinimide ester and the resulting 6-[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC-succinimido]hexanoic acid N-hydroxysuccinimide ester reacted with adenovirus. The modified adenovirus with the peptide-PEG hybrid exhibited high gene expression even in a CAR-negative cell line, DC2.4.

  19. Diminished Innate Antiviral Response to Adenovirus Vectors in cGAS/STING-Deficient Mice Minimally Impacts Adaptive Immunity

    PubMed Central

    Anghelina, Daniela; Lam, Eric

    2016-01-01

    ABSTRACT Infection by adenovirus, a nonenveloped DNA virus, induces antiviral innate and adaptive immune responses. Studies of transformed human and murine cell lines using short hairpin RNA (shRNA) knockdown strategies identified cyclic guanine adenine synthase (cGAS) as a pattern recognition receptor (PRR) that contributes to the antiadenovirus response. Here we demonstrate how the cGAS/STING cascade influences the antiviral innate and adaptive immune responses in a murine knockout model. Using knockout bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMMOs), we determined that cGAS and STING are essential to the induction of the antiadenovirus response in these antigen-presenting cells (APCs) in vitro. We next determined how the cGAS/STING cascade impacts the antiviral response following systemic administration of a recombinant adenovirus type 5 vector (rAd5V). Infection of cGAS−/− and STING−/− mice results in a compromised early antiviral innate response compared to that in wild-type (WT) controls: significantly lower levels of beta interferon (IFN-β) secretion, low levels of proinflammatory chemokine induction, and reduced levels of antiviral transcript induction in hepatic tissue. At 24 h postinfection, levels of viral DNA and reporter gene expression in the liver were similar in all strains. At 28 days postinfection, clearance of infected hepatocytes in cGAS or STING knockout mice was comparable to that in WT C57BL/6 mice. Levels of neutralizing anti-Ad5V antibody were modestly reduced in infected cGAS mice. These data support a dominant role for the cGAS/STING cascade in the early innate antiviral inflammatory response to adenovirus vectors. However, loss of the cGAS/STING pathway did not affect viral clearance, and cGAS deficiency had a modest influence on the magnitude of the antiviral humoral immune response to adenovirus infections. IMPORTANCE The detection of viral infection by host sentinel immune cells

  20. Differential effects of adenovirus-p16 on bladder cancer cell lines can be overcome by the addition of butyrate.

    PubMed

    Lee, C T; Seol, J Y; Park, K H; Yoo, C G; Kim, Y W; Ahn, C; Song, Y W; Han, S K; Han, J S; Kim, S; Lee, J S; Shim, Y S

    2001-01-01

    High frequency of p16 alteration and high local recurrence rate of bladder cancer make this cancer an ideal target for p16 gene therapy. However, a low transduction rate of p16 via adenoviral vector causes an inconsistent result. In this study, we have tested adenovirus-p16 in several bladder cancer cell lines and investigated a way of improving the low transduction rate. Adenovirus-p16 showed a strong antitumor effect on bladder cancer cell lines (253J and T24) with strong Coxackie-adenoviral receptor (CAR) expression but little antitumor effect on bladder cancer cell lines (J82 and HT1376) with little CAR expression. In this study, we suggest a simple way of overcoming the differential effects of the adenovirus. The addition of butyrate to media was found to increase the transduction rate of adenovirus remarkably and increase the antitumor effect of adenovirus-p16 in bladder cancer cell lines with little CAR expression. Butyrate effects were related with increased CAR expression on the cell surface as well as increased transgene expression from adenoviral vector. From these observations, application of adenovirus-p16 gene therapy with butyrate can overcome the obstacle of low gene transfer and enhance the antitumor effect of adenovirus-p16 in bladder cancer.

  1. Differential gene expression and clonal selection during cellular transformation induced by adhesion deprivation

    PubMed Central

    2010-01-01

    Background Anchorage independent growth is an important hallmark of oncogenic transformation. Previous studies have shown that when adhesion dependent fibroblasts were prevented from adhering to a substrate they underwent anoikis. In the present study we have demonstrated how anoikis resistant cells gain the transformation related properties with sequential selection of genes. We have proposed this process as a model system for selection of transformed cells from normal cells. Results This report demonstrates that some fibroblasts can survive during late stages of anoikis, at which time they exhibit transformation-associated properties such as in vitro colony formation in soft agar and in vivo subcutaneous tumour formation in nude mice. Cytogenetic characterisation of these cells revealed that they contained a t (2; 2) derivative chromosome and they have a selective survival advantage in non adherent conditions. Gene expression profile indicated that these cells over expressed genes related to hypoxia, glycolysis and tumor suppression/metastasis which could be helpful in their retaining a transformed phenotype. Conclusion Our results reveal some new links between anoikis and cell transformation and they provide a reproducible model system which can potentially be useful to study multistage cancer and to identify new targets for drug development. PMID:21122158

  2. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... identify adenoviruses directly from clinical specimens. The identification aids in the diagnosis of disease caused by adenoviruses and provides epidemiological information on these diseases. Adenovirus infections may cause pharyngitis (inflammation of the throat), acute respiratory diseases, and certain external...

  3. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... identify adenoviruses directly from clinical specimens. The identification aids in the diagnosis of disease caused by adenoviruses and provides epidemiological information on these diseases. Adenovirus infections may cause pharyngitis (inflammation of the throat), acute respiratory diseases, and certain external...

  4. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... identify adenoviruses directly from clinical specimens. The identification aids in the diagnosis of disease caused by adenoviruses and provides epidemiological information on these diseases. Adenovirus infections may cause pharyngitis (inflammation of the throat), acute respiratory diseases, and certain external...

  5. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... identify adenoviruses directly from clinical specimens. The identification aids in the diagnosis of disease caused by adenoviruses and provides epidemiological information on these diseases. Adenovirus infections may cause pharyngitis (inflammation of the throat), acute respiratory diseases, and certain external...

  6. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... identify adenoviruses directly from clinical specimens. The identification aids in the diagnosis of disease caused by adenoviruses and provides epidemiological information on these diseases. Adenovirus infections may cause pharyngitis (inflammation of the throat), acute respiratory diseases, and certain external...

  7. Winter Counts as Transformative Inquiry: The Role of Creative Imagery as an Expression of Adaptive Change

    ERIC Educational Resources Information Center

    Stanger, Nicholas R. G.; Tanaka, Michele T. D.; Tse, Vanessa V.; Starr, Lisa J.

    2013-01-01

    Pre-service teachers face a complex educational context and Transformative Inquiry is a useful approach for negotiating this terrain. We interpret the movement of students via the adaptive cycle put forth in panarchy theory as they engage in the inquiry process through "winter counts", a Plains First Nation tradition, as expressions of…

  8. Adenovirus vector delivery stimulates natural killer cell recognition

    PubMed Central

    Tomasec, Peter; Wang, Eddie C. Y.; Groh, Veronika; Spies, Thomas; McSharry, Brian P.; Aicheler, Rebecca J.; Stanton, Richard J.; Wilkinson, Gavin W. G.

    2007-01-01

    We report that delivery of first-generation replication-deficient adenovirus (RDAd) vectors into primary human fibroblasts is associated with the induction of natural killer (NK) cell-mediated cytolysis in vitro. RDAd vector delivery induced cytolysis by a range of NK cell populations including the NK cell clone NKL, primary polyclonal NK lines and a proportion of NK clones (36 %) in autologous HLA-matched assays. Adenovirus-induced cytolysis was inhibited by antibody blocking of the NK-activating receptor NKG2D, implicating this receptor in this function. NKG2D is ubiquitously expressed on NK cells and CD8+ T cells. Significantly, γ-irradiation of the vector eliminated the effect, suggesting that breakthrough expression from the vector induces at least some of the pro-inflammatory responses of unknown aetiology following the application of RDAd vectors during in vivo gene delivery. PMID:17374753

  9. Vaccination with recombinant adenovirus expressing multi-stage antigens of Toxoplasma gondii by the mucosal route induces higher systemic cellular and local mucosal immune responses than with other vaccination routes

    PubMed Central

    Wang, Ting; Yin, Huiquan; Li, Yan; Zhao, Lingxiao; Sun, Xiahui; Cong, Hua

    2017-01-01

    Toxoplasmosis caused by Toxoplasma gondii, an obligate intracellular protozoan, is a cause of congenital disease and abortion in humans and animals. Various vaccination strategies against toxoplasmosis in rodent models have been used in the past few decades; however, effective vaccines remain a challenge. A recombinant adenovirus vaccine expressing ubiquitin-conjugated multi-stage antigen segments (Ad-UMAS) derived from different life-cycle stages of T. gondii was constructed previously. Here, we compared the immune responses and protection effects in vaccination of mice with Ad-UMAS by five vaccination routes including intramuscular (i.m.), intravenous (i.v.), subcutaneous (s.c.), intraoral (i.o.), and intranasal (i.n.). Much higher levels of T. gondii-specific IgG and IgA antibodies were detected in the sera of the intraoral and intranasal vaccination groups on day 49 compared with controls (p < 0.05). The percentages of CD8+ T-cells in mice immunized intranasally and intraorally were larger than in mice immunized intramuscularly (p < 0.05). The highest level of IL-2 and IFN-γ was detected in the group with nasal immunization, and splenocyte proliferation activity was significantly enhanced in mice immunized via the oral and nasal routes. Furthermore, the higher survival rate (50%) and lower cyst numbers observed in the intraoral and intranasal groups all indicate that Ad-UMAS is far more effective in protecting mice against T. gondii infection via the mucosal route. Ad-UMAS could be an effective and safe mucosal candidate vaccine to protect animals and humans against T. gondii infection. PMID:28367800

  10. Vaccination with recombinant adenovirus expressing multi-stage antigens of Toxoplasma gondii by the mucosal route induces higher systemic cellular and local mucosal immune responses than with other vaccination routes.

    PubMed

    Wang, Ting; Yin, Huiquan; Li, Yan; Zhao, Lingxiao; Sun, Xiahui; Cong, Hua

    2017-01-01

    Toxoplasmosis caused by Toxoplasma gondii, an obligate intracellular protozoan, is a cause of congenital disease and abortion in humans and animals. Various vaccination strategies against toxoplasmosis in rodent models have been used in the past few decades; however, effective vaccines remain a challenge. A recombinant adenovirus vaccine expressing ubiquitin-conjugated multi-stage antigen segments (Ad-UMAS) derived from different life-cycle stages of T. gondii was constructed previously. Here, we compared the immune responses and protection effects in vaccination of mice with Ad-UMAS by five vaccination routes including intramuscular (i.m.), intravenous (i.v.), subcutaneous (s.c.), intraoral (i.o.), and intranasal (i.n.). Much higher levels of T. gondii-specific IgG and IgA antibodies were detected in the sera of the intraoral and intranasal vaccination groups on day 49 compared with controls (p < 0.05). The percentages of CD8(+) T-cells in mice immunized intranasally and intraorally were larger than in mice immunized intramuscularly (p < 0.05). The highest level of IL-2 and IFN-γ was detected in the group with nasal immunization, and splenocyte proliferation activity was significantly enhanced in mice immunized via the oral and nasal routes. Furthermore, the higher survival rate (50%) and lower cyst numbers observed in the intraoral and intranasal groups all indicate that Ad-UMAS is far more effective in protecting mice against T. gondii infection via the mucosal route. Ad-UMAS could be an effective and safe mucosal candidate vaccine to protect animals and humans against T. gondii infection.

  11. Towards a universal vaccine for avian influenza: protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus.

    PubMed

    Boyd, Amy C; Ruiz-Hernandez, Raul; Peroval, Marylene Y; Carson, Connor; Balkissoon, Devanand; Staines, Karen; Turner, Alison V; Hill, Adrian V S; Gilbert, Sarah C; Butter, Colin

    2013-01-11

    Current vaccines targeting surface proteins can drive antigenic variation resulting either in the emergence of more highly pathogenic viruses or of antigenically distinct viruses that escape control by vaccination and thereby persist in the host population. Influenza vaccines typically target the highly mutable surface proteins and do not provide protection against heterologous challenge. Vaccines which induce immune responses against conserved influenza epitopes may confer protection against heterologous challenge. We report here the results of vaccination with recombinant modified Vaccinia virus Ankara (MVA) and Adenovirus (Ad) expressing a fusion construct of nucleoprotein and matrix protein (NP+M1). Prime and boost vaccination regimes were trialled in different ages of chicken and were found to be safe and immunogenic. Interferon-γ (IFN-γ) ELISpot was used to assess the cellular immune response post secondary vaccination. In ovo Ad prime followed by a 4 week post hatch MVA boost was identified as the most immunogenic regime in one outbred and two inbred lines of chicken. Following vaccination, one inbred line (C15I) was challenged with low pathogenic avian influenza (LPAI) H7N7 (A/Turkey/England/1977). Birds receiving a primary vaccination with Ad-NP+M1 and a secondary vaccination with MVA-NP+M1 exhibited reduced cloacal shedding as measured by plaque assay at 7 days post infection compared with birds vaccinated with recombinant viruses containing irrelevant antigen. This preliminary indication of efficacy demonstrates proof of concept in birds; induction of T cell responses in chickens by viral vectors containing internal influenza antigens may be a productive strategy for the development of vaccines to induce heterologous protection against influenza in poultry.

  12. [Gene engineering of the adenovirus vector].

    PubMed

    Kondo, Saki; Terashima, Miho; Fukuda, Hiromitsu; Saito, Izumu; Kanegae, Yumi

    2007-06-01

    The adenovirus vector is very attractive tool not only for the gene therapy but also for the basic sciences. However, because a construction method of this vector had been complex, only limited scientists had constructed and enjoyed the benefits. Recently, various methods were developed and the researchers came to be able to choose an efficient method, which is the COS-TPC method, or a concise procedure, which is the intact-genome transfection method (in vitro ligation method). Here we described not only these methods but also new method to construct the various Ads simultaneously using the recombinase-mediated cassette exchange (RMCE) by the site-specific recombinase. And also we want to refer the possibility to the worth of the vector, especially the vector of the expression-switch.

  13. Adenovirus-mediated brain-derived neurotrophic factor expression regulated by hypoxia response element protects brain from injury of transient middle cerebral artery occlusion in mice.

    PubMed

    Shi, Qindong; Zhang, Pengbo; Zhang, Junfeng; Chen, Xinlin; Lu, Haixia; Tian, Yumei; Parker, T L; Liu, Yong

    2009-11-20

    Some gene expression may be regulated by hypoxia-responsive element (HRE) that is bound by hypoxia-inducible factor-1 (HIF-1) which is up-regulated during cerebral ischemia. To explore ischemia/hypoxia-controlled expression and the neuroprotective effects of brain-derived neurotrophic factor (BDNF) after ischemic brain injury, an adenoviral vector using five copies of hypoxia response element (HRE) in the vascular endothelial growth factor gene to regulate the expression of BDNF gene (Ad5HRE:BDNF) was constructed, and its efficacy was verified for driving BDNF expression in cultured Hela cells under hypoxic condition by ELISA. We found that the concentration of BDNF in the Ad5HRE:BDNF-transfected culture media was 28-fold greater in a hypoxic condition than under normoxia. To examine the effect of Ad5HRE:BDNF on ischemic brain injury in vivo, Ad5HRE:BDNF was injected into right caudate putamen of adult mice 7 days prior to 60 min transient middle cerebral artery occlusion (MCAO). It was found that exogenous BDNF expression was increased in the Ad5HRE-BDNF-treated group and infarct volume of the Ad5HRE:BDNF-treated group at 3 days after MCAO was significantly smaller than that of vehicle- or AdNull-treated groups. Moreover, Ad5HRE:BDNF injection resulted in significantly improved sensorimotor scores 7 days after MCAO and induced a reduction in the number of Fluoro-Jade B-positive neurons and TUNEL-positive cells, compared with vehicle- or AdNull-injection. Our findings suggest that BDNF expression could be regulated in hypoxia/ischemia condition with five copies of HRE and ameliorates ischemic brain injury in a mouse focal cerebral ischemia model.

  14. Replication-competent human adenovirus 11p vectors can propagate in Vero cells

    SciTech Connect

    Gokumakulapalle, Madhuri; Mei, Ya-Fang

    2016-08-15

    The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

  15. Adenovirus Early Proteins and Host Sumoylation

    PubMed Central

    Sohn, Sook-Young

    2016-01-01

    ABSTRACT The human adenovirus genome is transported into the nucleus, where viral gene transcription, viral DNA replication, and virion assembly take place. Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are implicated in the regulation of diverse cellular processes, particularly nuclear events. It is not surprising, therefore, that adenovirus modulates and utilizes the host sumoylation system. Adenovirus early proteins play an important role in establishing optimal host environments for virus replication within infected cells by stimulating the cell cycle and counteracting host antiviral defenses. Here, we review findings on the mechanisms and functional consequences of the interplay between human adenovirus early proteins and the host sumoylation system. PMID:27651358

  16. Adenovirus expressing dual c-Met-specific shRNA exhibits potent antitumor effect through autophagic cell death accompanied by senescence-like phenotypes in glioblastoma cells

    PubMed Central

    Yoo, Ji Young; Choi, Kyeong Sook; Yoon, Mi Jin; Yun, Chae-Ok

    2015-01-01

    c-Met, a cognate receptor tyrosine kinase of hepatocyte growth factor, is overexpressed and/or mutated in number of tumors. Therefore, abrogation of c-Met signaling may serve as potential therapeutic targets. In this study, we generated Ads expressing single shRNA specific to c-Met (shMet) (dl/shMet4 and dl/shMet5) or dual shRNAs specific to c-Met (dl/shMet4+5); and examined the therapeutic potential of these newly engineered Ads in targeting c-Met, and delineated their mechanism of action in vitro and in vivo. Ads expressing shMet induced knock-down in c-Met, and phenotypically resulted in autophagy-like features including appearance of membranousvacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein1 light chain 3 to autophagosomes. Ads expressing shMet also suppressed Akt phosphorylation and increased number of senescence-related gene products including SM22, TGase II, and PAI-1. These changes resulted in inhibition of cell proliferation and G2/M arrest of U343 cells. In vivo, intratumoral injection with dl/shMet4+5 resulted in a significant reduction of tumor growth with corresponding increasing overall survival. Histopathological analysis of these treated tumors revealed that Atg5 was highly up-regulated, indicating the therapeutic induction of autophagy. In sum, these results reveal that autophagic cell death induced by shMet-expressing Ads provide a novel strategy for targeting c-Met-expressing tumors through non-apoptotic mechanism of cell death. PMID:25726528

  17. Recombinant adenovirus-mediated overexpression of 3β-hydroxysteroid-Δ24 reductase

    PubMed Central

    Lu, Xiuli; Jia, Dan; Zhao, Chenguang; Wang, Weiqi; Liu, Ting; Chen, Shuchao; Quan, Xiaoping; Sun, Deliang; Gao, Bing

    2014-01-01

    3β-Hydroxysteroid-Δ24 reductase (DHCR24) is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid β deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human (h) and rat (r). Recombinant Ad-r(h)SYN1-DHCR24 was transfected into AD-293, N2A (mouse neuroblastoma), and MIN6 (mouse pancreatic carcinoma) cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunofluorescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer's disease. PMID:25206847

  18. Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis.

    PubMed Central

    Sheibani, N; Frazier, W A

    1995-01-01

    Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To determine the role of TS1 in regulation of endothelial cell phenotype, we stably transfected bEND.3 cells with a human TS1 expression vector. The cells expressing human TS1 were readily identified by their altered morphology and exhibited a slower growth rate and lower saturation density than the parental bEND.3 cells. The TS1-expressing cells also formed aligned cords of cells instead of clumps or cysts in Matrigel. Moreover, while the bEND.3 cells formed large tumors in nude mice within 48 hr, the TS1-expressing cells failed to form tumors even after 1 month. The TS1-transfected cells expressed transforming growth factor beta mRNA and bioactivity at levels similar to those of the parental or vector-transfected bEND.3 cells, indicating that the effects of TS1 expression are not due to the activation of transforming growth factor beta by TS1. TS1 expression resulted in a > 100-fold decrease in net fibrinolytic (urokinase-type plasminogen activator, uPA) activity due to more plasminogen-activator inhibitor 1 and less uPA secretion. TS1 thus appears to be an important regulator of endothelial cell phenotype required for maintaining the quiescent, differentiated state. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:7624320

  19. [Delivery of secreted placental alkaline phosphatase (SEAP) gene in vitro and in vivo as a component of recombinant avian adenovirus (CELO)].

    PubMed

    Logunov, D Iu; Cherenova, L V; Shmarov, M M; Shashkova, E V; Verkhovskaia, L V; Doronin, K K; Naroditskiĭ, B S

    2002-01-01

    Recombinant adenoviruses capable of expressing the gene of secreted placentary alkaline phosphatase (SEAP) under control of CMV-promoter was obtained on the basis of CELO avian adenovirus and human adenovirus-5 (Ad5) genomes. The efficiency of the CELO vector was determined in experiments with transduction of human (293, A549, and H1299), mouse (B16), and avian (LMH) cell cultures. It was shown in C57BL/6 mice in vivo that SEAP gene is expressed under conditions of intravenous, intranasal, and intratumoral application of recombinant adenovirus CELO-SEAP. The duration of expression of the alkaline phosphatase CELO = SEAP gene in immunocompetent mouse body was 21 days. The level of SEAP gene expression was measured in the allantois fluid of chicken embryo infected with recombinant adenovirus CELO-SEAP.

  20. Expression of immunoglobulin genes in the avian embryo bone marrow revealed by retroviral transformation.

    PubMed

    Benatar, T; Iacampo, S; Tkalec, L; Ratcliffe, M J

    1991-10-01

    Analysis of the early stages of avian B lymphocyte differentiation has been hampered by the low frequency of extra-bursal B lineage cells in sites of hematopoiesis. Consequently, little is known about B lineage precursors prior to their migration into the bursa of Fabricius. Colonization of the bursa typically occurs between about days 8 and 14 of embryonic (e) development, although cells which can colonize the bursa, functionally defined as pre-bursal stem cells, can be demonstrated in embryo bone marrow up until about the time of hatch. As a novel approach to analyzing early stages of avian B lymphocyte development, we show here that transformed B lineage cells can be derived from chick embryo bone marrow after infection in vitro with the replication-defective retrovirus REV-T produced in the context of the non-cytopathic CSV helper virus. Thus, exposure of day 14e-15e chick embryo bone marrow cells to REV-T (CSV) results in the generation of transformed, polyclonal lines of cells. From these lines, cells expressing cell surface immunoglobulin were readily isolated by flow cytometric cell sorting and single cell cloning. Analysis of the phenotype of REV-T(CSV)-transformed clones with a panel of monoclonal antibody reagents demonstrated that transformation by v-rel likely leads to marked changes in cell surface antigen expression. Nonetheless, clones expressing cell surface immunoglobulin expressed apparently normal mRNA for immunoglobulin mu and light chain and contained apparently normal immunoglobulin heavy and light chain gene rearrangements. Furthermore, no evidence for chromosomal deletions or aberrations of the Ig loci was detected among either sIg+ or sIg- REV-T(CSV)-transformed clones.

  1. Time-dependent biodistribution and transgene expression of a recombinant human adenovirus serotype 5-luciferase vector as a surrogate agent for rAd5-FMDV vaccines in cattle

    USDA-ARS?s Scientific Manuscript database

    Replication-defective recombinant adenovirus 5 (rAd5) vectors carrying foot-and-mouth disease virus (FMDV) transgenes elicit a robust immune response to FMDV challenge in cattle; however vaccine function mechanisms are incompletely understood. Recent efforts addressing critical interactions of rAd5 ...

  2. The amino-terminal portion of CD1 of the adenovirus E1A proteins is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse cells.

    PubMed Central

    Duerksen-Hughes, P J; Hermiston, T W; Wold, W S; Gooding, L R

    1991-01-01

    Previous work by our laboratory and others has shown that mouse cells normally resistant to tumor necrosis factor can be made sensitive to the cytokine by the expression of adenovirus E1A. The E1A gene can be introduced by either infection or transfection, and either of the two major E1A proteins, 289R or 243R, can induce this sensitivity. The E1A proteins are multifunctional and modular, with specific domains associated with specific functions. Here, we report that the CD1 domain of E1A is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse C3HA fibroblasts. Amino acids C terminal to residue 60 and N terminal to residue 36 are not necessary for this function. This conclusion is based on 51Cr-release assays for cytolysis in cells infected with adenovirus mutants with deletions in various portions of E1A. These E1A mutants are all in an H5dl309 background and therefore they lack the tumor necrosis factor protection function provided by the 14.7-kilodalton (14.7K) protein encoded by region E3. Western blot (immunoblot) analysis indicated that most of the mutant E1A proteins were stable in infected C3HA cells, although with certain large deletions the E1A proteins were unstable. The region between residues 36 and 60 is included within but does not precisely correlate with domains in E1A that have been implicated in nuclear localization, enhancer repression, cellular immortalization, cell transformation in cooperation with ras, induction of cellular DNA synthesis and proliferation, induction of DNA degradation, and binding to the 300K protein and the 105K retinoblastoma protein. Images PMID:1825340

  3. Adenovirus-mediated hAQP1 expression in irradiated mouse salivary glands causes recovery of saliva secretion by enhancing acinar cell volume decrease.

    PubMed

    Teos, L Y; Zheng, C-Y; Liu, X; Swaim, W D; Goldsmith, C M; Cotrim, A P; Baum, B J; Ambudkar, I S

    2016-07-01

    Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands

  4. Adenovirus infection elevates levels of cellular topoisomerase I.

    PubMed Central

    Chow, K C; Pearson, G D

    1985-01-01

    We have developed a specific, sensitive, and quantitative assay for topoisomerase I, which is based on the formation of a covalent enzyme-DNA intermediate. Our assay measures the quantitative transfer of 32P radioactivity from 32P-labeled DNA to topoisomerase I. Since 32P-labeled topoisomerase molecules are resolved by NaDodSO4/PAGE, HeLa topoisomerase I (100 kDa) and calf thymus topoisomerase I (82 kDa) can be quantitatively assayed in the same reaction mixture. The assay can detect at least 0.3 ng (3 fmol) of topoisomerase I. We have used our assay to measure the levels of topoisomerase I activity in crude extracts of nuclei prepared from uninfected, adenovirus-infected, and adenovirus-transformed human cells. The evidence suggests that an adenovirus early gene product, presumably a protein encoded in early region 1A (E1A), increases cellular topoisomerase I activity at least 10-fold. Immunoblotting analysis with antiserum against calf thymus topoisomerase I shows that the increase in activity is due to an increase in the amount of enzyme. Images PMID:2986107

  5. The Change of Immunoactivity of Dendritic Cells Induced by Mouse 4-1BBL Recombinant Adenovirus

    PubMed Central

    Youlin, Kuang; Xiaodong, Weng; Zhiyuan, Chen; Hengcheng, Zhu; Hui, Chen; Botao, Jiang

    2010-01-01

    Purpose The purpose of this study is to construct a recombinant adenovirus vector carrying mouse 4-1BBL and observe its effects in dendritic cells. Materials and Methods Mouse 4-1BBL cDNA was taken from the plasmid pcDNA3-m4-1BBL and subcloned into adenovirus shuttle plasmid pAdTrack-CMV, and then transformed into competent BJ5183 with plasmid pAdEasy-1. After recombination in E. coli, Ad-4-1BBL was packaged and amplified in HEK 293 cells. The expression of 4-1BBL in Ad-4-1BBL-transfected mouse prostate cancer cell line RM-1 was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. After the co-culture of dendritic cells (DCs) with Ad-4-1BBL-transfected RM-1 cells, interleukin (IL)-6 and IL-12 production were assessed by enzyme-linked immunosorbent assay (ELISA) and co-stimulatary moleculs (CD80 and CD86) on DCs were analyzed by flow cytometry. Results The levels of IL-6 (3,960 pg/mL) and IL-12 (249 pg/mL) production in Ad-m4-1BBL-pulsed DCs were more than those in none-pulsed DCs. The differences were statistically significant (p < 0.05). The expression of co-stimulatary molecules (CD80 and CD86) was up-regulated in Ad-m4-1BBL-pulsed DCs. Conclusion The results indicated the recombinant mouse 4-1BBL can effectively activate DCs. PMID:20499429

  6. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

    PubMed Central

    Gao, Meili; Li, Yongfei; Xue, Xiaochang; Wang, Xianfeng; Long, Jiangang

    2012-01-01

    Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed. PMID:23093835

  7. Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) expression is epigenetically regulated by one-carbon metabolism in invasive duct cell carcinoma of breast.

    PubMed

    Naushad, Shaik Mohammad; Prayaga, Aruna; Digumarti, Raghunadha Rao; Gottumukkala, Suryanarayana Raju; Kutala, Vijay Kumar

    2012-02-01

    In view of recent studies highlighting the prognostic relevance of expression and CpG island methylator phenotype (CIMP) of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) in invasive duct cell carcinoma (IDC), we hypothesized in this article that impaired one-carbon metabolism might influence CIMP phenotype of BNIP3. In order to substantiate the prognostic relevance of BNIP3, we explored its association with 8-oxo-2'deoxyguanosine (8-oxodG), a marker of oxidative stress with prognostic relevance. BNIP3 expression and CIMP phenotype were studied using semi-quantitative RT-PCR and combined bisulfite restriction analysis (COBRA), respectively, in 56 IDC tumors. Eight polymorphisms in one-carbon metabolism were studied using PCR-RFLP and PCR-AFLP approaches. 8-oxodG was measured using competitive ELISA kit. BNIP3 was found to be upregulated in IDC (cases vs. controls: 0.94 ± 0.05 vs. 0.18 ± 0.08, P < 0.0001). COBRA analysis confirmed hypomethylation of BNIP3 promoter CpG island in these cases. CIMP phenotype of BNIP3 showed positive association with tubule formation (P = 0.034) and methionine synthase reductase (MTRR) A66G (P = 0.002); inverse association with cytosolic serine hydroxyl methyltransferase (cSHMT) C1420T (P < 0.005) and 8-oxodG (<10% vs. >10% methylation: 7.24 ± 2.77 ng/ml vs. 4.42 ± 2.93 ng/ml, P < 0.0005); and no association with nuclear pleomorphism or mitotic index or ER, PR, and HER statuses. Synergistic effect of MTR A2756G and MTRR A66G variants on BNIP3 hypermethylator phenotype was clearly evident (P < 0.0007). MTRR A66G and cSHMT C1420T polymorphisms influence CIMP phenotype of BNIP3, thus epigenetically regulating BNIP3 in breast cancer. The linear association between BNIP3 and 8-oxodG substantiates the role of BNIP3 as redox sensor as well as prognostic marker in breast cancer.

  8. Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

    PubMed Central

    Li, Wenyan; Shen, Jun

    2016-01-01

    Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction. PMID:28116172

  9. Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

    PubMed Central

    Wang, Gui-Fang; Qi, Bing; Tu, Lei-Lei; Liu, Lian; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2016-01-01

    AIM To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively. RESULTS The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia. PMID:27672590

  10. Ganciclovir Inhibits Human Adenovirus Replication and Pathogenicity in Permissive Immunosuppressed Syrian Hamsters

    PubMed Central

    Ying, Baoling; Tollefson, Ann E.; Spencer, Jacqueline F.; Balakrishnan, Lata; Dewhurst, Stephen; Capella, Cristina; Buller, R. Mark L.; Wold, William S. M.

    2014-01-01

    Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment. PMID:25224011

  11. Ganciclovir inhibits human adenovirus replication and pathogenicity in permissive immunosuppressed Syrian hamsters.

    PubMed

    Ying, Baoling; Tollefson, Ann E; Spencer, Jacqueline F; Balakrishnan, Lata; Dewhurst, Stephen; Capella, Cristina; Buller, R Mark L; Toth, Karoly; Wold, William S M

    2014-12-01

    Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment.

  12. Purification and characterization of adenovirus core protein VII: a histone-like protein that is critical for adenovirus core formation.

    PubMed

    Sharma, Gaurav; Moria, Nithesh; Williams, Martin; Krishnarjuna, Bankala; Pouton, Colin W

    2017-07-01

    Adenovirus protein VII is a highly cationic core protein that forms a nucleosome-like structure in the adenovirus core by condensing DNA in combination with protein V and mu. It has been proposed that protein VII could condense DNA in a manner analogous to mammalian histones. Due to the lack of an expression and purification protocol, the interactions between protein VII and DNA are poorly understood. In this study we describe methods for the purification of biologically active recombinant protein VII using an E. coli expression system. We expressed a cleavable fusion of protein VII with thioredoxin and established methods for purification of this fusion protein in denatured form. We describe an efficient method for resolving the cleavage products to obtain pure protein VII using hydroxyapatite column chromatography. Mass spectroscopy data confirmed its mass and purity to be 19.4 kDa and >98 %, respectively. Purified recombinant protein VII spontaneously condensed dsDNA to form particles, as shown by dye exclusion assay, electrophoretic mobility shift assay and nuclease protection assay. Additionally, an in vitro bioluminescence assay revealed that protein VII can be used to enhance the transfection of mammalian cells with lipofectamine/DNA complexes. The availability of recombinant protein VII will facilitate future studies of the structure of the adenovirus core. Improved understanding of the structure and function of protein VII will be valuable in elucidating the mechanism of adenoviral DNA condensation, defining the morphology of the adenovirus core and establishing the mechanism by which adenoviral DNA enters the nucleus.

  13. Critical Role for Arginine Methylation in Adenovirus-Infected Cells▿

    PubMed Central

    Iacovides, Demetris C.; O'Shea, Clodagh C.; Oses-Prieto, Juan; Burlingame, Alma; McCormick, Frank

    2007-01-01

    During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection. PMID:17686851

  14. Infectivity-selective Oncolytic Adenovirus Developed by High-throughput Screening of Adenovirus-formatted Library

    PubMed Central

    Miura, Yoshiaki; Yamasaki, Satoshi; Davydova, Julia; Brown, Eric; Aoki, Kazunori; Vickers, Selwyn; Yamamoto, Masato

    2013-01-01

    Adenovirus (Ad) is a potent gene-delivery vehicle and has frequently been used for designing oncolytic viruses. However, lack of selectivity on infection has hampered the achievement of sufficient in vivo efficiency. Here, we developed a novel oncolytic virus system, infectivity-selective oncolytic adenovirus (ISOAd), via direct high-throughput screening of a high-diversity targeting-ligand library in adenoviral format. Through our newly designed rescue virus system, the high-diversity Ad library carrying the random seven amino acid sequences ligand-library in the AB-loop of its fiber-knob region (5 × 109 diversity) was successfully generated. During the screening of this library with the cells expressing the target molecule (mesothelin, MSLN), the AB-loop sequence of the virus clones converged to one dominant sequence and a novel MSLN-targeting sequence was isolated. The virus with the isolated motif showed selective infectivity to MSLN-positive cells in vitro. In vivo, it exhibited a selective and potent antitumor effect resulted from the viral replication in MSLN-positive xenografts. The ISOAd is a novel class of oncolytic Ad, which has selectivity at the step of transduction. The selectivity at the stage of infection can open new perspectives in oncolytic Ad therapy for various diseases. PMID:23032977

  15. An Update on Canine Adenovirus Type 2 and Its Vectors

    PubMed Central

    Bru, Thierry; Salinas, Sara; Kremer, Eric J.

    2010-01-01

    Adenovirus vectors have significant potential for long- or short-term gene transfer. Preclinical and clinical studies using human derived adenoviruses (HAd) have demonstrated the feasibility of flexible hybrid vector designs, robust expression and induction of protective immunity. However, clinical use of HAd vectors can, under some conditions, be limited by pre-existing vector immunity. Pre-existing humoral and cellular anti-capsid immunity limits the efficacy and duration of transgene expression and is poorly circumvented by injections of larger doses and immuno-suppressing drugs. This review updates canine adenovirus serotype 2 (CAV-2, also known as CAdV-2) biology and gives an overview of the generation of early region 1 (E1)-deleted to helper-dependent (HD) CAV-2 vectors. We also summarize the essential characteristics concerning their interaction with the anti-HAd memory immune responses in humans, the preferential transduction of neurons, and its high level of retrograde axonal transport in the central and peripheral nervous system. CAV-2 vectors are particularly interesting tools to study the pathophysiology and potential treatment of neurodegenerative diseases, as anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, oncolytic virus and as a platform to generate chimeric vectors. PMID:21994722

  16. Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression

    PubMed Central

    Zhao, Christopher Q.; Young, Matthew R.; Diwan, Bhalchandra A.; Coogan, Timothy P.; Waalkes, Michael P.

    1997-01-01

    Inorganic arsenic, a human carcinogen, is enzymatically methylated for detoxication, consuming S-adenosyl-methionine (SAM) in the process. The fact that DNA methyltransferases (MeTases) require this same methyl donor suggests a role for methylation in arsenic carcinogenesis. Here we test the hypothesis that arsenic-induced initiation results from DNA hypomethylation caused by continuous methyl depletion. The hypothesis was tested by first inducing transformation in a rat liver epithelial cell line by chronic exposure to low levels of arsenic, as confirmed by the development of highly aggressive, malignant tumors after inoculation of cells into Nude mice. Global DNA hypomethylation occurred concurrently with malignant transformation and in the presence of depressed levels of S-adenosyl-methionine. Arsenic-induced DNA hypomethylation was a function of dose and exposure duration, and remained constant even after withdrawal of arsenic. Hyperexpressibility of the MT gene, a gene for which expression is clearly controlled by DNA methylation, was also detected in transformed cells. Acute arsenic or arsenic at nontransforming levels did not induce global hypomethylation of DNA. Whereas transcription of DNA MeTase was elevated, the MeTase enzymatic activity was reduced with arsenic transformation. Taken together, these results indicate arsenic can act as a carcinogen by inducing DNA hypomethylation, which in turn facilitates aberrant gene expression, and they constitute a tenable theory of mechanism in arsenic carcinogenesis. PMID:9380733

  17. Prevalence and Quantitation of Species C Adenovirus DNA in Human Mucosal Lymphocytes

    PubMed Central

    Garnett, C. T.; Erdman, D.; Xu, W.; Gooding, Linda R.

    2002-01-01

    The common species C adenoviruses (serotypes Ad1, Ad2, Ad5, and Ad6) infect more than 80% of the human population early in life. Following primary infection, the virus can establish an asymptomatic persistent infection in which infectious virions are shed in feces for several years. The probable source of persistent virus is mucosa-associated lymphoid tissue, although the molecular details of persistence or latency of adenovirus are currently unknown. In this study, a sensitive real-time PCR assay was developed to quantitate species C adenovirus DNA in human tissues removed for routine tonsillectomy or adenoidectomy. Using this assay, species C DNA was detected in Ficoll-purified lymphocytes from 33 of 42 tissue specimens tested (79%). The levels varied from fewer than 10 to greater than 2 × 106 copies of the adenovirus genome/107 cells, depending on the donor. DNA from serotypes Ad1, Ad2, and Ad5 was detected, while the rarer serotype Ad6 was not. When analyzed as a function of donor age, the highest levels of adenovirus genomes were found among the youngest donors. Antibody-coated magnetic beads were used to purify lymphocytes into subpopulations and determine whether viral DNA could be enriched within any purified subpopulations. Separation of T cells (CD4/8- expressing and/or CD3-expressing cells) enriched viral DNA in each of nine donors tested. In contrast, B-cell purification (CD19-expressing cells) invariably depleted or eliminated viral DNA. Despite the frequent finding of significant quantities of adenovirus DNA in tonsil and adenoid tissues, infectious virus was rarely present, as measured by coculture with permissive cells. These findings suggest that human mucosal T lymphocytes may harbor species C adenoviruses in a quiescent, perhaps latent form. PMID:12368303

  18. A fatal case of neonatal adenovirus infection.

    PubMed

    Kelley, Cynthia J

    2010-01-01

    Adenovirus can produce severe disease and even death in the immunocompromised neonate. Symptoms of adenovirus infection are similar to those seen with bacterial infections in neonates, making early recognition and diagnosis difficult. Consideration of adenovirus as a causative agent is important to early diagnosis. Currently available culture techniques, particularly the shell vial culture technique, make more rapid identification of adenovirus infection possible. Early identification and treatment are necessary to improve patient outcomes and prevent the spread of infection to other neonates. Available agents for the treatment of adenovirus have had mixed results, yet their use is preferable to nontreatment of critical patients. This article presents the case of a preterm infant who became fatally ill from disseminated adenoviral infection.

  19. Enhancers of Agrobacterium-mediated Transformation of Tibouchina semidecandra Selected on the Basis of GFP Expression.

    PubMed

    Yong, Wilson Thau Lym; Henry, Erle Stanley; Abdullah, Janna Ong

    2010-12-01

    Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 μM galactose and 100 μM tyrosine supplemented with 600 μM AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 μM galactose and 50 μM tyrosine with 200 μM AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative T. semidecandra transformants was verified by PCR amplification with specific primers.

  20. Expression-invariant face recognition using depth and intensity dual-tree complex wavelet transform features

    NASA Astrophysics Data System (ADS)

    Ayatollahi, Fazael; Raie, Abolghasem A.; Hajati, Farshid

    2015-03-01

    A new multimodal expression-invariant face recognition method is proposed by extracting features of rigid and semirigid regions of the face which are less affected by facial expressions. Dual-tree complex wavelet transform is applied in one decomposition level to extract the desired feature from range and intensity images by transforming the regions into eight subimages, consisting of six band-pass subimages to represent face details and two low-pass subimages to represent face approximates. The support vector machine has been used to classify both feature fusion and score fusion modes. To test the algorithm, BU-3DFE and FRGC v2.0 datasets have been selected. The BU-3DFE dataset was tested by low intensity versus high intensity and high intensity versus low intensity strategies using all expressions in both training and testing stages in different levels. Findings include the best rank-1 identification rate of 99.8% and verification rate of 100% at a 0.1% false acceptance rate. The FRGC v2.0 was tested by the neutral versus non-neutral strategy, which applies images without expression in training and with expression in the testing stage, thereby achieving the best rank-1 identification rate of 93.5% and verification rate of 97.4% at a 0.1% false acceptance rate.

  1. Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants.

    PubMed

    Doyle, Timothy C; Nawotka, Kevin A; Purchio, Anthony F; Akin, Ali R; Francis, Kevin P; Contag, Pamela R

    2006-02-01

    The infectious yeast Candida albicans is a model organism for understanding the mechanisms of fungal pathogenicity. We describe the functional expression of the firefly luciferase gene, a reporter commonly used to tag genes in many other cellular systems. Due to a non-standard codon usage by this yeast, the CUG codons were first mutated to UUG to allow functional expression. When integrated into the chromosome of C. albicans with a strong constitutive promoter, cells bioluminesce when provided with luciferin substrate in their media. When fused to the inducible promoter from the HWP1 gene, expression and bioluminescence was only detected in cultures conditioning hyphal growth. We further used the luciferase gene as a selection to isolate transformed cell lines from clinical isolates of C. albicans, using a high-density screening strategy that purifies transformed colonies by virtue of light emission. This strategy requires no drug or auxotrophic selectable marker, and we were thus able to generate stable transformants of clinical isolates that are identical to the parental strain in all aspects tested, other than their bioluminescence. The firefly luciferase gene can, therefore, be used as a sensitive reporter to analyze gene function both in laboratory and clinical isolates of this medically important yeast.

  2. The adenovirus E4orf4 protein targets PP2A to the ACF chromatin-remodeling factor and induces cell death through regulation of SNF2h-containing complexes

    PubMed Central

    Brestovitsky, Anna; Sharf, Rakefet; Mittelman, Karin; Kleinberger, Tamar

    2011-01-01

    The adenovirus E4 open-reading-frame 4 (E4orf4) protein regulates the progression of viral infection and when expressed individually it induces non-classical apoptosis in transformed cells. Here we show that E4orf4 associates with the ATP-dependent chromatin-remodeling factor ACF that consists of a sucrose non fermenting-2h (SNF2h) ATPase and an Acf1 regulatory subunit. Furthermore, E4orf4 targets protein phosphatase 2A (PP2A) to this complex and to chromatin. Obstruction of SNF2h activity inhibits E4orf4-induced cell death, whereas knockdown of Acf1 results in enhanced E4orf4-induced toxicity in both mammalian and yeast cells, and Acf1 overexpression inhibits E4orf4′s ability to downregulate early adenovirus gene expression in the context of viral infection. Knockdown of the Acf1 homolog, WSTF, inhibits E4orf4-induced cell death. Based on these results we suggest that the E4orf4–PP2A complex inhibits ACF and facilitates enhanced chromatin-remodeling activities of other SNF2h-containing complexes, such as WSTF–SNF2h. The resulting switch in chromatin remodeling determines life versus death decisions and contributes to E4orf4 functions during adenovirus infection. PMID:21546548

  3. The adenovirus E4orf4 protein targets PP2A to the ACF chromatin-remodeling factor and induces cell death through regulation of SNF2h-containing complexes.

    PubMed

    Brestovitsky, Anna; Sharf, Rakefet; Mittelman, Karin; Kleinberger, Tamar

    2011-08-01

    The adenovirus E4 open-reading-frame 4 (E4orf4) protein regulates the progression of viral infection and when expressed individually it induces non-classical apoptosis in transformed cells. Here we show that E4orf4 associates with the ATP-dependent chromatin-remodeling factor ACF that consists of a sucrose non fermenting-2h (SNF2h) ATPase and an Acf1 regulatory subunit. Furthermore, E4orf4 targets protein phosphatase 2A (PP2A) to this complex and to chromatin. Obstruction of SNF2h activity inhibits E4orf4-induced cell death, whereas knockdown of Acf1 results in enhanced E4orf4-induced toxicity in both mammalian and yeast cells, and Acf1 overexpression inhibits E4orf4's ability to downregulate early adenovirus gene expression in the context of viral infection. Knockdown of the Acf1 homolog, WSTF, inhibits E4orf4-induced cell death. Based on these results we suggest that the E4orf4-PP2A complex inhibits ACF and facilitates enhanced chromatin-remodeling activities of other SNF2h-containing complexes, such as WSTF-SNF2h. The resulting switch in chromatin remodeling determines life versus death decisions and contributes to E4orf4 functions during adenovirus infection.

  4. Radioisotopic imaging allows optimization of adenovirus lung deposition for cystic fibrosis gene therapy.

    PubMed

    Lerondel, S; Le Pape, A; Sené, C; Faure, L; Bernard, S; Diot, P; Nicolis, E; Mehtali, M; Lusky, M; Cabrini, G; Pavirani, A

    2001-01-01

    Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

  5. Eradication of melanoma in vitro and in vivo via targeting with a Killer-Red-containing telomerase-dependent adenovirus.

    PubMed

    Takehara, Kiyoto; Yano, Shuya; Tazawa, Hiroshi; Kishimoto, Hiroyuki; Narii, Nobuhiro; Mizuguchi, Hiroyuki; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi; Hoffman, Robert M

    2017-01-05

    Melanoma is a highly recalcitrant cancer and transformative therapy is necessary for the cure of this disease. We recently developed a telomerase-dependent adenovirus containing the fluorescent protein Killer-Red. In the present report, we first determined the efficacy of Killer-Red adenovirus combined with laser irradiation on human melanoma cell lines in vitro. Cell viability of human melanoma cells was reduced in a dose-dependent and irradiation time-dependent manner. We used an intradermal xenografted melanoma model in nude mice to determine efficacy of the Killer-Red adenovirus. Intratumoral injection of Killer-Red adenovirus, combined with laser irradiation, eradicated the melanoma indicating the potential of a new paradigm of cancer therapy.

  6. Crystal structure of the fibre head domain of bovine adenovirus 4, a ruminant atadenovirus.

    PubMed

    Nguyen, Thanh H; Vidovszky, Márton Z; Ballmann, Mónika Z; Sanz-Gaitero, Marta; Singh, Abhimanyu K; Harrach, Balázs; Benkő, Mária; van Raaij, Mark J

    2015-05-22

    In adenoviruses, primary host cell recognition is generally performed by the head domains of their homo-trimeric fibre proteins. This first interaction is reversible. A secondary, irreversible interaction subsequently takes place via other adenovirus capsid proteins and leads to a productive infection. Although many fibre head structures are known for human mastadenoviruses, not many animal adenovirus fibre head structures have been determined, especially not from those belonging to adenovirus genera other than Mastadenovirus. We constructed an expression vector for the fibre head domain from a ruminant atadenovirus, bovine adenovirus 4 (BAdV-4), consisting of amino acids 414-535, expressed the protein in Escherichia coli, purified it by metal affinity and cation exchange chromatography and crystallized it. The structure was solved using single isomorphous replacement plus anomalous dispersion of a mercury derivative and refined against native data that extended to 1.2 Å resolution. Like in other adenoviruses, the BAdV-4 fibre head monomer contains a beta-sandwich consisting of ABCJ and GHID sheets. The topology is identical to the fibre head of the other studied atadenovirus, snake adenovirus 1 (SnAdV-1), including the alpha-helix in the DG-loop, despite of them having a sequence identity of only 15 %. There are also differences which may have implications for ligand binding. Beta-strands G and H are longer and differences in several surface-loops and surface charge are observed. Chimeric adenovirus fibres have been used to retarget adenovirus-based anti-cancer and gene therapy vectors. Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for such a vector. Here, we present the high-resolution atomic structure of the BAdV-4 fibre head domain, the second atadenovirus fibre head structure known and the first of an atadenovirus that infects a mammalian host. Future research should focus on the receptor-binding properties of

  7. Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors

    SciTech Connect

    Abbink, Peter; Maxfield, Lori F.; Ng'ang'a, David; Borducchi, Erica N.; Iampietro, M. Justin; Bricault, Christine A.; Teigler, Jeffrey E.; Blackmore, Stephen; Parenteau, Lily; Wagh, Kshitij; Handley, Scott A.; Zhao, Guoyan; Virgin, Herbert W.; Korber, Bette; Barouch, Dan H.

    2014-11-19

    Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. Furthermore, the phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. We describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved to have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.

  8. Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors

    DOE PAGES

    Abbink, Peter; Maxfield, Lori F.; Ng'ang'a, David; ...

    2014-11-19

    Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. Furthermore, the phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. We describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved tomore » have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.« less

  9. Involvement of template-activating factor I/SET in transcription of adenovirus early genes as a positive-acting factor.

    PubMed

    Haruki, Hirohito; Okuwaki, Mitsuru; Miyagishi, Makoto; Taira, Kazunari; Nagata, Kyosuke

    2006-01-01

    The adenovirus genome complexed with viral core protein VII (adenovirus DNA-protein VII complex) at least is the bona fide template for transcription of adenovirus early genes. It is believed that the highly basic protein VII, like cellular histones, is a negative regulator for genome functions. Analyses with in vitro replication and transcription systems using the adenovirus DNA-protein VII complex have revealed that remodeling of the complex is crucial for efficient DNA replication and transcription. We identified host acidic proteins, template-activating factor I (TAF-I), TAF-II, and TAF-III as stimulatory factors for replication from the adenovirus DNA-protein VII complex. Recently, it was reported that the adenovirus DNA interacts with TAF-I and pp32, another host acidic protein (Y. Xue, J. S. Johnson, D. A. Ornelles, J. Lieberman, and D. A. Engel, J. Virol. 79:2474-2483, 2005). We found that TAF-I interacts and colocalizes with protein VII in adenovirus-infected cells during the early phases of infection, but pp32 does not. Although pp32 had the potential ability to interact with protein VII, pp32 did not remodel the adenovirus DNA-protein VII complex in vitro. Small interfering RNA-mediated knockdown of TAF-I expression leads to the delay of the transcription timing of early genes. These results provide evidence that TAF-I plays an important role in the early stages of the adenovirus infection cycle.

  10. Differential transformation of C3H10T1/2 cells by v-mos: sequential expression of transformation parameters.

    PubMed Central

    van der Hoorn, F A; Müller, V

    1985-01-01

    Extremely small quantities of the product of the transforming gene v-mos of Moloney murine sarcoma virus are able to efficiently transform cells. Recent data indicate the existence of a threshold level for v-mos transformation of NIH3T3 cells. Using mouse mammary tumor virus long terminal repeat sequences or hybrid promoters consisting of mouse mammary tumor virus and Moloney murine sarcoma virus long terminal repeat elements to express v-mos in C3H10T1/2 cells, we established cell lines representing different stages of morphological transformation in vitro. The threshold level for v-mos transformation was considerably lower than that for NIH3T3 cells, because no treatment with dexamethasone or primary selection other than transformation was necessary during standard transfection procedures. Using the cell lines mentioned we established an association of the level of v-mos expression with the transformation parameters examined, but not with p53 levels. Furthermore, the characterization of the different promoters showed (i) that the distal binding site confers hormone responsiveness to Moloney murine sarcoma virus promoter elements and (ii) that artifactual transcription initiation sites can be detected in mouse mammary tumor virus-Moloney murine sarcoma virus hybrid promoters which are, however, not regulated by the hormone. Images PMID:3016522

  11. Assembly and cell surface expression of TAP-independent, chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin.

    PubMed

    Fromm, Sharon Vigodman; Duady-Ben Yaakov, Shirly; Schechter, Chana; Ehrlich, Rachel

    2002-02-01

    Antigen processing and presentation by class I MHC molecules generally require assembly with peptide epitopes generated by the proteasome and transported into the ER by the transporters associated with antigen presentation (TAP). Recently, TAP-independent pathways supporting class I MHC-mediated presentation of exogenous antigens, as well as of endogenously synthesized viral antigens, were described. We now characterize a TAP-independent pathway that is operative in both TAP1- and TAP2-deficient Adenovirus (Ad)-transformed fibroblast cell lines. To the best of our knowledge, this is the first time that the existence of such a pathway has been described in non-infected cells that do not belong to the hematopoietic lineage. We show that this pathway is proteasome-independent and chloroquine-sensitive. Cell surface expression of these TAP-independent class I complexes is modulated by tapasin levels and is enhanced by IFN-gamma. The data imply that IFN-gamma increases the relative level of TAP-independent high affinity class I complexes that exit the ER on their way to the cell surface and to vacuolar compartments where peptide cleavage/exchange might take place before recycling to the cell surface. Since both TAP and tapasin expression are altered in numerous tumors and in virus-infected cells, TAP-independent class I complexes may be a valuable target source for immune responses.

  12. Transforming growth factor-β1 induces cell cycle arrest by activating atypical cyclin-dependent kinase 5 through up-regulation of Smad3-dependent p35 expression in human MCF10A mammary epithelial cells.

    PubMed

    Park, Seong Ji; Yang, Sun Woo; Kim, Byung-Chul

    2016-04-08

    Cyclin-dependent kinases (Cdks) play important roles in control of cell division. Cdk5 is an atypical member of Cdk family with non-cyclin-like regulatory subunit, p35, but its role in cell cycle progression is still unclear. In the present study, we investigated the role of Cdk5/p35 on transforming growth factor-β1 (TGF-β1)-induced cell cycle arrest. In human MCF10A mammary epithelial cells, TGF-β1 induced cell cycle arrest at G1 phase and increased p27KIP1 expression. Interestingly, pretreatment with roscovitine, an inhibitor of Cdk5, or transfection with small interfering (si) RNAs specific to Cdk5 and p35 significantly attenuated the TGF-β1-induced p27KIP1 expression and cell cycle arrest. TGF-β1 increased Cdk5 activity via up-regulation of p35 gene at transcriptional level, and these effects were abolished by transfection with Smad3 siRNA or infection of adenovirus carrying Smad3 mutant at the C-tail (3SA). Chromatin immunoprecipitation assay further revealed that wild type Smad3, but not mutant Smad3 (3SA), binds to the region of the p35 promoter region (-1000--755) in a TGF-β1-dependent manner. These results for the first time demonstrate a role of Cdk5/p35 in the regulation of cell cycle progression modulated by TGF-β1. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Phylogenetic analysis of adenovirus sequences.

    PubMed

    Harrach, Balázs; Benko, Mária

    2007-01-01

    Members of the family Adenoviridae have been isolated from a large variety of hosts, including representatives from every major vertebrate class from fish to mammals. The high prevalence, together with the fairly conserved organization of the central part of their genomes, make the adenoviruses one of (if not the) best models for studying viral evolution on a larger time scale. Phylogenetic calculation can infer the evolutionary distance among adenovirus strains on serotype, species, and genus levels, thus helping the establishment of a correct taxonomy on the one hand, and speeding up the process of typing new isolates on the other. Initially, four major lineages corresponding to four genera were recognized. Later, the demarcation criteria of lower taxon levels, such as species or types, could also be defined with phylogenetic calculations. A limited number of possible host switches have been hypothesized and convincingly supported. Application of the web-based BLAST and MultAlin programs and the freely available PHYLIP package, along with the TreeView program, enables everyone to make correct calculations. In addition to step-by-step instruction on how to perform phylogenetic analysis, critical points where typical mistakes or misinterpretation of the results might occur will be identified and hints for their avoidance will be provided.

  14. Cellular localization of transforming growth factor-beta expression in bleomycin-induced pulmonary fibrosis.

    PubMed Central

    Zhang, K.; Flanders, K. C.; Phan, S. H.

    1995-01-01

    Bleomycin-induced pulmonary fibrosis is associated with increased lung transforming growth factor-beta (TGF-beta) gene expression, but cellular localization of the source of this expression has not been unequivocally established. In this study, lung fibrosis was induced in rats by endotracheal bleomycin injection on day 0 and, on selected days afterwards, lungs were harvested for in situ hybridization, immunohistochemical and histochemical analyses for TGF-beta 1 mRNA and protein expression, and cell identification. The results show that control lungs express essentially no detectable TGF-beta 1 mRNA or protein in the parenchyma. Before day 3 after bleomycin treatment, scattered bronchiolar epithelial cells, mononuclear cells, and eosinophils expressed elevated levels of TGF-beta 1. Between days 3 and 14, there was a major increase in the number of eosinophils, myofibroblasts, and fibroblasts strongly expressing TGF-beta 1 mRNA and protein. TGF-beta 1-producing cells were predominantly localized within areas of injury and active fibrosis. After day 14, the intensity and number of TGF-beta 1-expressing cells significantly declined and were predominantly found in fibroblasts in fibrotic areas. The expression of TGF-beta 1 protein was generally coincident with that for mRNA with the exception of bronchiolar epithelial cells in which strong protein expression was unaccompanied by a commensurate increase in mRNA. The study demonstrates that myofibroblasts, fibroblasts, and eosinophils represent the major sources of increased lung TGF-beta 1 expression in this model of pulmonary fibrosis. Images Figure 2 Figure 3 Figure 4 PMID:7543734

  15. Molecular Characterization of a Lizard Adenovirus Reveals the First Atadenovirus with Two Fiber Genes and the First Adenovirus with Either One Short or Three Long Fibers per Penton

    PubMed Central

    Pénzes, Judit J.; Menéndez-Conejero, Rosa; Condezo, Gabriela N.; Ball, Inna; Papp, Tibor; Doszpoly, Andor; Paradela, Alberto; Pérez-Berná, Ana J.; López-Sanz, María; Nguyen, Thanh H.; van Raaij, Mark J.; Marschang, Rachel E.; Harrach, Balázs; Benkő, Mária

    2014-01-01

    ABSTRACT Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCE Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins—one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton

  16. Expression of the small T antigen of Lymphotropic Papovavirus is sufficient to transform primary mouse embryo fibroblasts

    SciTech Connect

    Gupta, Tushar; Robles, Maria Teresa Sáenz; Schowalter, Rachel M.; Buck, Christopher B.; Pipas, James M.

    2016-01-15

    Polyomaviruses induce cell proliferation and transformation through different oncoproteins encoded within the early region (ER): large T antigen (LT), small T antigen (sT) and, in some cases, additional components. Each virus utilizes different mechanisms to achieve transformation. For instance, the LTs of Simian virus 40 (SV40), BK and/or JC virus can induce transformation; but Merkel Cell Polyomavirus (MCPyV) requires expression of sT. Lymphotropic Papovavirus (LPV) is closely related to Human Polyomavirus 9 (HuPyV9) and, under similar conditions, mice expressing LPV.ER exhibit higher rates of tumor formation than mice expressing SV40.ER. We have investigated the contributions of individual LPV.ER components to cell transformation. In contrast to SV40, LPV.ER transforms mouse embryonic fibroblasts (MEFs), but expression of LPV LT is insufficient to transform MEFs. Furthermore, LPV sT induces immortalization and transformation of MEFs. Thus, in the case of LPV, sT is the main mediator of oncogenesis. - Highlights: • Characterization of early region products from the Lymphotropic Polyomavirus (LPV). • On its own, sT immortalizes and transforms mouse primary cells, and is able to block p53 activation. • Combined LT and sT expression induces a greater rate of proliferation than either LT or sT alone.

  17. Multistep nature of X-ray-induced neoplastic transformation in golden hamster embryo cells: expression of transformed phenotypes and stepwise changes in karyotypes

    SciTech Connect

    Suzuki, K.; Suzuki, F.; Watanabe, M.; Nikaido, O.

    1989-04-15

    We have examined the expression of transformed phenotypes and genetic changes associated with the expression of each transformed phenotype after X-ray irradiation. Unirradiated cells grown at a constant growth rate until 8 passages (population doubling number, 15) exhibited little morphological change and ceased to divide thereafter. X-irradiated cells escaped from senescence and showed morphological alteration and anchorage independence after a population doubling number of 20. The acquisition of tumorigenicity in nude mice was observed much later (35 population doublings after irradiation). From cytogenetic analysis, all anchorage-independent clones were consistently found to have trisomy of chromosome 7. Furthermore, cells derived from tumors contained three copies of chromosome 9q in addition to the trisomy of chromosome 7. We have not detected any augmented expression of v-Ha-ras- and v-myc-related oncogenes with RNA dot-blot analysis and could not find activation of any type of oncogenes by NIH3T3 transfection experiments. Our studies demonstrated that X-ray-induced neoplastic transformation is a multistep phenomenon and that the numerical change of specific chromosomes may play an important role in the expression of each transformed phenotype. The results suggest that different endogenous oncogenes, other than the ras gene family and myc oncogene, could be responsible for the progressive nature of neoplastic transformation.

  18. Oncolytic adenovirus-mediated therapy for prostate cancer

    PubMed Central

    Sweeney, Katrina; Halldén, Gunnel

    2016-01-01

    Prostate cancer is a leading cause of cancer-related death and morbidity in men in the Western world. Tumor progression is dependent on functioning androgen receptor signaling, and initial administration of antiandrogens and hormone therapy (androgen-deprivation therapy) prevent growth and spread. Tumors frequently develop escape mechanisms to androgen-deprivation therapy and progress to castration-resistant late-stage metastatic disease that, in turn, inevitably leads to resistance to all current therapeutics, including chemotherapy. In spite of the recent development of more effective inhibitors of androgen–androgen receptor signaling such as enzalutamide and abiraterone, patient survival benefits are still limited. Oncolytic adenoviruses have proven efficacy in prostate cancer cells and cause regression of tumors in preclinical models of numerous drug-resistant cancers. Data from clinical trials demonstrate that adenoviral mutants have limited toxicity to normal tissues and are safe when administered to patients with various solid cancers, including prostate cancer. While efficacy in response to adenovirus administration alone is marginal, findings from early-phase trials targeting local-ized and metastatic prostate cancer suggest improved efficacy in combination with cytotoxic drugs and radiation therapy. Here, we review recent progress in the development of multimodal oncolytic adenoviruses as biological therapeutics to improve on tumor elimination in prostate cancer patients. These optimized mutants target cancer cells by several mechanisms including viral lysis and by expression of cytotoxic transgenes and immune-stimulatory factors that activate the host immune system to destroy both infected and noninfected prostate cancer cells. Additional modifications of the viral capsid proteins may support future systemic delivery of oncolytic adenoviruses. PMID:27579296

  19. Cell surface expression of v-fms-coded glycoproteins is required for transformation.

    PubMed Central

    Roussel, M F; Rettenmier, C W; Look, A T; Sherr, C J

    1984-01-01

    The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane. Images PMID:6390182

  20. STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus

    PubMed Central

    Spurrell, Emma; Gangeswaran, Rathi; Wang, Pengju; Cao, Fengyu; Gao, Dongling; Feng, Baisui; Wold, William; Tollefson, Ann

    2014-01-01

    Oncolytic viruses based on adenovirus type 5 (Ad5) have been developed as a new class of therapeutic agents for cancers that are resistant to conventional therapies. Clinical experience shows that these agents are safe, but virotherapy alone has not achieved long-term cure in cancer patients. The vast majority of oncolytic adenoviruses used in clinical trials to date have deletion of the E3B genes. It has been demonstrated that the antitumor potency of the E3B-deleted mutant (dl309) is inferior to adenovirus with E3B genes intact. Tumors treated with dl309 show markedly greater macrophage infiltration than E3B-intact adenovirus. However, the functional mechanisms for this were not previously known. Here, we demonstrate that deletion of E3B genes increases production of chemokines by monocytes after adenovirus infection and increases monocyte migration. The E3B 14,700-Da protein (E3B-14.7K) inhibits STAT1 function by preventing its phosphorylation and nuclear translocation. The STAT1 inhibitor, fludarabine, rescues the effect of E3B-14.7K deletion by downregulating target chemokine expression in human and murine monocytes and results in an enhanced antitumor efficacy with dl309 in vivo. These findings have important implications for clinical use of E3B-deleted oncolytic adenovirus and other E3B-deleted adenovirus vector-based therapy. PMID:24335311

  1. Characterization of an upstream regulatory element of adenovirus L1 poly (A) site.

    PubMed

    Liu, Li

    2005-06-20

    The transition from early to late stage infection by adenovirus involves a change in mRNA expression from the adenovirus major late transcription unit (AdMLTU). This early to late switch centers around alternative selection of one of five poly (A) sites (L1-L5) that code for the major structural proteins of Adenovirus. During the early stage of infection, steady state mRNA is primarily derived from the L1 poly (A) site. During the late stage of infection, each of the MLTU poly (A) sites is represented in the steady state mRNA pool (Falck-Pedersen, E., Logan, J., 1989. Regulation of poly(A) site selection in adenovirus. J. Virol. 63 (2), 532-541.). Using transient transfection of a plasmid expressing Chloramphenicol Acetyl Transferase with a tandem poly (A) minigene system (L13) (DeZazzo, J.D., Falck-Pedersen, E., Imperiale, M.J., 1991. Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit. Mol. Cell. Biol. 11 (12), 5977-5984; Prescott, J., Falck-Pedersen, E., 1994. Sequence elements upstream of the 3' cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites. Mol. Cell. Biol. 14 (7), 4682-4693.), it has been demonstrated that the promoter-proximal L1 poly (A) site which is poorly recognized by the 3' end processing machinery, contains an upstream repressor element (URE) that influences steady state levels of mRNA (Prescott, J.C., Liu, L., Falck-Pedersen, E., 1997. Sequence-mediated regulation of adenovirus gene expression by repression of mRNA accumulation. Mol. Cell. Biol. 17 (4), 2207-2216.). In this study, we have further characterized the elements that mediate L1URE function. These studies indicate that the L1 upstream regulatory element (L1 URE) contains a complex RNA architecture that serves to repress gene expression through multiple sub-effectors. The L1URE functions when located upstream of a heterologous poly (A) site, and is able to strongly suppress steady state m

  2. Studies on the Interaction of Tumor-Derived HD5 Alpha Defensins with Adenoviruses and Implications for Oncolytic Adenovirus Therapy.

    PubMed

    Vragniau, Charles; Hübner, Jens-Martin; Beidler, Peter; Gil, Sucheol; Saydaminova, Kamola; Lu, Zhuo-Zhuang; Yumul, Roma; Wang, Hongjie; Richter, Maximilian; Sova, Pavel; Drescher, Charles; Fender, Pascal; Lieber, André

    2017-03-15

    Defensins are small antimicrobial peptides capable of neutralizing human adenovirus (HAdV) in vitro by binding capsid proteins and blocking endosomal escape of virus. In humans, the alpha defensin HD5 is produced by specialized epithelial cells of the gastrointestinal and genito-urinary tracts. Here, we demonstrate, using patient biopsy specimens, that HD5 is also expressed as an active, secreted peptide by epithelial ovarian and lung cancer cells in situ This finding prompted us to study the role of HD5 in infection and spread of replication-competent, oncolytic HAdV type 3 (HAdV3). HAdV3 produces large amounts of penton-dodecahedra (PtDd), virus-like particles, during replication. We have previously shown that PtDd are involved in opening epithelial junctions, thus facilitating lateral spread of de novo-produced virions. Here, we describe a second function of PtDd, namely, the blocking of HD5. A central tool to prove that viral PtDd neutralize HD5 and support spread of progeny virus was an HAdV3 mutant virus in which formation of PtDd was disabled (mut-Ad3GFP, where GFP is green fluorescent protein). We demonstrated that viral spread of mut-Ad3GFP was blocked by synthetic HD5 whereas that of the wild-type (wt) form (wt-Ad3GFP) was only minimally impacted. In human colon cancer Caco-2 cells, induction of cellular HD5 expression by fibroblast growth factor 9 (FGF9) significantly inhibited viral spread and progeny virus production of mut-Ad3GFP but not of wt-Ad3GFP. Finally, the ectopic expression of HD5 in tumor cells diminished the in vivo oncolytic activity of mut-Ad3GFP but not of wt-Ad3GFP. These data suggest a new mechanism of HAdV3 to overcome innate antiviral host responses. Our study has implications for oncolytic adenovirus therapy.IMPORTANCE Previously, it has been reported that human defensin HD5 inactivates specific human adenoviruses by binding to capsid proteins and blocking endosomal escape of virus. The central new findings described in our

  3. Model-driven Approaches for in Vitro Combination Therapy using ONYX-015 Replicating Oncolytic Adenovirus 1

    PubMed Central

    Zurakowski, Ryan; Wodarz, Dominik

    2009-01-01

    Replicating genetically modified adenoviruses have shown promise as a new treatment approach against cancer. Recombinant adenoviruses replicate only in cancer cells which contain certain mutations, such as the loss of functional p53, as is the case in the virus ONYX-015. The successful entry of the viral particle into target cells is strongly dependent on the presence of the main receptor for adenovirus, the coxsackie- and adeno-virus receptor (CAR). This receptor is frequently down-regulated in highly malignant cells, rendering this population less vulnerable to viral attack. It has been shown that use of MEK inhibitors can up-regulate CAR expression, resulting in enhanced adenovirus entry into the cells. However, inhibition of MEK results in G1 cell cycle arrest, rendering infected cells temporarily unable to produce virus. This forces a tradeo1. While drug mediated up-regulation of CAR enhances virus entry into cancer cells, the consequent cell cycle arrest inhibits production of new virus particles and the replication of the virus. Optimal control-based schedules of MEK inhibitor application should increase the efficacy of this treatment, maximizing the overall tumor toxicity by exploiting the dynamics of CAR expression and viral production. We introduce a mathematical model of these dynamics and show simple optimal control based strategies which motivate this approach. PMID:17095020

  4. [Expression analysis of a transformer gene in Daphnia pulex after RNAi].

    PubMed

    Guo, C Y; Chen, P; Zhang, M M; Ning, J J; Wang, С L; Wang, D L; Zhao, Y L

    2016-01-01

    In order to explore the importance of the transformer (tra) gene in reproductive mode switching in Daphnia pulex, we studied the effect of silencing of this gene using RNA interference (RNAi). We obtained Dptra dsRNA by constructing and using a dsRNA expression vector and transcription method in vitro. D. pulex individuals in different reproductive modes were treated by soaking in a solution of Dptra dsRNA. We then assayed the expression of the endogenous Dptra mRNA after RNAi treatment using RT-PCR and obtained the suppression ratio. Expression of the tra gene in the RNAi groups was down-regulated compared with the controls after 16 h (p < 0.05). We also analyzed the effect of RNAi on the expression of the TRA protein using Western blot, which showed that the expression level of the TRA protein was reduced after RNAi treatment. Our experimental results showed that soaking of D. pulex adults in tra-specific dsRNA transcribed in vitro can specifically reduce the level of tra mRNA and also reduce the expression of the TRA protein, demonstrating effective in vivo silencing of the tra gene.

  5. Epiregulin expression by Ets-1 and ERK signaling pathway in Ki-ras-transformed cells.

    PubMed

    Cho, Min-Chul; Choi, Hee-Sook; Lee, Sojung; Kim, Bo Yeon; Jung, Mira; Park, Sue Nie; Yoon, Do-Young

    2008-12-19

    Epiregulin belongs to the epidermal growth factor family, binds to the epidermal growth factor receptor, and its expression is upregulated in various cancer cells, but the regulatory mechanism is unclear. We investigated the regulatory mechanism of epiregulin expression in Ki-ras-transformed cancer cells. In 267B1/Ki-ras cells, the RAF/MEK/ERK pathway was constitutively activated, epiregulin was up-regulated, and the expression and phosphorylation of Ets-1 were augmented. The inhibition of ERK by PD98059 decreased epiregulin and Ets-1 expression and suppressed the growth of 267B1/Ki-ras cells. A chromatin immunoprecipitation assay demonstrated that Ets-1 was bound to human epiregulin promoter, and this binding was abolished by PD98059. Silencing of Ets-1 by RNA interference decreased cellular epiregulin transcript expression. We suggest that the Ki-ras mutation in 267B1 prostate cells constitutively activates the RAF/MEK/ERK pathway and induces the activation of the Ets-1 transcription factor, ultimately leading to the increased expression of epiregulin.

  6. A rapid Q-PCR titration protocol for adenovirus and helper-dependent adenovirus vectors that produces biologically relevant results

    PubMed Central

    Gallaher, Sean D.; Berk, Arnold J.

    2013-01-01

    Adenoviruses are employed in the study of cellular processes and as expression vectors used in gene therapy. The success and reproducibility of these studies is dependent in part on having accurate and meaningful titers of replication competent and helper-dependent adenovirus stocks, which is problematic due to the use of varied and divergent titration protocols. Physical titration methods, which quantify the total number of viral particles, are used by many, but are poor at estimating activity. Biological titration methods, such as plaque assays, are more biologically relevant, but are time consuming and not applicable to helper-dependent gene therapy vectors. To address this, a protocol was developed called “infectious genome titration” in which viral DNA is isolated from the nuclei of cells ~3 h post-infection, and then quantified by Q-PCR. This approach ensures that only biologically active virions are counted as part of the titer determination. This approach is rapid, robust, sensitive, reproducible, and applicable to all forms of adenovirus. Unlike other Q-PCR-based methods, titers determined by this protocol are well correlated with biological activity. PMID:23624118

  7. High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts.

    PubMed Central

    Boast, S; La Mantia, G; Lania, L; Blasi, F

    1983-01-01

    Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells. Images Fig. 2. Figure 3. PMID:6321161

  8. Tumor Associated Stromal Cells Play a Critical Role on the Outcome of the Oncolytic Efficacy of Conditionally Replicative Adenoviruses

    PubMed Central

    Lopez, M. Verónica; Viale, Diego L.; Cafferata, Eduardo G. A.; Bravo, Alicia I.; Carbone, Cecilia; Gould, David; Chernajovsky, Yuti; Podhajcer, Osvaldo L.

    2009-01-01

    The clinical efficacy of conditionally replicative oncolytic adenoviruses (CRAd) is still limited by the inefficient infection of the tumor mass. Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter-based CRAds since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC. Interestingly, both CRAds displayed cytotoxic activity on SPARC positive-transformed human microendothelial HMEC-1 cells and WI-38 fetal fibroblasts. Both CRAds were therapeutically effective on SPARC positive-human melanoma tumors growing in nude mice but exhibited restricted efficacy in the presence of co-administered HMEC-1 or WI-38 cells. Conversely, co-administration of HMEC-1 cells enhanced the oncolytic efficacy of Ad(I)-F512-TK on SPARC-negative MIA PaCa-2 pancreatic cancer cells in vivo. Moreover, conditioned media produced by stromal cells pre-infected with the CRAds enhanced the in vitro viral oncolytic activity on pancreatic cancer cells, but not on melanoma cells. The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses. PMID:19337591

  9. A new component of the Nasonia sex determining cascade is maternally silenced and regulates transformer expression.

    PubMed

    Verhulst, Eveline C; Lynch, Jeremy A; Bopp, Daniel; Beukeboom, Leo W; van de Zande, Louis

    2013-01-01

    Although sex determination is a universal process in sexually reproducing organisms, sex determination pathways are among the most highly variable genetic systems found in nature. Nevertheless, general principles can be identified among the diversity, like the central role of transformer (tra) in insects. When a functional TRA protein is produced in early embryogenesis, the female sex determining route is activated, while prevention of TRA production leads to male development. In dipterans, male development is achieved by prevention of female-specific splicing of tra mRNA, either mediated by X-chromosome dose or masculinizing factors. In Hymenoptera, which have haplodiploid sex determination, complementary sex determination and maternal imprinting have been identified to regulate timely TRA production. In the parasitoid Nasonia, zygotic transformer (Nvtra) expression and splicing is regulated by a combination of maternal provision of Nvtra mRNA and silencing of Nvtra expression in unfertilized eggs. It is unclear, however, if this silencing is directly on the tra locus or whether it is mediated through maternal silencing of a trans-acting factor. Here we show that in Nasonia, female sex determination is dependent on zygotic activation of Nvtra expression by an as yet unknown factor. This factor, which we propose to term womanizer (wom), is maternally silenced during oogenesis to ensure male development in unfertilized eggs. This finding implicates the upstream recruitment of a novel gene in the Nasonia sex determining cascade and supports the notion that sex determining cascades can rapidly change by adding new components on top of existing regulators.

  10. Regulation of pituitary tumor transforming gene (PTTG) expression and phosphorylation in thyroid cells.

    PubMed

    Lewy, Gregory D; Ryan, Gavin A; Read, Martin L; Fong, Jim C W; Poole, Vikki; Seed, Robert I; Sharma, Neil; Smith, Vicki E; Kwan, Perkin P K; Stewart, Sarah L; Bacon, Andrea; Warfield, Adrian; Franklyn, Jayne A; McCabe, Christopher J; Boelaert, Kristien

    2013-11-01

    Human pituitary tumor transforming gene (hPTTG) is a multifunctional proto-oncogene implicated in the initiation and progression of several tumors. Phosphorylation of hPTTG is mediated by cyclin-dependent kinase 2 (CDC2), whereas cellular expression is regulated by specificity protein 1 (SP1). The mechanisms underlying hPTTG propagation of aberrant thyroid cell growth have not been fully defined. We set out to investigate the interplay between hPTTG and growth factors, as well as the effects of phosphorylation and SP1 regulation on hPTTG expression and function. In our study, epidermal growth factor (EGF), TGFα, and IGF-1 induced hPTTG expression and phosphorylation in thyroid cells, which was associated with activation of MAPK and phosphoinositide 3-kinase. Growth factors induced hPTTG independently of CDC2 and SP1 in thyroid carcinoma cells. Strikingly, CDC2 depletion in TPC-1 cells resulted in enhanced expression and phosphorylation of hPTTG and reduced cellular proliferation. In reciprocal experiments, hPTTG overexpression induced EGF, IGF-1, and TGFα mRNAs in primary human thyrocytes. Treatment of primary human thyrocytes with conditioned media derived from hPTTG-transfected cells resulted in autocrine upregulation of hPTTG protein, which was ameliorated by growth factor depletion or growth factor receptor tyrosine kinase inhibitors. A transgenic murine model of thyroid targeted hPTTG overexpression (hPTTG-Tg) (FVB/N strain, both sexes) demonstrated smaller thyroids with reduced cellular proliferation and enhanced secretion of Egf. In contrast, Pttg(-/-) knockout mice (c57BL6 strain, both sexes) showed reduced thyroidal Egf mRNA expression. These results define hPTTG as having a central role in thyroid autocrine signaling mechanisms via growth factors, with profound implications for promotion of transformed cell growth.

  11. Adenovirus sensing by the immune system.

    PubMed

    Atasheva, Svetlana; Shayakhmetov, Dmitry M

    2016-12-01

    The host immune system developed multiple ways for recognition of viral pathogens. Upon disseminated adenovirus infection, the immune system senses adenovirus invasion from the moment it enters the bloodstream. The soluble blood factors, FX, antibodies, and complement, can bind and activate plethora of host-protective immune responses. Adenovirus binding to the cellular β3 integrin and endosomal membrane rupture trigger activation of IL-1α/IL-1R1 proinflammatory cascade leading to attraction of cytotoxic immune cells to the site of infection. Upon cell entry, adenovirus exposes its DNA genome in the cytoplasm and triggers DNA sensors signaling. Even when inside the nucleus, the specialized cellular machinery that recognizes the double-strand DNA breaks become activated and triggers viral DNA replication arrest. Thus, the host employs very diverse mechanisms to prevent viral dissemination.

  12. Gene Transcript Abundance Profiles Distinguish Kawasaki Disease from Adenovirus Infection

    PubMed Central

    Popper, Stephen J.; Watson, Virginia E.; Shimizu, Chisato; Kanegaye, John T.; Burns, Jane C.; Relman, David A.

    2010-01-01

    Background Acute Kawasaki disease (KD) is difficult to distinguish from other illnesses that involve acute rash or fever, in part because the etiologic agent(s) and pathophysiology remain poorly characterized. As a result, diagnosis and critical therapies may be delayed. Methods We used DNA microarrays to identify possible diagnostic features of KD. We compared gene expression patterns in the blood of 23 children with acute KD and 18 age-matched febrile children with 3 illnesses that resemble KD. Results Genes associated with platelet and neutrophil activation were expressed at higher levels in patients with KD than in patients with acute adenovirus infections or systemic adverse drug reactions, but levels in patients with KD were not higher than those in patients with scarlet fever. Genes associated with B cell activation were also expressed at higher levels in patients with KD than in control subjects. A striking absence of interferon-stimulated gene expression in patients with KD was confirmed in an independent cohort of patients with KD. Using a set of 38 gene transcripts, we successfully predicted the diagnosis for 21 of 23 patients with KD and 7 of 8 patients with adenovirus infection. Conclusions These findings provide insight into the molecular features that distinguish KD from other febrile illnesses and support the feasibility of developing novel diagnostic reagents for KD based on the host response. PMID:19583510

  13. Tubulin alpha 8 is expressed in hepatic stellate cells and is induced in transformed hepatocytes.

    PubMed

    Rein-Fischboeck, Lisa; Pohl, Rebekka; Haberl, Elisabeth M; Zimny, Sebastian; Neumann, Maximilian; Eisinger, Kristina; Weiss, Thomas S; Krautbauer, Sabrina; Buechler, Christa

    2017-04-01

    Tubulin alpha 8 (TUBA8) is highly abundant in murine liver tumors suggesting a role in hepatocellular carcinoma (HCC). Non-alcoholic steatohepatitis (NASH) is a risk factor for HCC. In mice that are fed with a methionine-choline deficient diet for two weeks to induce advanced murine NASH, we do see increased hepatic levels of TUBA8 protein. In animals given a high-fat diet for 14 weeks or an atherogenic diet for 12 weeks, hepatic TUBA8 is unchanged. TUBA8 is highly expressed in human hepatic stellate cells (HSC) and co-localizes with the HSC marker desmin in the murine liver. Inflammatory (TNF, LPS, IL-6) and profibrotic mediators (TGF-beta) do not regulate TUBA8 in HepG2 cells, primary HSC and the HSC cell line LX-2, when stimulated for 24 h. Agonists of the farnesoid X receptor and peroxisome proliferator activated receptor gamma, which are nuclear receptors involved in NASH and HCC pathophysiology, have no effect on TUBA8 in HepG2 and LX-2 cells. In human HCC tissues of 18 patients TUBA8 is significantly upregulated when compared to the corresponding non-tumorous tissues. Compared to non-transformed hepatocytes, TUBA8 protein is strongly expressed in transformed cells. Thus, TUBA8 is a marker of HSC whose cell number is increased in NASH, while higher levels in HCC may be related to induction of TUBA8 in parenchymal cells.

  14. Expression of the Small T antigen of Lymphotropic Papovavirus is Sufficient to Transform Primary Mouse Embryo Fibroblasts

    PubMed Central

    Gupta, Tushar; Robles, Maria Teresa Sáenz; Schowalter, Rachel M.; Buck, Christopher B.; Pipas, James M.

    2015-01-01

    Polyomaviruses induce cell proliferation and transformation through different oncoproteins encoded within the early region (ER): large T antigen (LT), small T antigen (sT) and, in some cases, additional components. Each virus utilizes different mechanisms to achieve transformation. For instance, the LTs of Simian virus 40 (SV40), BK and/or JC virus can induce transformation; but Merkel Cell Polyomavirus (MCPyV) requires expression of sT. Lymphotropic Papovavirus (LPV) is closely related to Human Polyomavirus 9 (HuPyV9) and, under similar conditions, mice expressing LPV.ER exhibit higher rates of tumor formation than mice expressing SV40.ER. We have investigated the contributions of individual LPV.ER components to cell transformation. In contrast to SV40, LPV.ER transforms mouse embryonic fibroblasts (MEFs), but expression of LPV LT is insufficient to transform MEFs. Furthermore, LPV sT induces immortalization and transformation of MEFs. Thus, in the case of LPV, sT is the main mediator of oncogenesis. PMID:26517398

  15. Co-expression of Erns and E2 genes of classical swine fever virus by replication-defective recombinant adenovirus completely protects pigs against virulent challenge with classical swine fever virus.

    PubMed

    Sun, Yongke; Yang, Yuai; Zheng, Huanli; Xi, Dongmei; Lin, Mingxing; Zhang, Xiaomin; Yang, Linfu; Yan, Yulin; Chu, Xiaohui; Bi, Baoliang

    2013-04-01

    The objective of this study was to construct a recombinant adenovirus for future CSFV vaccines used in the pig industry for the reduction of losses involved in CSF outbreaks. The Erns and E2 genes of classical swine fever virus (CSFV), which encode the two main protective glycoproteins from the "Shimen" strain of CSFV, were combined and inserted into the replication-defective human adenovirus type-5 and named the rAd-Erns-E2. Nine pigs were randomly assigned to three treatment groups (three pigs in each group) including the rAd-Erns-E2, hAd-CMV control and DMEM control. Intramuscular vaccination with 2×10(6) TCID(50) of the rAd-Erns-E2 was administered two times with an interval of 21 days. At 42 days post inoculation, pigs in all groups were challenged with a lethal dose of 1×10(3) TCID(50) CSFV "Shimen" strain. Observation of clinical signs was made and the existence of CSFV RNA was detected. Animals in the hAd-CMV and DMEM groups showed severe clinical CSF symptoms and were euthanized from 7 to 10 days after the challenge. However, no adverse clinical CSF signs were observed in vaccinated pigs after the administration of rAd-Erns-E2 and even after CSFV challenge. Neither CSFV RNA nor pathological changes were detected in the tissues of interest of the above vaccinated pigs. These results implied that the recombination adenovirus carrying the Erns-E2 genes could be used to prevent swine from classical swine fever. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Isoform-Specific Regulation and Localization of the Coxsackie and Adenovirus Receptor in Human Airway Epithelia

    PubMed Central

    Excoffon, Katherine J. D. A.; Gansemer, Nicholas D.; Mobily, Matthew E.; Karp, Philip H.; Parekh, Kalpaj R.; Zabner, Joseph

    2010-01-01

    Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR) to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAREx7 and CAREx8). We found low-level expression of the CAREx8 isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAREx7, CAREx8 localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAREx7, CAREx8 differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAREx8 providing a potential mechanism for the apical localization of CAREx8 in airway epithelial. In summary, apical localization of CAREx8 may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins. PMID:20361046

  17. Functional Effects of Coxsackievirus and Adenovirus Receptor Glycosylation on Homophilic Adhesion and Adenoviral Infection▿

    PubMed Central

    Excoffon, Katherine J. D. Ashbourne; Gansemer, Nicholas; Traver, Geri; Zabner, Joseph

    2007-01-01

    The coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and homophilic adhesion protein. The extracellular portion of CAR consists of two immunoglobulin (Ig)-like domains, each with a consensus sequence for N-glycosylation. We used chemical, genetic, and biochemical studies to show that both sites are glycosylated and contribute to the function of CAR. Although the glycosylation of CAR does not alter cell surface levels or junctional localization, it affects both adhesion and adenovirus infection in unique ways. CAR-mediated adhesion appears to require at least one site of glycosylation since cells expressing CAR without glycosylation do not cluster with each other. In contrast, glycosylation of the Ig-like domain proximal to the membrane is key to the cooperative behavior of adenovirus binding and infection. Contrary to the hypothesis that cooperativity improves viral infection, our data show that although glycosylation of the D2 domain is required for adenovirus cooperative binding, it has a negative consequence upon infection. This is the first report dissecting the adhesion and receptor activities of CAR, revealing that factors other than the binding interface play a significant role in the function of CAR. These data have important implications for both cancers with altered glycosylation states and cancer treatments using oncolytic adenovirus. PMID:17376928

  18. Localization of the N-terminus of minor coat protein IIIa in the adenovirus capsid

    PubMed Central

    San Martín, Carmen; Glasgow, Joel N.; Borovjagin, Anton; Beatty, Matthew S.; Kashentseva, Elena A.; T. Curiel, David; Marabini, Roberto; Dmitriev, Igor P.

    2008-01-01

    Summary Minor coat protein IIIa is conserved in all adenoviruses and required for correct viral assembly, but its precise function in capsid organization is unknown. The latest adenovirus capsid model proposes that IIIa is located underneath the vertex region. To obtain experimental evidence on the location of IIIa and further define its role, we engineered the IIIa gene to encode heterologous N-terminal peptide extensions. Recombinant adenovirus variants with IIIa encoding six-histidine tag (6-His), 6-His and FLAG peptides, or 6-His linked to FLAG with a (Gly4Ser)3 linker were rescued and analyzed for virus yield, capsid incorporation of heterologous peptides, and capsid stability. Longer extensions could not be rescued. Western blot analysis confirmed that the modified IIIa proteins were expressed in infected cells and incorporated into virions. In the adenovirus encoding the 6-His-linker-FLAG-IIIa gene, the 6-His tag was present in light particles but not in mature virions. Immuno-electron microscopy of this virus showed that the FLAG epitope is not accessible to antibodies on the viral particles. Three-dimensional electron microscopy (3DEM) and difference mapping located the IIIa N-terminal extension beneath the vertex complex, wedged at the interface between penton base and the peripentonal hexons, therefore supporting the latest proposed model. The position of the IIIa N-terminus and its low tolerance for modification provide new clues for understanding the role of this minor coat protein in adenovirus capsid assembly and disassembly. PMID:18786542

  19. Cancer-targeted oncolytic adenoviruses for modulation of the immune system.

    PubMed

    Cerullo, Vincenzo; Capasso, Cristian; Vähä-Koskela, Markus; Hemminki, Otto; Hemminki, Akseli

    2017-05-02

    Adenovirus is one of the most commonly used vectors for gene therapy and it is the first approved virus-derived drug for treatment of cancer. As an oncolytic agent, it can induce lysis of infected cells, but it can also engage the immune system, promoting activation and maturation of antigen-presenting cells (APCs). In essence, oncolysis combined with the associated immunostimulatory actions result in a "personalized in situ vaccine" for each patient. In order to take full advantage of these features, we should try to understand how adenovirus interacts with the immune system, what are the receptors involved in triggering subsequent signals and which kind of responses they elicit. Tackling these questions will give us further insight in how to manipulate adenovirus-mediated immune responses for enhancement of anti-tumor efficacy. In this review, we first highlight how oncolytic adenovirus interacts with the innate immune system and its receptors such as Toll-like receptors, nucleotide-binding and oligomerization domain (NOD)-like receptors and other immune sensors. Then we describe the effect of these interactions on the adaptive immune system and its cells, especially B and T lymphocytes. Finally, we summarize the most significant preclinical and clinical results in the field of gene therapy where researchers have engineered adenovirus to manipulate the host immune system by expressing cytokines and signaling mediators. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Correction of a deletion mutant by gene targeting with an adenovirus vector.

    PubMed Central

    Wang, Q; Taylor, M W

    1993-01-01

    The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter. Images PMID:8423811

  1. Protein expression in a transformed trabecular meshwork cell line: proteome analysis.

    PubMed

    Steely, H Thomas; Dillow, Glen W; Bian, Liangqian; Grundstad, Jason; Braun, Terry A; Casavant, Thomas L; McCartney, Mitchell D; Clark, Abbot F

    2006-04-18

    Characterization of the human trabecular meshwork (TM) proteome is hindered by the small mass of intact tissue and the slow growth of cultured cell strains. We have previously characterized a transformed TM cell strain (GTM3) that demonstrates many of the same protein expression and cell signaling systems of nontransformed cell strains. The aim of this study was to initiate a proteomic survey of GTM3 cells as the initial step toward characterization of the complete human TM proteome. GTM3 cells were cultured to confluence, harvested and solubilized in urea/Nonidet. The protein extract (600 mug) was focused in immobilized isoelectric focusing (IEF) strips, separated by 10% SDS PAGE, and visualized with colloidal Coomassie Blue. Spots of interest were excised, destained, and the contained proteins subjected to in-gel reduction, derivatization, and tryptic digestion. Tryptic peptides were extracted and analyzed by electrospray LC/MS/MS. Protein identification was made using the TurboSequest search algorithm and a recent version of the nonredundant human protein database downloaded from the National Center for Biotechnology Information (NCBI). Eighty-seven (87) primary proteins and 93 variants of these proteins were identified. A website was created (TM proteome) that combines data such as graphic spot location within the gel, peptide sequence, apparent and calculated pI, apparent and calculated mass, percentage of coverage, and protein informatic website links. Proteomic analysis of a transformed human TM cell line has been initiated combining preparative two-dimensional PAGE separation, LC/MS/MS analysis of major proteins, and bioinformatic cataloging of the data. Further investigation of data from the transformed cell strain will be used in a comparative fashion for spot identification of analytical proteomic gels of human TM tissue and cultured normal cells. These initial data will form the base from which the characterization of protein expression in the normal

  2. Agrobacterium-mediated transformation of polyploid cereals. The efficiency of selection and transgene expression in wheat.

    PubMed

    Przetakiewicz, Anna; Karaś, Agnieszka; Orczyk, Wacław; Nadolska-Orczyk, Anna

    2004-01-01

    Three combinations of Agrobacterium tumefaciens strains and vectors were used in the transformation of selected Polish wheat cultivars. The combinations were: two hypervirulent strains, AGL1, containing the pDM805 binary plasmid, and EHA101, containing pGAH; and the common Agro strain LBA4404, harboring the super-binary pTOK233 vector. pDM805 contained bar under the control of Ubi1 promoter, pGAH had nptII under nos, and pTOK233 had hpt under 35S. Additionally, pDM805 and pTOK233 carried the gus reporter gene under the Act1 promoter or 35S promoter, respectively. The highest selection rate was 12.6% and was obtained with EHA101(pGAH) on a kanamycin-containing medium. Sixty-five of the plants grown on that medium were PCR positive. The second best combination was LBA4404(pTOK233) and kanamycin selection, which gave an average transformation rate of 2.3%. Phosphinothricin selection gave 1.0% transformation efficiency, while hygromycin, depending on the strain/vector used, gave from 0.2 to 0.4%. PCR tests in T1 revealed that 67% of the lines showed a 3:1 segregation ratio, and 11% a 15:1 ratio, while in 22%, segregation was non-Mendelian. The high number of T0 transgenic plants containing one copy of the transgene was confirmed via Southern blot analysis. Kanamycin resistance in the T1 generation was very low; in some lines, all the progeny were kanamycin sensitive. GUS expression, only tested in young T1 plants, was in agreement with Mendelian segregation in three out of the twelve tested. The factors influencing the efficiency of selection and transgene expression are discussed in this paper.

  3. The Underlying Bases of Gene Expression Differences in Stable Transformants of the ROSY Locus in DROSOPHILA MELANOGASTER

    PubMed Central

    Daniels, Stephen B.; McCarron, Margaret; Love, Carol; Clark, Stephen H.; Chovnick, Arthur

    1986-01-01

    This report represents a continuation of our laboratory's effort to understand the major phenomena associated with P-M dysgenesis-mediated transformation in Drosophila. A group of stable transformants are characterized with respect to rosy gene expression. Stable, true-breeding, line-specific variants in gene expression are described. These are shown to be associated with single transposons present in each line, and the lines are free of functional P elements. The effects on expression are cis-acting, and there are no identifiable rosy DNA sequence lesions associated with these transposons. Evidence is presented that demonstrates that two features of the transformation experimental system are responsible for such variation. The first relates to the fact that the transposons insert at numerous genomic sites. Both heterochromatic and euchromatic position effects are characterized. The second relates to the fact that transformation involves dysgenic mobilization of a P-element transposon. This process is mutagenic, and such a mutation is characterized. PMID:3013723

  4. Adenovirus E1A specifically blocks SWI/SNF-dependent transcriptional activation.

    PubMed Central

    Miller, M E; Cairns, B R; Levinson, R S; Yamamoto, K R; Engel, D A; Smith, M M

    1996-01-01

    Expression of the adenovirus E1A243 oncoprotein in Saccharomyces cerevisiae produces a slow-growth phenotype with accumulation of cells in the G1 phase of the cell cycle. This effect is due to the N-terminal and CR1 domains of E1A243, which in rodent cells are involved in triggering cellular transformation and also in binding to the cellular transcriptional coactivator p300. A genetic screen was undertaken to identify genes required for the function of E1A243 in S. cerevisiae. This screen identified SNF12, a gene encoding the 73-kDa subunit of the SWI/SNF transcriptional regulatory complex. Mutation of genes encoding known members of the SWI/SNF complex also led to loss of E1A function, suggesting that the SWI/SNF complex is a target of E1A243. Moreover, expression of E1A in wild-type cells specifically blocked transcriptional activation of the INO1 and SUC2 genes, whose activation pathways are distinct but have a common requirement for the SWI/SNF complex. These data demonstrate a specific functional interaction between E1A and the SWI/SNF complex and suggest that a similar interaction takes place in rodent and human cells. PMID:8816487

  5. Protection of non-human primates against rabies with an adenovirus recombinant vaccine

    SciTech Connect

    Xiang, Z.Q.; Greenberg, L.; Ertl, H.C.; Rupprecht, C.E.

    2014-02-15

    Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. - Highlights: • Pre-exposure vaccination with vaccine based on a chimpanzee derived adenovirus protects against rabies. • Protection is sustained. • Protection is achieved with single low-dose of vaccine given intramuscularly. • Protection is not affected by pre-existing antibodies to common human serotypes of adenovirus.

  6. Chapter eight--Oncolytic adenoviruses for cancer immunotherapy: data from mice, hamsters, and humans.

    PubMed

    Cerullo, Vincenzo; Koski, Anniina; Vähä-Koskela, Markus; Hemminki, Akseli

    2012-01-01

    Adenovirus is one of the most commonly used vectors for gene therapy and two products have already been approved for treatment of cancer in China (Gendicine(R) and Oncorine(R)). An intriguing aspect of oncolytic adenoviruses is that by their very nature they potently stimulate multiple arms of the immune system. Thus, combined tumor killing via oncolysis and inherent immunostimulatory properties in fact make these viruses in situ tumor vaccines. When further engineered to express cytokines, chemokines, tumor-associated antigens, or other immunomodulatory elements, they have been shown in various preclinical models to induce antigen-specific effector and memory responses, resulting both in full therapeutic cures and even induction of life-long tumor immunity. Here, we review the state of the art of oncolytic adenovirus, in the context of their capability to stimulate innate and adaptive arms of the immune system and finally how we can modify these viruses to direct the immune response toward cancer.

  7. Transient HEXA expression in a transformed human fetal Tay-Sachs disease neuroglial cell line

    SciTech Connect

    Fernandes, M.J.; Hechtman, P.; Kaplan, F.

    1994-09-01

    Tay-Sachs disease (TSD) is a severe neurodegenerative disorder characterized by the accumulation of GM{sub 2} ganglioside in the neurons of the central cortex. The recessively inherited disorder results from deficiency of hexosaminidase A (Hex A), a heterodimer of an {alpha} and {beta} subunit encoded by the HEXA and HEXB genes. Expression of HEXA mutations in COS cells has several disadvantages including high endogenous hexosaminidase activity. We report a new transient expression system with very low endogenous Hex A activity. An SV40-transformed fetal TSD neuroglial cell line was assessed for transient expression of the HEXA gene. pCMV{alpha}, a vector incorporating the cytomegalovirus promoter with the human {alpha}-subunit cDNA insert, proved to be the most efficient expression vector. Transfection of 4x10{sup 6} cells with 5-20 {mu}g of plasmid resulted in 100 to 500-fold Hex A activity (4MUGS hydrolysis) relative to mock-transfected cells. Use of pCMV{beta}-Gal as a control for transfection efficiency indicated that 10-20% of cells were transfected. Hex A specific activity increased for at least 72 h post-transfection. This new transient expression system should greatly improve the characterization of mutations in which low levels of HEXA expression result in milder clinical phenotypes and permit studies on enzymatic properties of mutant forms of Hex A. Since the cells used are of CNS origin and synthesize gangliosides, it should also be possible to study, in culture, the metabolic phenotype associated with TSD.

  8. Prognostic significance of human pituitary tumor-transforming gene immunohistochemical expression in differentiated thyroid cancer.

    PubMed

    Sáez, Carmen; Martínez-Brocca, M Asunción; Castilla, Carolina; Soto, Alfonso; Navarro, Elena; Tortolero, María; Pintor-Toro, José A; Japón, Miguel A

    2006-04-01

    Human securin pituitary tumor-transforming gene (hPTTG) is overexpressed in a variety of primary neoplasias, including differentiated thyroid cancer (DTC). The objective of this study was to examine the immunohistochemical expression of hPTTG in DTC and its association with known prognostic factors. hPTTG expression was analyzed by immunostaining on paraffin-embedded tissues. Clinical data were used to determine any associations between the expression of hPTTG and prognostic variables of DTC. A median follow-up of 43 months allowed us to analyze the persistence of disease and the response to radioiodine therapy. The study was conducted at a tertiary university hospital. Ninety-five patients undergoing surgical resection for DTC (n = 60) or benign nodular thyroid disease (n = 35) were studied. The main outcome measure was the association between hPTTG expression and prognostic factors in DTC. Among DTC cases, 21 (35%) had low and 39 (65%) had high hPTTG immunostaining. Adjacent nonneoplastic thyroid tissue was largely unstained. Among benign nodular thyroid disease cases, immunostaining was detected focally in eight (22.8%). A significant association was found between hPTTG expression and the presence of nodal (P < 0.01) or distant metastases (P < 0.05). A significant association with TNM was also found, because 83.3% of advanced TNM stages showed elevated hPTTG (P < 0.05). The association between hPTTG overexpression and decreased radioiodine uptake during follow-up was also significant (P < 0.05). The expression levels of hPTTG were confirmed as an independent prognostic factor for persistent disease (relative risk, 3.0; 95% confidence interval, 1.1-8.7; P < 0.05). Immunohistochemical analysis of hPTTG is of potential value in the determination of tumor aggressiveness in DTC.

  9. Pulmonary vasculature directed adenovirus increases epithelial lining fluid alpha-1 antitrypsin levels.

    PubMed

    Buggio, Maurizio; Towe, Christopher; Annan, Anand; Kaliberov, Sergey; Lu, Zhi Hong; Stephens, Calvin; Arbeit, Jeffrey M; Curiel, David T

    2016-01-01

    Gene therapy for inherited serum deficiency disorders has previously been limited by the balance between obtaining adequate expression and causing hepatic toxicity. Our group has previously described modifications of a replication deficient human adenovirus serotype 5 that increase pulmonary vasculature transgene expression. In the present study, we use a modified pulmonary targeted adenovirus to express human alpha-1 antitrypsin (A1AT) in C57BL/6 J mice. Using the targeted adenovirus, we were able to achieve similar increases in serum A1AT levels with less liver viral uptake. We also increased pulmonary epithelial lining fluid A1AT levels by more than an order of magnitude compared to that of untargeted adenovirus expressing A1AT in a mouse model. These gains are achieved along with evidence of decreased systemic inflammation and no evidence for increased inflammation within the vector-targeted end organ. In addition to comprising a step towards clinically viable gene therapy for A1AT, maximization of protein production at the site of action represents a significant technical advancement in the field of systemically delivered pulmonary targeted gene therapy. It also provides an alternative to the previous limitations of hepatic viral transduction and associated toxicities. Copyright © 2016 John Wiley & Sons, Ltd.

  10. Mutant IDH1 Expression Drives TERT Promoter Reactivation as Part of the Cellular Transformation Process.

    PubMed

    Ohba, Shigeo; Mukherjee, Joydeep; Johannessen, Tor-Christian; Mancini, Andrew; Chow, Tracy T; Wood, Matthew; Jones, Lindsey; Mazor, Tali; Marshall, Roxanne E; Viswanath, Pavithra; Walsh, Kyle M; Perry, Arie; Bell, Robert J A; Phillips, Joanna J; Costello, Joseph F; Ronen, Sabrina M; Pieper, Russell O

    2016-11-15

    Mutations in the isocitrate dehydrogenase gene IDH1 are common in low-grade glioma, where they result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, and gliomagenesis. IDH1 mutations also cosegregate with mutations in the ATRX gene and the TERT promoter, suggesting that IDH mutation may drive the creation or selection of telomere-stabilizing events as part of immortalization/transformation process. To determine whether and how this may occur, we investigated the phenotype of pRb-/p53-deficient human astrocytes engineered with IDH1 wild-type (WT) or R132H-mutant (IDH1(mut)) genes as they progressed through their lifespan. IDH1(mut) expression promoted 2HG production and altered histone methylation within 20 population doublings (PD) but had no effect on telomerase expression or telomere length. Accordingly, cells expressing either IDH1(WT) or IDH1(mut) entered a telomere-induced crisis at PD 70. In contrast, only IDH1(mut) cells emerged from crisis, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo Clonal populations of postcrisis IDH1(mut) cells displayed shared genetic alterations, but no mutations in ATRX or the TERT promoter were detected. Instead, these cells reactivated telomerase and stabilized their telomeres in association with increased histone lysine methylation (H3K4me3) and c-Myc/Max binding at the TERT promoter. Overall, these results show that although IDH1(mut) does not create or select for ATRX or TERT promoter mutations, it can indirectly reactivate TERT, and in doing so contribute to astrocytic immortalization and transformation. Cancer Res; 76(22); 6680-9. ©2016 AACR.

  11. A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression

    PubMed Central

    Bopp, Daniel; Beukeboom, Leo W.; van de Zande, Louis

    2013-01-01

    Although sex determination is a universal process in sexually reproducing organisms, sex determination pathways are among the most highly variable genetic systems found in nature. Nevertheless, general principles can be identified among the diversity, like the central role of transformer (tra) in insects. When a functional TRA protein is produced in early embryogenesis, the female sex determining route is activated, while prevention of TRA production leads to male development. In dipterans, male development is achieved by prevention of female-specific splicing of tra mRNA, either mediated by X-chromosome dose or masculinizing factors. In Hymenoptera, which have haplodiploid sex determination, complementary sex determination and maternal imprinting have been identified to regulate timely TRA production. In the parasitoid Nasonia, zygotic transformer (Nvtra) expression and splicing is regulated by a combination of maternal provision of Nvtra mRNA and silencing of Nvtra expression in unfertilized eggs. It is unclear, however, if this silencing is directly on the tra locus or whether it is mediated through maternal silencing of a trans-acting factor. Here we show that in Nasonia, female sex determination is dependent on zygotic activation of Nvtra expression by an as yet unknown factor. This factor, which we propose to term womanizer (wom), is maternally silenced during oogenesis to ensure male development in unfertilized eggs. This finding implicates the upstream recruitment of a novel gene in the Nasonia sex determining cascade and supports the notion that sex determining cascades can rapidly change by adding new components on top of existing regulators. PMID:23717455

  12. Increased expression of transforming growth factor α precursors in acute experimental colitis in rats

    PubMed Central

    Hoffmann, P; Zeeh, J; Lakshmanan, J; Wu, V; Procaccino, F; Reinshagen, M; McRoberts, J; Eysselein, V

    1997-01-01

    Background and aim—Epidermal growth factor (EGF) and transforming growth factor α (TGF-α), members of the EGF family of growth factors, protect rat gastric and colonic mucosa against injury. Having shown previously that exogenously applied EGF protects rat colonic mucosa against injury, the aim of the present study was to evaluate the endogenously expressed ligand mediating the protective effect of EGF/TGF-α in vivo. 
Methods—In an experimental model of trinitrobenzene sulphonic acid (TNBS)/ ethanol induced colitis in rats EGF and TGF-α expression was evaluated using a ribonuclease protection assay, northern blot analysis, western blot analysis, and immunohistochemistry. 
Results—TGF-α mRNA increased 3-4 times at 4-8 hours after induction of colitis and returned to control levels within 24 hours. TGF-α immunoreactive protein with a molecular size of about 28kDa representing TGF-α precursors increased markedly after induction of colitis with a peak at 8-12 hours. No fully processed 5.6 kDa TGF-α protein was detected in normal or inflamed colon tissue. Only a weak signal for EGF mRNA expression was detected in the rat colon and no EGF protein was observed by immunohistochemistry or western blot analysis. 
Conclusions—TGF-α precursors are the main ligands for the EGF receptor in acute colitis. It is hypothesised that TGF-α precursors convey the biological activity of endogenous TGF-α peptides during mucosal defence and repair. 

 Keywords: transforming growth factor alpha (TGF-α); epidermal growth factor (EGF); precursor molecules; colitis; rat PMID:9301498

  13. The expression of a viral microRNA is regulated by clustering to allow optimal B cell transformation

    PubMed Central

    Haar, Janina; Contrant, Maud; Bernhardt, Katharina; Feederle, Regina; Diederichs, Sven; Pfeffer, Sébastien; Delecluse, Henri-Jacques

    2016-01-01

    The Epstein-Barr virus (EBV) transforms B cells by expressing latent proteins and the BHRF1 microRNA cluster. MiR-BHRF1–3, its most transforming member, belongs to the recently identified group of weakly expressed microRNAs. We show here that miR-BHRF1–3 displays an unusually low propensity to form a stem–loop structure, an effect potentiated by miR-BHRF1–3's proximity to the BHRF1 polyA site. Cloning miR-BHRF1–2 or a cellular microRNA, but not a ribozyme, 5′ of miR-BHRF1–3 markedly enhanced its expression. However, a virus carrying mutated miR-BHRF1–2 seed regions expressed miR-BHRF1–3 at normal levels and was fully transforming. Therefore, miR-BHRF1–2's role during transformation is independent of its seed regions, revealing a new microRNA function. Increasing the distance between miR-BHRF1–2 and miR-BHRF1–3 in EBV enhanced miR-BHRF1–3's expression but decreased its transforming potential. Thus, the expression of some microRNAs must be restricted to a narrow range, as achieved by placing miR-BHRF1–3 under the control of miR-BHRF1–2. PMID:26635399

  14. Trehalose expression confers desiccation tolerance on human cells.

    PubMed

    Guo, N; Puhlev, I; Brown, D R; Mansbridge, J; Levine, F

    2000-02-01

    Many organisms that withstand desiccation express the disaccharide trehalose. We have now expressed the otsA and otsB genes of Escherichia coli, which encode trehalose biosynthetic enzymes, in human primary fibroblasts using a recombinant adenovirus vector. Infected cells produced increased amounts of trehalose with increasing multiplicity of infection (MOI). Human primary fibroblasts expressing trehalose could be maintained in the dry state for up to five days. Fourier transform infrared spectroscopy indicated that dry, but viable, human cells contained no detectable water. This study shows that mammalian cells can be engineered to retain viability in the absence of water.

  15. Selective targeting of human cells by a chimeric adenovirus vector containing a modified fiber protein.

    PubMed Central

    Stevenson, S C; Rollence, M; Marshall-Neff, J; McClelland, A

    1997-01-01

    The adenovirus fiber protein is responsible for attachment of the virion to unidentified cell surface receptors. There are at least two distinct adenovirus fiber receptors which interact with the group B (Ad3) and group C (Ad5) adenoviruses. We have previously shown by using expressed adenovirus fiber proteins that it is possible to change the specificity of the fiber protein by exchanging the head domain with another serotype which recognizes a different receptor (S. C. Stevenson et al., J. Virol. 69:2850-2857, 1995). A chimeric fiber cDNA containing the Ad3 fiber head domain fused to the Ad5 fiber tail and shaft was incorporated into the genome of an adenovirus vector with E1 and E3 deleted encoding beta-galactosidase to generate Av9LacZ4, an adenovirus particle which contains a chimeric fiber protein. Western blot analysis of the chimeric fiber vector confirmed expression of the chimeric fiber protein and its association with the adenovirus capsid. Transduction experiments with fiber protein competitors demonstrated the altered receptor tropism of the chimeric fiber vector compared to that of the parental Av1LacZ4 vector. Transduction of a panel of human cell lines with the chimeric and parental vectors provided evidence for a different cellular distribution of the Ad5 and Ad3 receptors. Three cell lines (THP-1, MRC-5, and FaDu) were more efficiently transduced by the vector containing the Ad3 fiber head than by the Ad5 fiber vector. In contrast, human coronary artery endothelial cells were transduced more readily with the vector containing the Ad5 fiber than with the chimeric fiber vector. HeLa and human umbilical vein endothelial cells were transduced at equivalent levels compared with human diploid fibroblasts, which were refractory to transduction with both vectors. These results provide evidence for the differential expression of the Ad5 and Ad3 receptors on human cell lines derived from clinically relevant target tissues. Furthermore, we show that exchange

  16. The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in human tumors in vitro.

    PubMed

    Grill, Jacques; Lamfers, Martine L M; van Beusechem, Victor W; Dirven, Clemens M; Pherai, D Shareen; Kater, Mathijs; Van der Valk, Paul; Vogels, Ronald; Vandertop, W Peter; Pinedo, Herbert M; Curiel, David T; Gerritsen, Winald R

    2002-11-01

    The use of adenoviruses for gene transfer and as oncolytic agents is currently receiving widespread attention. As specific constraints to adenovirus distribution and spread cannot be studied in cell cultures, there is a need for an in vitro three-dimensional (3D) model mimicking the in vivo biology of tumors. We studied the interactions between tumor and adenoviruses using multicellular spheroids grown from primary brain tumor material. Using beta-galactosidase and luciferase reporter genes expressed by replication-defective adenoviruses, we showed that infection was restricted to the first layer of cells. Using a replication-competent adenovirus expressing the luciferase gene, we showed that transgene expression in the spheroid was considerably enhanced and that viral spreading deep into the 3D structure took place. In addition, a tetrazolium salt-based metabolic assay could be used to compare the oncolytic activity of different concentrations of replication-competent adenoviruses. We can conclude that organotypic spheroids offer a versatile in vitro system for studying distribution, spread, and oncolysis by adenoviruses in a clinically relevant model.

  17. Cloning and expression analysis of a transformer gene in Daphnia pulex during different reproduction stages.

    PubMed

    Chen, Ping; Xu, Shan-Liang; Zhou, Wei; Guo, Xiao-Ge; Wang, Chun-Lin; Wang, Dan-Li; Zhao, Yun-Long

    2014-05-01

    The full-length cDNA of a transformer gene (Dptra) was cloned from the cladoceran Daphnia pulex using RACE. Dptra expression was assessed by qPCR and whole-mount in situ hybridization in different reproductive stages. The Dptra cDNA, 1652bp in length, has a 1158-bp open reading frame that encodes a 385 amino acid polypeptide containing one Sex determination protein N terminal (SDP_N) superfamily, eight putative phosphorylation sites, and an arginine-serine (RS)-rich domain at the N-terminus. Dptra showed 81%, 53%, 51% and 45% identity to orthologous genes in Daphnia magna, Apis mellifera, Apis cerana and Bombus terrestris, respectively. Phylogenetic analysis based on deduced amino acid sequences revealed that Dptra clustered in the hymenopteran clade and was most closely related to D. magna and A. mellifera. qPCR showed that Dptra expression increased significantly (P<0.05) in different reproductive stages in the following order: male, ephippial female, parthenogenetic female, resting egg and juvenile female. Dptra expression is significantly different between males and females and it is significantly greater in ephippial females and males than in parthenogenetic D. pulex (with summer eggs). Whole-mount in situ hybridization revealed that Dptra was expressed at different levels between males and females. In males, hybridization signals were found in the first antennae, second antennae and thoracic limb, whereas expression levels in the corresponding sites of parthenogenetic and ephippial females were relatively weak. This suggests that the Dptra gene plays significant roles in switching modes of reproduction and in sexual differentiation. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Inhibition of spermidine synthase gene expression by transforming growth factor-beta 1 in hepatoma cells.

    PubMed Central

    Nishikawa, Y; Kar, S; Wiest, L; Pegg, A E; Carr, B I

    1997-01-01

    We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression. PMID:9020892

  19. Dynamic Epstein-Barr virus gene expression on the path to B-cell transformation.

    PubMed

    Price, Alexander M; Luftig, Micah A

    2014-01-01

    Epstein-Barr virus (EBV) is an oncogenic human herpesvirus in the γ-herpesvirinae subfamily that contains a 170-180kb double-stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B-cell compartment of the peripheral blood. EBV can be reactivated from its latent state, leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome and structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady-state viral gene expression within EBV-immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection, EBV only expressed the well-characterized latency-associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation and delayed responses in the known latency genes. This chapter summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, the inhibition of apoptosis, and innate and adaptive immune responses.

  20. Pattern of galectins expression in actinic cheilitis with different risks of malignant transformation.

    PubMed

    Lopes, Maria Luiza Diniz de Sousa; Nonaka, Cassiano Francisco Weege; Queiroz, Lélia Maria Guedes; de Souza, Lélia Batista; Miguel, Márcia Cristina da Costa; da Silveira, Éricka Janine Dantas

    2016-09-01

    Actinic cheilitis (AC) is a chronic inflammatory lesion that in some situations can turn into squamous cell carcinoma of the lip. The molecular mechanisms involved in this process are not yet completely understood. This study aimed to investigate the expression pattern of galectins in actinic cheilitis according to the histopathological grading. Immunoexpression of galectin-1, galectin-3, galectin-7, and galectin-9 was semiquantitatively analyzed in 65 cases of actinic cheilitis graded as low risk (n = 40) or high risk (n = 25) of malignant transformation. Association between the location of the galectins in the cellular compartments and histopathological grading was analyzed. Galectin-1 was mainly observed in the cell cytoplasm, and was elevated (score 3) in 60% of cases, regardless of the histopathological grade (P > 0.05). Galectin-3 expression was higher in high-risk group than in the low-risk group (P < 0.05), with a predominant expression in the cytoplasm and nucleus of low-risk (67.5%), and only in the cytoplasm of high-risk cases (60%) (P < 0.05). Galectin-7 expression did not show significant differences between low-risk and high-risk groups (P > 0.05). With respect to galectin-9, 89.2% of cases were positive, showing decrease in median of scores as there was an increase in histological grade (P < 0.001), with predominant expression in the nucleus and cytoplasm. This study is the first indication of galectins involvement in the pathogenesis and morphologic progression of actinic cheilitis, particularly galectin-3 and galectin-9. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. The expression of transforming growth factor beta in pregnant rat myometrium is hormone and stretch dependent.

    PubMed

    Shynlova, Oksana; Tsui, Prudence; Dorogin, Anna; Langille, B Lowell; Lye, Stephen J

    2007-09-01

    From a quiescent state in early pregnancy to a highly contractile state in labor, the myometrium displays tremendous growth and remodeling. We hypothesize that the transforming growth factor beta (TGFbeta) system is involved in the differentiation of pregnant myometrium throughout gestation and labor. Furthermore, we propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial TGFbetas. The expression of TGFbeta1-3 mRNAs and proteins was examined by real-time PCR, Western immunoblot, and localized with immunohistochemistry in the rat uterus throughout pregnancy and labor. Tgfbeta1-3 genes were expressed differentially in pregnant myometrium. Tgfbeta2 gene was not affected by pregnancy, whereas the Tgfbeta1 gene showed a threefold increase during the second half of gestation. In contrast, we observed a dramatic bimodal change in Tgfbeta3 gene expression throughout pregnancy. Tgfbeta3 mRNA levels first transiently increased at mid-gestation (11-fold on day 14) and later at term (45-fold at labor, day 23). Protein expression levels paralleled the changes in mRNA. Treatment of pregnant rats with the progesterone (P4) receptor antagonist RU486 induced premature labor on day 19 and increased Tgfbeta3 mRNA, whereas artificial maintenance of elevated P4 levels at late gestation (days 20-23) caused a significant decrease in the expression of Tgfbeta3 gene. In addition, Tgfbeta3 was up-regulated specifically in the gravid horn of unilaterally pregnant rats subjected to a passive biological stretch imposed by the growing fetuses, but not in the empty horn. Collectively, these data indicate that the TGFbeta family contributes in the regulation of myometrial activation at term integrating mechanical and endocrine signals for successful labor contraction.

  2. Molecular Analysis of Adenovirus Isolates from Previously Vaccinated Young Adults

    DTIC Science & Technology

    2004-04-01

    NAVAL HEALTH RESEARCH CENTER MOLECULAR ANALYSIS OF ADENOVIRUS ISOLATES FROM PREVIOUSLY VACCINATED YOUNG ADULTS D. A. Blasiole...Molecular Analysis of Adenovirus Isolates From Previously Vaccinated Young Adults . 6. AUTHORS Daniel A Blasiole, David Metzgar, Luke T Daum, Margaret AK

  3. Transforming growth factor beta-1 expression in macrophages of human chronic periapical diseases.

    PubMed

    Liang, Z-Z; Li, J; Huang, S-G

    2017-03-30

    The objective of this study was to observe the distribution of macrophages (MPs) expressing transforming growth factor beta-1 (TGF-β1) in tissue samples from patients with different human chronic periapical diseases. In this study, samples were collected from 75 volunteers, who were divided into three groups according to classified standards, namely, healthy control (N = 25), periapical granuloma (N = 25), and periapical cyst (N = 25). The samples were fixed in 10% buffered formalin for more than 48 h, dehydrated, embedded, and stained with hematoxylin and eosin for histopathology. Double immunofluorescence was conducted to analyze the expression of TGF-β-CD14 double-positive MPs in periapical tissues. The number of double-positive cells (cells/mm(2)) were significantly higher in the chronic periapical disease tissues (P < 0.01) compared to that in the control tissue; in addition, the density of TGF-β1-CD14 double positive cells was significantly higher in the periapical cyst group than in the periapical granuloma group (P < 0.01). The number of TGF-β1 expressing macrophages varied with human chronic periapical diseases. The TGF-β1-CD14 double-positive cells might play an important role in the pathology of human chronic periapical diseases.

  4. Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression.

    PubMed

    Maximova, S; Miller, C; Antúnez de Mayolo, G; Pishak, S; Young, A; Guiltinan, M J

    2003-06-01

    We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L. using cotyledonary explants from primary somatic embryos (SEs) and A. tumefaciens strain AGL1. Transgenic plants carrying the visible marker, gene green fluorescent protein ( EGFP), the selectable marker gene neomycin phosphotransferase II ( NPTII), the class I chitinase gene from cacao ( Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs. The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone. However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics. Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis. Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity. Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants. Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression-a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA.

  5. Transforming growth factor beta enhances integrin expression and type IV collagenase secretion in human monocytes.

    PubMed Central

    Wahl, S M; Allen, J B; Weeks, B S; Wong, H L; Klotman, P E

    1993-01-01

    Transforming growth factor beta (TGF-beta), secreted within an inflammatory site or injected locally, induces leukocyte margination, chemotaxis, and accumulation. In addition to its potent direct chemotactic activity, TGF-beta may promote this leukocyte response by influencing cell surface integrin expression. At picomolar concentrations, TGF-beta increases steady-state mRNA levels for both the alpha 5 and the beta 1 chain of the fibronectin receptor in human blood monocytes. This increase in gene expression is reflected by selectively enhanced expression of alpha 5 (CDw49e), beta 1 (CDw29), and also alpha 3 (CDw49c) adhesion molecules on the cell surface. Functionally, TGF-beta promotes, in a dose- and time-dependent fashion, monocyte adhesion to type IV collagen, laminin, and fibronectin. Potentially facilitating the movement of monocytes through the extracellular matrix, TGF-beta triggers transcriptional and posttranscriptional regulation of both the 92-kDa and the 72-kDa gelatinase/type IV collagenase. Thus, TGF-beta may play a pivotal role in the early phases of inflammation and repair through its ability to mediate monocyte adhesion, chemotaxis, and enzymatic digestion of extracellular matrix, whereas in chronic lesions, excess TGF-beta may contribute to persistent leukocyte accumulation. Images Fig. 3 Fig. 5 Fig. 6 PMID:8506302

  6. [Study on human aFGF fusion gene transformation with soybean 24 kDa oleosin and expression in safflower].

    PubMed

    Gai, Yu-Hong; Wang, Wang; Jin, Xiao; Wang, Jia-Qi; Li, Hong-Bo; Wang, Gang; Yang, Jing; Wei, Jian

    2013-06-01

    To investigate the expression of acidic fibroblast growth factor (aFGF) in transgenic safflower and lay the foundation for the use of the plant bioreactor large-scale production aFGF. The haFGF gene was transformed into plant preference of the aFGF sequence as a basis for design of primers, plant preferences aFGF gene sequences was amplified by PCR. The vegetable body expression vector was constructed by using digested connection method and then transferred to Agrobacterium tumefaciens EHA105 by the freeze-thaw method. It transferred to safflowers by agrobacterium-mediated transformation method, and identified by PCR, southern blot and RT-PCR. The full-length aFGF gene sequences were amplified through PCR and constructed into plant expression vector with soybean oleosin and promoter, and transformed into safflower. Three independently transformed safflower plant units with point insertion were successfully obtained, which showed the same size of aFGF expression at the transcriptional level. The plant oil body expression vectors were successfully constructed, and the optimal condition for genetic transformation was selected. The transgenic safflower plants were obtained.

  7. Ion beam transformation with corn DNA alters proteinase expression in rice seedling roots.

    PubMed

    Li, W C; Ji, S D; Wang, X C; Li, Z K; Zhang, H C; Tian, C Z; Liu, Y L; Duan, C X

    2015-06-29

    Corn DNA was introduced into dry seeds of rice (cv. 'YuJing-6') by ion beam irradiation. Proteinase activities in rice seedling roots were subsequently analyzed by renaturation electrophoresis at pH 4.5, 7.0, and 8.5. Proteinase activity was more pronounced on gels at higher pH. Irradiation of rice seedling roots caused the loss of some proteinase bands at all pH conditions although a novel 50-kDa band was found at both pH 7.0 and 8.5. No new proteinase activity was detected at pH 4.5. However, novel bands and bands showing stronger activity were observed at pH 7.0 and 8.5. The data indicate that the expression of proteinases in rice seedling roots was altered following low energy ion beam mediated transformation with corn DNA.

  8. Immunocytochemical study of transforming growth factor expression in benign and malignant gliomas.

    PubMed Central

    Samuels, V.; Barrett, J. M.; Bockman, S.; Pantazis, C. G.; Allen, M. B.

    1989-01-01

    Immunocytochemical studies using polyclonal antibodies to epidermal growth factor (EGF) and transforming growth factor (TGF) alpha and beta were performed on 20 cases of human gliomas. EGF immunoreactive material was detected in both benign and malignant glial tumors. In addition, EGF immunoreactive material was detected in normal brain. TGF-beta was detected in both benign and malignant tumors, but was not detected in normal brain. In contrast, TGF-alpha was highly conserved in its expression, occurring predominantly in malignant compared with benign or normal brain tissue (P less than 0.0001). In malignant gliomas, glioblastomas contained 76% TGF-alpha reactivity (immunoreactive product), and anaplastic types contained 85% reactivity. Benign gliomas contained only 13% TGF-alpha reactivity. These findings support the role of TGF-alpha as an oncoprotein marker in brain neoplasms. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2705509

  9. Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells.

    PubMed

    Wang, Haili; Kroeber, Markus; Hanke, Michael; Ries, Rainer; Schmid, Carsten; Poller, Wolfgang; Richter, Wiltrud

    2004-02-01

    To develop new therapeutic options for the treatment of disc degeneration we tested the possibility of overexpression of active growth and differentiation factor (GDF) 5 and of transforming growth factor (TGF) beta(1) by adenoviral gene transfer and characterized its effect on cell proliferation and matrix synthesis of cultured rabbit and human intervertebral disc cells. Recombinant adenovirus encoding for GDF-5 or TGF-beta(1) was developed and transgene expression characterized by RT-PCR, western blot and ELISA. Growth and matrix synthesis of transduced cells was measured by [(3)H]thymidine or [(35)S]sulfate incorporation. Disc cells expressed the receptors BMPR1A, BMPR1B, and BMPR2, which are relevant for GDF-5 action. Adenovirus efficiently transferred the GDF-5 gene or the TGF-beta(1) gene to rabbit and human intervertebral disc cells. About 50 ng GDF-5 protein/10(6 )cells per 24 h or 7 ng TGF-beta(1) protein/10(6 )cells per 24 h was produced. According to western blotting, two GDF-5 forms, with molecular weights consistent with the activated GDF-5 dimer and the proform, were secreted over the 3 weeks following gene transfer. Overexpressed GDF-5 and TGF-beta(1) were bioactive and promoted growth of rabbit disc cells in monolayer culture. Our results suggest that ex vivo gene delivery of GDF-5 and TGF-beta(1) is an attractive approach for the release of mature and pre-GDF-5 in surrounding tissue. This leads us to hope that it will prove possible to improve the treatment of degenerative disc disease by means of ex vivo gene transfer of single or multiple growth factors.

  10. Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine.

    PubMed

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

    2012-01-01

    The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines.

  11. Radicicol suppresses transformation and restores tropomyosin-2 expression in both ras- and MEK-transformed cells without inhibiting the Raf/MEK/ERK signaling cascade.

    PubMed

    Kim, P N; Jonasch, E; Mosterman, B C; Mier, J W; Janssen, R A

    2001-11-01

    The antibiotic radicicol suppresses transformation in a variety of transformed cells. The antineoplastic effects of the drug have been attributed to the degradation of Raf and the inactivation of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade. Here we demonstrate that radicicol induces cell spreading, suppresses anchorage-independent cell growth, and increases the expression of the high-molecular weight tropomyosin isoform TM-2 in cells stably expressing a constitutively active form of MEK-1 as well as in ras-transformed cells. Furthermore, the reverting effects of the drug are achieved at concentrations below those required to deplete Raf from the cell or to inhibit the phosphorylation of ERK or its substrates Elk and pp90(RSK). In contrast, low concentrations of radicicol significantly inhibited activator protein (AP-1) and serum response factor (SRF)-mediated transcription. The lack of correlation between the effects of radicicol on cell phenotype and on the signaling activities of the Raf/MEK/ERK pathway indicate that Raf depletion or disruption of proximal signaling events in the mitogen-activated protein kinase pathway are not the predominant mechanisms by which the drug suppresses the transformed phenotype. Our observation that low concentrations of radicicol block transcriptional activities mediated by AP-1 and SRF suggests that interference with signaling upstream of these transcription factors may contribute to the reverting effects of the drug.

  12. Construction and uses of cell lines containing integrated adenovirus E2 promoters.

    PubMed

    Strair, R K

    1991-11-01

    The adenovirus E1a gene encodes polypeptides which regulate the expression of adenovirus early genes as well as a variety of cellular genes. Although it is likely that the E1a encoded polypeptides regulate the expression of these genes by interaction with a variety of cellular transcription factors, the precise mechanism by which this occurs is currently unknown. This report describes the development of cell lines which contain integrated copies of the E2 promoter driving the expression of the Tn5 neo gene or the Escherichia coli beta-galactosidase gene. In each case phenotypic changes concurrent with expression of the E1a 289 amino acid polypeptide are demonstrated. The use of these cell lines to detect rare events in the activation of the E2 promoter is demonstrated in transfection experiments. These cell lines are also used to study the effects of c-myc expression on integrated E2 promoters.

  13. [Gene expression of transforming growth factor beta receptor II in the epidermis of pathological scar].

    PubMed

    Chen, Ming-Rui; An, Gang; Liu, Shun-Li; Wei, Feng-Cai

    2012-08-01

    To study the gene expression of transforming growth factor beta receptor II (TbetaR II) in pathological scar. Twenty samples of pathological scar were collected from 20 burn or trauma patients hospitalized in the General Hospital of Ji'nan Military Command from 2007 to 2009. Twenty specimens of epidermal layer were obtained from the middle portion and the edge of pathological scars. Twenty normal skin specimens which were located more than 10 cm away from the lesion sites of 20 patients were collected as self-controls. Serum from 1-2 mL whole blood were obtained from each of the 20 patients for second self-control. Eight normal skin specimens from 8 patients without pathological scar, discarded from un-related operations, were also collected as negative-control. Positive expressions of TbetaR II in three different skin specimens were determined with biotin-streptavidin-peroxidase staining. Gene expressions of TbetaR II in all specimens were compared with PCR-single strand conformation polymorphism analysis and gene sequencing. Data were processed with Fisher's exact test. Positive expression of TbetaR II in pathological scar epidermis was lower than that in normal skin specimen of patients with pathological scar or normal skin specimen of patients without pathological scar, and TbetaR II was mainly located in the basal layer of epidermis. Positive expressions of TbetaR II were seldom found in acanthocytes, granular cells, and cuticle or even non-existing. No abnormality of TbetaR II was found in normal skin epidermis or serum samples of pathological scar patients or normal skin epidermis of patients without pathological scar. TbetaR II expressing in 8 specimens of epidermis of pathological scar showed abnormal electrophoresis pattern at poly A fragments hand and loss of one A base in DNA fragment (P = 0.044). There may he abnormal gene expression of TbetaR II in pathological scar epidermis. Replantation of epidermis of scar may increase the risk of scar recurrence

  14. Rotavirus and adenovirus gastroenteritis: time series analysis.

    PubMed

    Celik, Cem; Gozel, Mustafa Gokhan; Turkay, Hakan; Bakici, Mustafa Zahir; Güven, Ahmet Sami; Elaldi, Nazif

    2015-08-01

    This study investigated the effects of changes in weather conditions (monthly average temperature, monthly minimum temperature, monthly average humidity) on rotavirus and adenovirus gastroenteritis frequency and whether there was a seasonal correlation. Between 2006 and 2012, 4702 fecal samples were taken from patients ≤ 5 years of age with acute gastroenteritis; these samples were analyzed in terms of rotavirus group A and adenovirus serotype 40-41 antigens using time-series and negative binomial regression analysis. Rotavirus antigens were found in 797 samples (17.0%), adenovirus antigens in 113 samples (2.4%), and rotavirus and adenovirus antigens together in 16 samples (0.3%). There was a seasonal change in rotavirus gastroenteritis (P < 0.001), and a 1°C decrease in average temperature increased the ratio of rotavirus cases in those with diarrhea by 0.523%. In addition, compared with data from other years, the number of patients was lower in the first month of 2008 and in the second month of 2012, when the temperature was below -20°C (monthly minimum temperature). There was no statistically significant relationship between adenovirus infection and change in weather conditions. Various factors such as change in weather conditions, as well as the population's sensitivity and associated changes in activity, play a role in the spread of rotavirus infection. © 2015 Japan Pediatric Society.

  15. Components of Adenovirus Genome Packaging

    PubMed Central

    Ahi, Yadvinder S.; Mittal, Suresh K.

    2016-01-01

    Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging. PMID:27721809

  16. Expression of gamma-glutamyl transpeptidase in adult rat liver cells after transformation with SV40 virus.

    PubMed

    Lafarge-Frayssinet, C; Estrade, S; Rosa-Loridon, B; Frayssinet, C; Cassingena, R

    1984-02-01

    By the use of histochemical techniques we have shown that the transformation