Adenovirus Vector (adenovirus + vector)

Distribution by Scientific Domains


Selected Abstracts


Possible Involvement of I,B Kinase 2 and MKK7 in Osteoclastogenesis Induced by Receptor Activator of Nuclear Factor ,B Ligand,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2002
Aiichiro Yamamoto
Abstract Recent studies have revealed the essential role of the receptor activator of nuclear factor ,B (NF-,B) ligand (RANKL) in osteoclast differentiation and activation. Adenovirus vector could efficiently transduce genes into RAW264.7 cells, which differentiate into osteoclast-like multinucleated cells in the presence of RANKL. The role of NF-,B and c- jun N-terminal kinase (JNK) activation in RANKL-induced osteoclast differentiation was investigated using an adenovirus vector carrying the dominant negative I,B kinase 2 gene (AxIKK2DN) or dominant negative MKK7 gene (AxMKK7DN). IKK2DN and MKK7DN overexpression in RAW cells specifically suppressed the NF-,B activation and JNK activation in response to RANKL, respectively, without affecting other signaling pathways. Either inhibition of NF-,B or JNK pathways dose-dependently inhibited osteoclast formation induced by RANKL. These results suggest that both NF-,B and JNK activation are independently required for osteoclast differentiation. [source]


In Vitro and In Vivo Transfer of bcl-2 Gene into Keratinocytes Suppresses UVB-induced Apoptosis,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2001
Hidetoshi Takahashi
ABSTRACT Bcl-2 is a member of the large Bcl-2 family and protects cells from apoptosis. Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes that is known as "sunburn cells." Previously we reported that UVB irradiation induces apoptosis accompanied by sequential activation of caspase 8, 3 and 1 in keratinocytes, and that the process is inhibited by various caspase inhibitors. Using bcl-2,expressing adenovirus vector we investigated the effect of Bcl-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bcl-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (1 × 106) were transfected at 1 × 108 plaque-forming unit (PFU)/mL. Bcl-2,transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2,transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 × 109 PFU in 200 ,L. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m2) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2,transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo. [source]


Simultaneous administration of a low-dose mixture of donor bone marrow cells and splenocytes plus adenovirus containing the CTLA4Ig gene result in stable mixed chimerism and long-term survival of cardiac allograft in rats

IMMUNOLOGY, Issue 2 2003
Yongzhu Jin
Summary T-cell costimulatory blockade combined with donor bone marrow transfusion may induce mixed chimerism, rendering robust tolerance in transplanted organs and cells. However, most protocols entail high doses of donor bone marrow cells (BMCs) or repeated administration of costly agents that block costimulatory pathways, thus delaying clinical development. To circumvent these shortcomings, we developed a strategy in which the dosage of donor BMCs was reduced but compensated by donor splenocytes (SPLCs). Furthermore, repeated administration of costly agents was replaced with a single injection of adenovirus expressing a gene of interest. In rat cardiac transplantation models, cardiac allografts from DA (RT-1a) rats were transplanted heterotopically into the abdomen of LEW (RT-11) recipient rats. Immediately after cardiac transplantation, an adenovirus vector (AdCTLA4Ig; 5 × 109 plaque-forming units) containing the gene for CTLA4Ig was administered to recipients (n = 6) simultaneously with a low dose of donor BMCs (1 × 108/rat) and SPLCs (5 × 107/rat) via the portal vein. The treated LEW recipient rats developed long-lasting mixed chimerism (>10% at >100 days) and exhibited long-term cardiac allografts (mean survival time of > 200 days) compared with control recipients. Moreover, recipients displaying long-lasting mixed chimerism accepted subsequent donor skin allografts while promptly rejecting third-party skin allografts. These results suggest that blockade of the CD28-B7 pathway, using adenovirus-mediated CTLA4Ig gene transfer, in concert with a low dosage of donor BMCs and SPLCs, may represent a feasible strategy to induce stable mixed chimerism and permit long-term survival of cardiac allografts. [source]


Immune enhancement of nitroreductase-induced cytotoxicity: Studies using a bicistronic adenovirus vector

INTERNATIONAL JOURNAL OF CANCER, Issue 1 2003
Nicola K. Green
Abstract The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in preclinical studies and is currently being assessed in phase I clinical trials. It is well established that there is an immune component to the bystander effect observed with other systems such as thymidine kinase and cytosine deaminase; however, such an effect has not previously been described using NR. We have preliminary data suggesting an immune bystander effect with NR to further examine these effects and their potential enhancement by cytokines, an adenoviral vector containing CMV-NR, an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF) was constructed. The NR-GM-CSF virus was validated in 2 experimental models and demonstrated increased therapeutic efficacy in the MC26 murine colorectal tumour model. These data illustrate that the combination of suicide gene therapy using NR and CB1954 with immune stimulation via GM-CSF gives an improved response compared to either modality alone and suggests that the immune component of this response may be beneficial in combating unresectable, metastatic disease and preventing tumour recurrence. © 2002 Wiley-Liss, Inc. [source]


Possible Involvement of I,B Kinase 2 and MKK7 in Osteoclastogenesis Induced by Receptor Activator of Nuclear Factor ,B Ligand,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2002
Aiichiro Yamamoto
Abstract Recent studies have revealed the essential role of the receptor activator of nuclear factor ,B (NF-,B) ligand (RANKL) in osteoclast differentiation and activation. Adenovirus vector could efficiently transduce genes into RAW264.7 cells, which differentiate into osteoclast-like multinucleated cells in the presence of RANKL. The role of NF-,B and c- jun N-terminal kinase (JNK) activation in RANKL-induced osteoclast differentiation was investigated using an adenovirus vector carrying the dominant negative I,B kinase 2 gene (AxIKK2DN) or dominant negative MKK7 gene (AxMKK7DN). IKK2DN and MKK7DN overexpression in RAW cells specifically suppressed the NF-,B activation and JNK activation in response to RANKL, respectively, without affecting other signaling pathways. Either inhibition of NF-,B or JNK pathways dose-dependently inhibited osteoclast formation induced by RANKL. These results suggest that both NF-,B and JNK activation are independently required for osteoclast differentiation. [source]


Enhancement of anchorage-independent growth of human pancreatic carcinoma MIA PaCa-2 cells by signaling from protein kinase C to mitogen-activated protein kinase

MOLECULAR CARCINOGENESIS, Issue 4 2002
Keiko Ishino
Abstract We found that 12- O -tetradecanoylphorbol-13-acetate (TPA) promoted anchorage-independent growth but did not affect anchorage-dependent growth of MIA PaCa-2 human pancreatic carcinoma cells. TPA markedly activated mitogen-activated protein kinase (MAPK)/extracellular signal,regulated kinase in an anchorage-independent manner. Two protein kinase C (PKC) isoforms, conventional PKC (cPKC) and novel PKC (nPKC), but not apical PKC, translocated from the cytosolic to the particulate fraction upon TPA treatment. To identify the PKC isoforms involved in the regulation of anchorage-independent growth, four PKC isoforms (,, ,, ,, and ,) were forced to be expressed in MIA PaCa-2 cells with an adenovirus vector. Overexpression of nPKC, or nPKC, activated MAPK and promoted anchorage-independent growth. Overexpression of cPKC, alone did not influence anchorage-independent growth but lowered the concentration of TPA that was required to enhance such growth. Expression of constitutively active MAPK kinase-1 (MEK1) also promoted anchorage-independent growth. Furthermore, PKC inhibitors or an MEK inhibitor completely suppressed both TPA-induced activation of MAPK and promotion of anchorage-independent growth, but a cPKC-selective inhibitor partially suppressed TPA-induced promotion of the growth. Based on these results, we suggest that MAPK activation, mediated by certain isoforms of PKC, plays a part in oncogenic growth of MIA PaCa-2 cells. In summary, our data indicated that specific inhibitors of the cPKC and nPKC signaling pathway might be selective anti-oncogenic growth agents for some types of human pancreatic cancer. © 2002 Wiley-Liss, Inc. [source]


In Vitro and In Vivo Transfer of bcl-2 Gene into Keratinocytes Suppresses UVB-induced Apoptosis,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2001
Hidetoshi Takahashi
ABSTRACT Bcl-2 is a member of the large Bcl-2 family and protects cells from apoptosis. Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes that is known as "sunburn cells." Previously we reported that UVB irradiation induces apoptosis accompanied by sequential activation of caspase 8, 3 and 1 in keratinocytes, and that the process is inhibited by various caspase inhibitors. Using bcl-2,expressing adenovirus vector we investigated the effect of Bcl-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bcl-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (1 × 106) were transfected at 1 × 108 plaque-forming unit (PFU)/mL. Bcl-2,transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2,transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 × 109 PFU in 200 ,L. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m2) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2,transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo. [source]


Infection of replication-deficient adenoviral vector enhances interleukin-8 production in small airway epithelial cells more than in large airway epithelial cells

RESPIROLOGY, Issue 4 2001
YUZO KODAMA
Objective: In clinical trials or experiments of gene therapy, airway administration of an adenoviral-based vector (E1A-deleted) elicits a dose-dependent inflammatory response with limitation in the duration of transgene expression. The purpose of this study was to evaluate the possibility that the adenoviral-based vector directly enhances IL-8 production independent of adenoviral E1A in normal human airway epithelial cells and to examine the different responses between primary human bronchial epithelial cells (HBE) and primary human small airway epithelial cells (HSAE) in production of IL-8 following exposure to an adenovirus vector. Methodology: Interleukin (IL)-8 levels were evaluated in the culture medium from HBE and HSAE treated with increasing doses of E1A-deleted adenoviral vector contained the Escherichia coli LacZ reporter gene (AdCMVLacZ). To clarify the mechanism of enhancing IL-8 production in airway epithelial cells by infection with adenovirus vector, ,v,5 agonistic antibody as an analogue of adenoviral capsid and adenoviral capsid vector denatured by exposure to ultraviolet (UV) light were used in the present study. Results: Inoculation of HBE with AdCMVLacZ at a multiplicity of infection (MOI) of between 1 and 200 resulted in a dose-dependent expression of LacZ, and maximal expression was observed at a MOI of 100. In contrast, inoculation of HSAE with AdCMVLacZ resulted in maximum expression of LacZ at a MOI of 10. Interleukin-8 levels in culture media from the same experiments revealed significantly greater production of IL-8 in HSAE inoculated with AdCMVLacZ at a MOI of 50, compared to HBE under the same conditions. The capsid-denatured adenoviral vector did not enhance IL-8 production, and ,v,5 agonistic antibody induced IL-8 enhancement. Conclusion: These results suggest that the adenoviral vector directly induces the expression of airway epithelial inflammatory cytokines in the pathogenesis of inflammation and that small airway cells have a greater affinity for adenovirus than other airway epithelial cells. [source]


Bimodal role of conventional protein kinase C in insulin secretion from rat pancreatic , cells

THE JOURNAL OF PHYSIOLOGY, Issue 1 2004
Hui Zhang
The present study was conducted to evaluate the role of conventional protein kinase C (PKC) in calcium-evoked insulin secretion. In rat , cells transfected with green fluorescent protein-tagged PKC-, (PKC-,,EGFP), a depolarizing concentration of potassium induced transient elevation of cytoplasmic free calcium ([Ca2+]c), which was accompanied by transient translocation of PKC-,,EGFP from the cytosol to the plasma membrane. Potassium also induced transient translocation of PKC-,,EGFP, the C1 domain of PKC-, and PKC-,,GFP. A high concentration of glucose induced repetitive elevation of [Ca2+]c and repetitive translocation of PKC-,,EGFP. Diazoxide completely blocked both elevation of [Ca2+]c and translocation of PKC-,,EGFP. We then studied the role of conventional PKC in calcium-evoked insulin secretion using rat islets. When islets were incubated for 10 min with high potassium, Gö-6976, an inhibitor of conventional PKC, and PKC-, pseudosubstrate fused to antennapedia peptide (Antp-PKC19,31) increased potassium induced secretion. Similarly, insulin release induced by high glucose for 10 min was enhanced by Gö-6976 and Antp-PKC19,31. However, when islets were stimulated for 60 min with high glucose, both Gö-6976 and Antp-PKC19,31 reduced glucose-induced insulin secretion. Similar results were obtained by transfection of dominant-negative PKC-, using adenovirus vector. Taken together, PKC-, is activated when cells are depolarized by a high concentration of potassium or glucose. Conventional PKC is inhibitory on depolarization-induced insulin secretion per se, but it also augments glucose-induced secretion. [source]


In vivo inhibition of angiogenesis by interleukin-13 gene therapy in a rat model of rheumatoid arthritis

ARTHRITIS & RHEUMATISM, Issue 8 2007
Christian S. Haas
Objective Interleukin-13 (IL-13) is a pleiotropic cytokine that can affect vessel formation, an important component of the rheumatoid arthritis (RA) synovial tissue pannus. The purpose of this study was to use a gene therapy approach to investigate the role of IL-13 in angiogenesis in vivo, using a rat adjuvant-induced arthritis model of RA. Methods Ankle joints of female rats were injected preventatively with an adenovirus vector containing human IL-13 (AxCAIL-13), a control vector with no insert (AxCANI), or phosphate buffered saline (PBS). Joints were harvested at the peak of arthritis, and histologic and biochemical features were evaluated. Results AxCAIL-13,treated joint homogenates had lower hemoglobin levels, suggesting reduced joint vascularity, and both endothelial cell migration and tube formation were significantly inhibited (P < 0.05). Similarly, AxCAIL-13 inhibited capillary sprouting in the rat aortic ring assay and vessel growth in the Matrigel plug in vivo assay. IL-13 gene delivery resulted in up-regulation and association of phosphorylated ERK-1/2 and protein kinase C,/,II, suggesting a novel pathway in IL-13,mediated angiostasis. The angiostatic effect of AxCAIL-13 was associated with down-regulation of proangiogenic cytokines (IL-18, cytokine-induced neutrophil chemoattractant 1/CXCL1, lipopolysaccharide-induced CXC chemokine/CXCL5) and up-regulation of the angiogenesis inhibitor endostatin. The expression and activity of matrix metalloproteinases 2 and 9, which participate in angiogenesis, was impaired in response to IL-13 as compared with AxCANI and PBS treatment. Conclusion Our findings support a role for IL-13 as an in vivo antiangiogenic factor and provide a rationale for its use in RA to control pathologic neovascularization. [source]


Induction of prolonged infiltration of T lymphocytes and transient T lymphocyte,dependent collagen deposition in mouse lungs following adenoviral gene transfer of CCL18

ARTHRITIS & RHEUMATISM, Issue 8 2006
Irina G. Luzina
Objective Levels of CCL18 are elevated in patients with scleroderma lung disease and other fibrotic pulmonary diseases associated with T lymphocyte involvement. We sought to determine whether CCL18 alone can induce pulmonary T lymphocytic infiltration and fibrosis in mouse lungs. Methods An adenovirus vector was constructed and used for CCL18 delivery to mouse lungs in vivo. Immunohistochemical, flow cytometric, and enzyme-linked immunosorbent assay analyses were used to assess the resulting changes. Results Overexpression of CCL18 led to massive perivascular and peribronchial infiltration of T lymphocytes. Although the expression of CCL18 peaked on day 7, the infiltration persisted up to day 64 after infection. The infiltrates were negative for proliferating cell nuclear antigen and TUNEL, suggesting the role of cell trafficking, rather than proliferation and apoptosis, in the infiltration dynamics. Patchy destruction of the alveolar architecture and collagen accumulation in association with the infiltrates were also noticed. These changes were infiltration-dependent, rather than CCL18-dependent, since treatment with antilymphocyte serum completely abrogated the CCL18-induced changes. The infiltrates consisted almost exclusively of T lymphocytes that were minimally activated, with a minimal increase in the expression of CD69 and no changes in the expression of CD25, Fas, FasL, or CD40L. There was no increase in total pulmonary levels of profibrotic cytokines transforming growth factor ,1 (TGF,1) or interleukin-13, although active TGF,1 was present locally in association with the infiltrates and areas of distorted alveolar architecture. Prestimulation of primary T lymphocytes with CCL18 in vitro caused an up-regulation of TGF,1 and collagen production in T lymphocyte/fibroblast cocultures. Conclusion CCL18 promotes selective, long-term pulmonary infiltration of T lymphocytes and infiltration-dependent accumulation of collagen through a TGF,1-dependent mechanism. [source]


Optimal amount of monocyte chemoattractant protein-1 enhances antitumor effects of suicide gene therapy against hepatocellular carcinoma by M1 macrophage activation

CANCER SCIENCE, Issue 10 2008
Tomoya Tsuchiyama
Suicide gene therapy combined with chemokines provides significant antitumor efficacy. Coexpression of suicide gene and monocyte chemoattractant protein-1 (MCP-1) increases antitumor effects in murine models of hepatocellular carcinoma (HCC) and colon cancer. However, it is unclear whether the doses administered achieved the maximum antitumor effects. We evaluated antitumor effects of various amounts of recombinant adenovirus vector (rAd) expressing MCP-1 in the presence of a suicide gene in a murine model of HCC. HCC cells were transplanted subcutaneously into BALB/c nude mice, and transduced with a fixed amount of Ad-tk harboring the suicide gene, HSV-tk, and various doses of Ad-MCP1 harboring MCP-1 (ratios of 1:1, 0.1:1, and 0.01:1 relative to Ad-tk). Growth of primary tumors was suppressed when treated with Ad-tk plus Ad-MCP1 (1:1 and 1:0.1) as compared with Ad-tk alone. The antitumor effects against tumor rechallenge tended to be high in the Ad-tk plus Ad-MCP1 group (1:0.1). The effects were dependent on production of Th1 type-cytokines. Delivery of an optimal amount of rAd expressing MCP-1 enhanced the antitumor effects of suicide gene therapy against HCC by M1 macrophage activation, suggesting that this is a plausible form of cancer gene therapy to prevent HCC progression and recurrence. (Cancer Sci 2008; 99: 2075,2082) [source]


Osteoclast Differentiation by RANKL Requires NF-,B-Mediated Downregulation of Cyclin-Dependent Kinase 6 (Cdk6),

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2004
Toru Ogasawara
Abstract This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-,B signaling. Introduction: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. Materials and Methods: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-,B and c- jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the I,B kinase 2 (IKKDN) gene and mitogen-activated protein kinase kinase 7 (MKK7DN) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. Results and Conclusion: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-,B pathway by IKKDN overexpression, but not that of the JNK pathway by MKK7DN overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-,B-mediated downregulation is essential for efficient osteoclast differentiation. [source]


BIT/SHPS-1 Enhances Brain-Derived Neurotrophic Factor-Promoted Neuronal Survival in Cultured Cerebral Cortical Neurons

JOURNAL OF NEUROCHEMISTRY, Issue 4 2000
Toshiyuki Araki
Abstract: Brain-derived neurotrophic factor (BDNF) activates a variety of signaling molecules to exert various functions in the nervous system, including neuronal differentiation, survival, and regulation of synaptic plasticity. Previously, we have suggested that BIT/SHPS-1 (brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1) is a substrate of Shp-2 and is involved in BDNF signaling in cultured cerebral cortical neurons. To elucidate the biological function of BIT/SHPS-1 in cultured cerebral cortical neurons in connection with its role in BDNF signaling, we generated recombinant adenovirus vectors expressing the wild type of rat BIT/SHPS-1 and its 4F mutant in which all tyrosine residues in the cytoplasmic domain of BIT/SHPS-1 were replaced with phenylalanine. Overexpression of wild-type BIT/SHPS-1, but not the 4F mutant, in cultured cerebral cortical neurons induced tyrosine phosphorylation of BIT/SHPS-1 itself and an association of Shp-2 with BIT/SHPS-1 even without addition of BDNF. We found that BDNF-promoted survival of cultured cerebral cortical neurons was enhanced by expression of the wild type and also 4F mutant, indicating that this enhancement by BIT/SHPS-1 does not depend on its tyrosine phosphorylation. BDNF-induced activation of mitogen-activated protein kinase was not altered by the expression of these proteins. In contrast, BDNF-induced activation of Akt was enhanced in neurons expressing wild-type or 4F mutant BIT/SHPS-1. In addition, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase, blocked the enhancement of BDNF-promoted neuronal survival in both neurons expressing wild-type and 4F mutant BIT/SHPS-1. These results indicate that BIT/SHPS-1 contributes to BDNF-promoted survival of cultured cerebral cortical neurons, and that its effect depends on the phosphatidylinositol 3-kinase-Akt pathway. Our results suggest that a novel action of BIT/SHPS-1 does not occur through tyrosine phosphorylation of BIT/SHPS-1 in cultured cerebral cortical neurons. [source]


Selective gene transfer into neurons via Na,K-ATPase ,1.

THE JOURNAL OF GENE MEDICINE, Issue 6 2008
Targeting gene transfer with monoclonal antibody, adenovirus vector
Abstract Background Neuron-selective gene transfer is an attractive therapeutic strategy for neurological disorders. However, optimal targets and gene delivery systems remain to be determined. Methods Following immunization of mice with PC12 cells, hybridomas were screened by ,-Gal reporter gene assay using FZ33 fiber-modified adenovirus vectors. Subsequently, the efficacy and specificity of monoclonal antibody (mAb)-mediated gene transfer via FZ33 and FdZ adenovirus vectors were evaluated by flow cytometry, chemiluminescent ,-Gal reporter gene assay, and immunocytochemistry. Finally, the antigen recognized by the mAb was identified by mass spectrometry and transfection analysis. Results A hybridoma clone 6E3 producing monoclonal antibody, mAb6E3, was screened. Flow cytometry, chemiluminescent ,-Gal reporter gene assay, and immunocytochemistry with mAb6E3 and the fiber mutant adenovirus demonstrated efficient gene transfer into the PC12 cells. Treatment of neuron,glia cocultures with mAb6E3 and FdZ adenovirus resulted in neuron-selective gene transfer. Immunohistochemical images of rat spinal cord tissue showed that mAb6E3 reacts specifically with neurons. Finally, Na,K-ATPase ,1 was identified as the antigen of mAb6E3. Conclusions Hybridoma screening using FZ33 fiber-modified adenovirus vectors serves as an efficient approach to detect antigens in mAb-targeted gene transfer. Neuronal tropism in the central nervous system through mAb6E3 represents an important initial step towards neuron-selective gene transfer in the treatment of local neurological disorders, such as spinal cord injury. Copyright © 2008 John Wiley & Sons, Ltd. [source]


CAR chasing: canine adenovirus vectors,all bite and no bark?

THE JOURNAL OF GENE MEDICINE, Issue S1 2004
Eric J. Kremer
Abstract This review deals primarily with canine adenovirus serotype 2 (CAV-2) vectors and gives a simplified overview of how the various domains of virology, cellular and molecular biology, as well as immunology, come into play when trying to understand and ameliorate adenovirus (Ad)-mediated gene transfer. The generation of early region 1 (E1)-deleted (,E1) CAV-2 vectors, the lack of pre-existing humoral immunity, trafficking, the use of the coxsackie B adenovirus receptor (CAR), the surprising neuronal tropism, and the ability to migrate via axons to afferent regions of the central and peripheral nervous system, are described. Due to these intrinsic properties, CAV-2 vectors may be powerful tools for the study of the pathophysiology and potential treatment of neurodegenerative diseases like lysosomal storage disorders, Parkinson's, Alzheimer's, Huntington's, amyotrophic lateral sclerosis, and others. Other potential uses include anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, pain therapy, cancer therapy (e.g. K9 CRAds), and gene transfer to other somatic tissues. Copyright © 2004 John Wiley & Sons, Ltd. [source]


Generation of fiber-modified adenovirus vectors containing heterologous peptides in both the HI loop and C terminus of the fiber knob

THE JOURNAL OF GENE MEDICINE, Issue 4 2003
Naoya Koizumi
Abstract Background Fiber-modified adenovirus (Ad) vectors can be effective in overcoming the limitations of conventional Ad vectors, specifically their inefficient gene transfer into cells lacking the primary receptor, the coxsackievirus and adenovirus receptor (CAR). Several types of fiber-modified Ad vectors have been developed. In this study, we evaluated the functionality of several fiber-modified Ad vectors. Methods We developed a simple method based on in vitro ligation to construct Ad vectors containing heterologous foreign peptides in both the HI loop and C terminus of the fiber knob. A functional comparison of Ad vectors containing RGD and/or K7 (KKKKKKK) peptide in the HI loop or C terminus of the fiber knob was performed in several types of human, mouse, and rat cells, including CAR-positive and -negative cells, and tumor cells in mice in vivo. Results In the case of the in vitro experiment, Ad vectors containing RGD peptide in the HI loop of the fiber knob showed a higher level of gene transfer than vectors containing RGD peptide at the C terminus of the fiber knob. Ad vectors containing K7 peptide at the C terminus of the fiber knob showed levels of gene transfer similar to those of Ad vectors containing RGD peptide in the HI loop of the fiber knob, depending on the cell type. Ad vectors containing both peptides in the HI loop or C terminus of the fiber knob showed the highest levels of gene transfer and a broader tropism. For gene transfer into tumor cells in vivo, the Ad vectors containing RGD peptide were the most efficient. Conclusions In the experiment using cultured cells, Ad vectors containing both RGD and K7 peptides were the most efficient with a broader tropism. In contrast, in the experiment in vivo, Ad vectors containing RGD peptide in the HI loop of the fiber knob were more efficient than the vectors containing K7 peptide (including double-modified vectors containing both the RGD and K7 peptides). These comparative analyses could provide a systemic reference for the use of fiber-modified Ad vectors. Our simple method, in which the peptide of interest can be expressed in Ad vectors in either the HI loop or the C terminus of the fiber knob, or both, could be a powerful tool for gene transfer into mammalian cells in studies of gene function as well as in gene therapy. Copyright © 2002 John Wiley & Sons, Ltd. [source]


Feline immunodeficiency virus vectors.

THE JOURNAL OF GENE MEDICINE, Issue 5 2002
Gene transfer to mouse retina following intravitreal injection
Abstract Background Transduction of the murine retinal pigmented epithelium (RPE) with adenovirus vectors requires technically difficult and invasive subretinal injections. This study tested the hypothesis that recombinant vectors based on feline immunodeficiency virus (FIV) could access the retina following intravitreal injection. Methods FIV vectors expressing E. coli ,-galactosidase (FIV,gal) were injected alone, or in combination with adenovirus vectors expressing eGFP, into the vitreous of normal mice and eyes evaluated for transgene expression. In further studies, the utility of FIV-mediated gene transfer to correct lysosomal storage defects in the anterior and posterior chambers of eyes was tested using recombinant FIV vectors expressing ,-glucuronidase. FIV,gluc vectors were injected into ,-glucuronidase-deficient mice, an animal model of mucopolysacharridoses type VII. Results The results of this study show that similar to adenovirus, both corneal endothelium and cells of the iris could be transduced following intravitreal injection of FIV,gal. However, in contrast to adenovirus, intravitreal injection of FIV,gal also resulted in transduction of the RPE. Immunohistochemistry following an intravitreal injection of an AdeGFP (adenovirus expressing green fluorescent protein) and FIV,gal mixture confirmed that both viruses mediated transduction of corneal endothelium and cells of the iris, while only FIV,gal transduced cells in the retina. Using the ,-glucuronidase-deficient mouse, the therapeutic efficacy of intravitreal injection of FIV,gluc (FIV expressing ,-glucuronidase) was tested. Intravitreal injection of FIV,gluc to the eyes of ,-glucuronidase-deficient mice resulted in rapid reduction (within 2,weeks) of the lysosomal storage defect within the RPE, corneal endothelium, and the non-pigmented epithelium of the ciliary process. Transgene expression and correction of the lysosomal storage defect remained for at least 12,weeks, the latest time point tested. Conclusion These studies demonstrate that intravitreal injection of FIV-based vectors can mediate efficient and lasting transduction of cells in the cornea, iris, and retina. Copyright © 2002 John Wiley & Sons, Ltd. [source]


Interleukin-1, and tumor necrosis factor , inhibit chondrogenesis by human mesenchymal stem cells through NF-,B,dependent pathways,

ARTHRITIS & RHEUMATISM, Issue 3 2009
N. Wehling
Objective The differentiation of mesenchymal stem cells (MSCs) into chondrocytes provides an attractive basis for the repair and regeneration of articular cartilage. Under clinical conditions, chondrogenesis will often need to occur in the presence of mediators of inflammation produced in response to injury or disease. The purpose of this study was to examine the effects of 2 important inflammatory cytokines, interleukin-1, (IL-1,) and tumor necrosis factor , (TNF,), on the chondrogenic behavior of human MSCs. Methods Aggregate cultures of MSCs recovered from the femoral intermedullary canal were used. Chondrogenesis was assessed by the expression of relevant transcripts by quantitative reverse transcription,polymerase chain reaction analysis and examination of aggregates by histologic and immunohistochemical analyses. The possible involvement of NF-,B in mediating the effects of IL-1, was examined by delivering a luciferase reporter construct and a dominant-negative inhibitor of NF-,B (suppressor-repressor form of I,B [srI,B]) with adenovirus vectors. Results Both IL-1, and TNF, inhibited chondrogenesis in a dose-dependent manner. This was associated with a marked activation of NF-,B. Delivery of srI,B abrogated the activation of NF-,B and rescued the chondrogenic response. Although expression of type X collagen followed this pattern, other markers of hypertrophic differentiation responded differently. Matrix metalloproteinase 13 was induced by IL-1, in a NF-,B,dependent manner. Alkaline phosphatase activity, in contrast, was inhibited by IL-1, regardless of srI,B delivery. Conclusion Cell-based repair of lesions in articular cartilage will be compromised in inflamed joints. Strategies for enabling repair under these conditions include the use of specific antagonists of individual pyrogens, such as IL-1, and TNF,, or the targeting of important intracellular mediators, such as NF-,B. [source]


293 cell cycle synchronisation adenovirus vector production

BIOTECHNOLOGY PROGRESS, Issue 1 2009
Tiago B. Ferreira
Abstract As the market requirements for adenovirus vectors (AdV) increase, the maximisation of the virus titer per culture volume per unit time is a key requirement. However, despite the fact that 293 cells can grow up to 8 × 106 cell/mL in simple batch mode operations, for optimal AdV infection a maximum cell density of 1 × 106 cell/mL at infection time has usually been utilized due to the so called "cell density effect". In addition, AdV titer appears to be dependent upon cell cycle phase at the time of infection. To evaluate the dependence of AdV production upon cell cycle phase, 293 cells were chemically synchronised at each phase of the cell cycle; a 2.6-fold increase on AdV cell specific titer was obtained when the percentage of cells at the S phase of the cell cycle was increased from 36 to 47%; a mathematical equation was used to relate AdV cell specific productivities with cell synchronisation at the S phase using this data. To avoid the use of chemical inhibitors, a temperature shift strategy was also used for synchronisation at the S phase. S phase synchronisation was obtained by decreasing the culture temperature to 31°C during 67 h and restoring it to 37°C during 72 h. By using this strategy we were able to synchronise 57% of the population in the S phase of the cell cycle obtaining an increase of 7.3-fold on AdV cell specific titer after infection. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]


Level of reactive oxygen species induced by p21WAF(1)/CIP(1) is critical for the determination of cell fate

CANCER SCIENCE, Issue 7 2009
Takafumi Inoue
p21WAF(1)/CIP(1) is a well-known cell cycle regulatory protein which is overexpressed in several cancer cell lines, and known to determine cell fate. We generated three recombinant adenovirus vectors that expressed either the full-length p21 (Ad-p21F), a p21 mutant with a deletion of the C-terminal proliferative cell nuclear antigen (PCNA) binding domain (Ad-p21N), or a p21 mutant with a deletion of the N-terminal cyclin-dependent kinase binding domain (Ad-p21C). We transfected these vectors into five cancer cell lines. Premature senescence was induced in all of the lines only following transfection with Ad-p21N and Ad-p21F. In addition, apoptosis was also induced in LoVo and HCT116 cells that harbored wild-type p53 and the reactive oxygen species (ROS) level was higher than in senescent cells. Finally, the induction of apoptosis was inhibited by using siRNA to downregulate p53. This observation implies that there is a feedback signaling loop involving p21/ROS/p53 in apoptotic responses. It appears to be, at least in part, driven by high levels of p21 protein. Next, we investigated the cell death effect of endogenous p21 protein on cell fate using sodium butyrate (NaB). Treatment with 1 mM NaB or 2 to 5 mM NaB induced senescence or apoptosis, respectively. The level of intracellular ROS in 5 mM NaB treated cells was 2-fold higher, compared with that in 1 mM NaB treated cells. We also demonstrated that DNA damage response signals including ataxia telangiectasia mutated, ,H2AX, and p38 MAPK were involved in NaB-induced cell death. The magnitude of intracellular ROS levels in response to p21 elicited either senescence or apoptosis in the cancer cell lines. (Cancer Sci 2009; 100: 1275,1283) [source]