Adenovirus-mediated Delivery Research Articles

Herpes Simplex Virus VP22 Enhances Adenovirus-Mediated Microdystrophin Gene Transfer to Skeletal Muscles in Dystrophin-Deficient (mdx) Mice

One of the obstacles to efficient vector-mediated gene therapy for Duchenne's muscular dystrophy (DMD) is its limited transduction efficiency. The VP22 tegument protein of herpes simplex virus type 1 (HSV-1) is able to cross biological membranes and translocate the VP22 fusion protein from transfected primary cells to surrounding cells and improve the outcome of gene transfer. To improve the efficiency of vector-mediated gene therapy and to investigate the utility of the intercellular trafficking properties of VP22-linked protein for the treatment for DMD, the recombinant adenoviruses Ad-VP22, Ad-MICDYS, and Ad-VP22-MICDYS were constructed and the VP22-mediated shuttle effect was evaluated both in vitro and in vivo. About 92 +/- 3.6% of cells were microdystrophin positive 48 hr postinfection with Ad-VP22-MICDYS. The number of centralized nuclei in Ad-VP22-MICDYS-transduced tibialis anterior (TA) muscle was significantly reduced, from 78 +/- 5.2 to 20 +/- 2.5%, by 2 weeks postinjection. By 2 months postinjection, the average number of microdystrophin-positive fibers in TA muscle injected with Ad-VP22-MICDYS was 2.2 times more than that of TA muscle injected with Ad-MICDYS. Ad-VP22-MICDYS led to significant recovery of force-producing capabilities in TA muscle. These results demonstrate that VP22 greatly augmented adenovirus-mediated microdystrophin delivery to C2C12 cells and to the skeletal muscles of dystrophin-deficient (mdx) mice. These results highlight the efficiency of VP22-mediated intercellular protein delivery for the potential therapy of DMD and suggest that VP22 may be a promising tool with which to enhance the efficacy of adenoviral gene transfer for somatic gene therapy of DMD.

Eradication of Therapy-Resistant Human Prostate Tumors Using a Cancer Terminator Virus

Terminal prostate cancer is refractory to conventional anticancer treatments because of frequent overexpression of antiapoptotic proteins Bcl-2 and/or Bcl-x(L). Adenovirus-mediated delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), a secreted cytokine having cancer-selective apoptosis-inducing properties, profoundly inhibits prostate cancer cell growth. However, forced overexpression of Bcl-2 or Bcl-x(L) renders prostate cancer cells resistant to Ad.mda-7. We constructed a conditionally replication-competent adenovirus in which expression of the adenoviral E1A gene, necessary for replication, is driven by the cancer-specific promoter of progression elevated gene-3 (PEG-3) and which simultaneously expresses mda-7/IL-24 in the E3 region of the adenovirus (Ad.PEG-E1A-mda-7), a cancer terminator virus (CTV). This CTV generates large quantities of MDA-7/IL-24 as a function of adenovirus replication uniquely in cancer cells. Infection of Ad.PEG-E1A-mda-7 (CTV) in normal prostate epithelial cells and parental and Bcl-2- or Bcl-x(L)-overexpressing prostate cancer cells confirmed cancer cell-selective adenoviral replication, mda-7/IL-24 expression, growth inhibition, and apoptosis induction. Injecting Ad.PEG-E1A-mda-7 (CTV) into xenografts derived from DU-145-Bcl-x(L) cells in athymic nude mice completely eradicated not only primary tumors but also distant tumors (established in the opposite flank), thereby implementing a cure. These provocative findings advocate potential therapeutic applications of this novel virus for advanced prostate cancer patients with metastatic disease.

High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells

Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at improvement of neovascularization of engineered tissues are a key issue in tissue engineering applications. This in vitro study aimed at exploring the usability of osteogenically differentiated human mesenchymal stem cells (MSCs) as carriers of the angiogenic growth factor genes vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) for therapeutic angiogenesis in bone tissue engineering. The ex vivo adenoviral vector mediated transduction into osteogenically differentiated MSCs revealed a highly efficient and long lasting expression of the transgenes. Biological activity of VEGF and Ang-1 secreted from transduced cells was confirmed by analyzing the sprouting, proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) in response to conditioned medium obtained from transduced cells. The transduced osteogenically differentiated MSCs described in this report may be suitable for inducing neovascularization in bone tissue engineering applications.

275. Effect of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) on Transgene Expression Controlled by the Human Synapsin 1 Gene Promoter Following Adenoviral-Mediated Gene Delivery

275. Effect of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) on Transgene Expression Controlled by the Human Synapsin 1 Gene Promoter Following Adenoviral-Mediated Gene Delivery

758. Adenoviral-Mediated Delivery of Pseudomonas Exotoxin (PE) Fused to IL-13 Induces Regression of Intracranial Human Glioblastoma Xenografts in Mice

758. Adenoviral-Mediated Delivery of Pseudomonas Exotoxin (PE) Fused to IL-13 Induces Regression of Intracranial Human Glioblastoma Xenografts in Mice

100. Detection of CFTR Activity in Individual Epithelial Cells Following Adenovirus-Mediated Delivery of a cDNA Encoding Yellow Fluorescent Protein

100. Detection of CFTR Activity in Individual Epithelial Cells Following Adenovirus-Mediated Delivery of a cDNA Encoding Yellow Fluorescent Protein

Toward gene therapy of endometriosis: adenovirus-mediated delivery of dominant negative estrogen receptor genes inhibits cell proliferation, reduces cytokine production, and induces apoptosis of endometriotic cells

To use dominant negative mutants of estrogen receptor genes delivered to endometriosis cells via an adenovirus vector (Ad-DN-ER) to abrogate estrogen action on these cells. Experimental in vitro study. University research laboratory. Patients with ovarian endometriomas provided endometriotic cells, and patients with uterine prolapse or subserous leiomyoma provided control endometrial cells. Transfection of endometriotic cells by dominant negative estrogen receptor genes via adenovirus vector (Ad-DN-ER). The main outcome measures were cellular proliferation, cytokine production, and induction of apoptosis in endometriotic cells. Coxsackievirus-adenovirus receptor mRNA expression and adenovirus transduction efficiency were significantly higher in endometriotic than normal endometrial cells. Ad-DN-ER-treated endometriotic cells, as compared with control virus-treated cells, showed cell rounding and detachment (cell death), a 72% reduction in the number of viable cells 5 days after transduction, significantly less production of monocyte chemotactic protein-1 (7.8 +/- 0.5 vs. 152.8 +/- 1.9 pg/mL, respectively), vascular endothelial growth factor (356.2 +/- 11.6 vs. 997.3 +/- 16.5 pg/mL, respectively), and interleukin-6 (268.7 +/- 2.6 vs. 414.5 +/- 3.6 pg/mL, respectively), and a significantly higher percentage of apoptotic cells (51.2 +/- 7.8 vs. 23.8 +/- 1.7, respectively). An adenovirus can effectively transfect endometriotic cells in vitro. The DN-ER delivered to endometriotic cells via an adenovirus decreases cell proliferation, induces apoptosis, and decreases cytokine production. Adenovirus-mediated gene therapy may represent a potential therapeutic option for endometriosis in the future.

Phosphorylation of a Wiscott-Aldrich Syndrome Protein-associated Signal Complex Is Critical in Osteoclast Bone Resorption

The activities of different kinases have been correlated to the phosphorylation of Wiscott-Aldrich syndrome protein (WASP) by studies in multiple cell systems. The purpose of this study was to elucidate the regulatory mechanisms involved in WASP phosphorylation and the resulting sealing ring formation in osteoclasts. The phosphorylation state of WASP and WASP-interacting proteins was determined in osteoclasts treated with osteopontin or expressing either constitutively active or kinase-defective Src by adenovirus-mediated delivery. In vitro kinase analysis of WASP immunoprecipitates exhibited phosphorylation of c-Src, PYK2, WASP, protein-tyrosine phosphatase (PTP)-PEST, and Pro-Ser-Thr phosphatase-interacting protein (PSTPIP). Phosphorylation of these proteins was increased in osteopontin-treated and constitutively active Src-expressing osteoclasts. Pulldown analysis with glutathione S-transferase-fused proline-rich regions of PTP-PEST revealed coprecipitation of WASP, PYK2, c-Src, and PSTPIP proteins with the N-terminal region (amino acids 294-497) of PTP-PEST. Similarly, interaction of the same signaling proteins, as well as PTP-PEST, was observed with glutathione S-transferase-fused proline-rich regions of WASP. Furthermore, osteopontin stimulation or constitutively active Src expression resulted in serine phosphorylation and inhibition of WASP-associated PTP-PEST. The inhibition of PTP-PEST was accompanied by an increase in tyrosine phosphorylation of WASP and other associated signaling proteins. Experiments with an inhibitor to phosphatase and small interference RNA to PTP-PEST confirmed the involvement of PTP-PEST in sealing ring formation and bone resorption. WASP, which is identified in the sealing ring of resorbing osteoclasts, also demonstrates colocalization with c-Src, PYK2, PSTPIP, and PTP-PEST in immunostaining analyses. Our findings suggest that both tyrosine kinase(s) and the tyrosine phosphatase PTP-PEST coordinate the formation of the sealing ring and thus the bone-resorbing function of osteoclasts.

Adenovirus-mediated gene delivery into mouse spermatogonial stem cells

Spermatogonial stem cells represent a self-renewing population of spermatogonia, and continuous division of these cells supports spermatogenesis throughout the life of adult male animals. Previous attempts to introduce adenovirus vectors into spermatogenic cells, including spermatogonial stem cells, have failed to yield evidence of infection, suggesting that male germ cells may be resistant to adenovirus infection. In this study we show the feasibility of transducing spermatogonial stem cells by adenovirus vectors. When testis cells from ROSA26 Cre reporter mice were incubated in vitro with a Cre-expressing adenovirus vector, Cre-mediated recombination occurred at an efficiency of 49-76%, and the infected spermatogonial stem cells could reinitiate spermatogenesis after transplantation into seminiferous tubules of infertile recipient testes. No evidence of germ-line integration of adenovirus vector could be found in offspring from infected stem cells that underwent Cre-mediated recombination, which suggests that the adenovirus vector infected the cells but did not stably integrate into the germ line. Nevertheless, these results suggest that adenovirus may inadvertently integrate into the patient's germ line and indicate that there is no barrier to adenovirus infection in spermatogonial stem cells.

Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor

LPL mediates the uptake of lipoproteins into different cell types independent of its catalytic activity. The mechanism of this process and its physiological relevance are not clear. Taking into account the importance of the endothelial barrier for lipoprotein uptake, in vitro studies with primary aortic endothelial cells from wild-type and low density lipoprotein receptor (LDLR)-deficient (LDLR(-/-)) mice were performed. Addition of LPL almost doubled the uptake of LDL into wild-type cells. However, there was virtually no LPL-mediated change of LDL uptake into LDLR(-/-) cells. Upregulation of LDLR by lipoprotein-deficient serum/lovastatin in wild-type cells resulted in a 7-fold increase of LPL-mediated LDL uptake. Uptake of chylomicron remnants was not affected by LDLR expression. In proteoglycan-deficient cells, LPL did not increase the uptake of lipoproteins. The physiological relevance of this pathway was studied in mice that were both LDLR(-/-) and transgenic for catalytically inactive LPL in muscle. In the presence of LDLR, inactive LPL reduced LDL cholesterol significantly (13-24%). In the absence of LDLR, LDL cholesterol was not affected by transgenic LPL. Metabolic studies showed that in the presence of LDLR, LPL increased the muscular uptake of LDL by 77%. In the absence of LDLR, transgenic LPL did not augment LDL uptake. Chylomicron uptake was not affected by the LDLR genotype. We conclude that LPL-mediated cellular uptake of LDL, but not of chylomicrons, is dependent on the presence of both LDLR and proteoglycans.

Hepatic lipid accumulation in apolipoprotein C-I-deficient mice is potentiated by cholesteryl ester transfer protein

The impact of apolipoprotein C-I (apoC-I) deficiency on hepatic lipid metabolism was addressed in mice in the presence or the absence of cholesteryl ester transfer protein (CETP). In addition to the expected moderate reduction in plasma cholesterol levels, apoCIKO mice showed significant increases in the hepatic content of cholesteryl esters (+58%) and triglycerides (+118%) and in biliary cholesterol concentration (+35%) as compared with wild-type mice. In the presence of CETP, hepatic alterations resulting from apoC-I deficiency were enforced, with up to 58% and 302% increases in hepatic levels of cholesteryl esters and triglycerides in CETPTg/apoCIKO mice versus CETPTg mice, respectively. Biliary levels of cholesterol, phospholipids, and bile acids were increased by 88, 77, and 20%, respectively, whereas total cholesterol, HDL cholesterol, and triglyceride concentrations in plasma were further reduced in CETPTg/apoCIKO mice versus CETPTg mice. Finally, apoC-I deficiency was not associated with altered VLDL production rate. In line with the previously recognized inhibition of lipoprotein clearance by apoC-I, apoC-I deficiency led to decreased plasma lipid concentration, hepatic lipid accumulation, and increased biliary excretion of cholesterol. The effect was even greater when the alternate reverse cholesterol transport pathway via VLDL/LDL was boosted in the presence of CETP.

Protoporphyrin IX Interacts with Wild-type p53 Protein in Vitro and Induces Cell Death of Human Colon Cancer Cells in a p53-dependent and -independent Manner

Photodynamic therapy (PDT) of cancer is an alternative treatment for tumors resistant to chemo- and radiotherapy. It induces cancer cell death mainly through generation of reactive oxygen species by a laser light-activated photosensitizer. It has been suggested that the p53 tumor suppressor protein sensitizes some human cancer cells to PDT. However, there is still no direct evidence for this. We have demonstrated here for the first time that the photosensitizer protoporphyrin IX (PpIX) binds to p53 and disrupts the interaction between p53 tumor suppressor protein and its negative regulator HDM2 in vitro and in cells. Moreover, HCT116 colon cancer cells exhibited a p53-dependent sensitivity to PpIX in a dose-dependent manner, as was demonstrated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and fluorescence-activated cell sorter (FACS) analysis of cell cycle profiles. We have also observed induction of p53 target pro-apoptotic genes, e.g. puma (p53-up-regulated modulator of apoptosis), and bak in PpIX-treated cells. In addition, p53-independent growth suppression by PpIX was detected in p53-negative cells. PDT treatment (2 J/cm2) of HCT116 cells induced p53-dependent activation of pro-apoptotic gene expression followed by growth suppression and induction of apoptosis.

Both Nox2‐ and Nox4‐containing NAD(P)H oxidases are required for the full vasopressor effects of angiotensin‐II (Ang‐II) in the central nervous system

Recently we showed that Nox2 and Nox4 are abundantly expressed in forebrain circumventricular organs (CVOs), and identified Nox2 as a key player in central Ang-II-induced cardiovascular (CV) responses. However, the contribution of Nox4 to these responses is unknown. Here we used adenovirus-mediated delivery of siRNA targeted to Nox4 (AdsiNox4) to test the hypothesis that this homologue is also important in central Ang-II CV effects. Adult C57 mice underwent CVO gene transfer with AdsiNox4 alone (n=5), AdsiNox 4 + AdsiNox2 (AdsiNox2/4, n=3) or a control siRNA (AdsiGFP, n=3). Telemeters were also implanted for conscious recording of blood pressure (BP) and heart rate (HR). After 1wk recovery, BP and HR effects of ICV injection of Ang-II (200ng, 200nl) were recorded. Baseline BP and HR were not different between groups (BP / HR: AdsiNox4 102±3 / 590±32, AdsiNox2/4 108±5 / 619±58, AdsiGFP 103±2 mmHg / 592±11 bpm, p>.05). Ang-II caused a robust pressor and bradycardic response in AdsiGFP mice (ΔBP / ΔHR: 16±2 / −124±17), but these responses were attenuated in AdsiNox4 mice (ΔBP / ΔHR: 8±1 / −54±24, p<.05). More importantly, combined treatment with AdsiNox2 + AdsiNox4 nearly abolished Ang-II-induced CV responses (ΔBP / ΔHR: 2±1 / −21±15, p<.05). These data establish Nox4 as a key molecule in central Ang-II signaling, and suggest that both Nox4 and Nox2 are necessary for the full CV response profile elicited by Ang-II in the brain.

Effect of gene delivery of NOS isoforms on intimal hyperplasia and endothelial regeneration after balloon injury

Endothelial cell loss is a critical event in the pathological repair of the injured blood vessel. Impaired endothelial function results in reduced production of key vascular mediators such as nitric oxide (NO) within the vessel wall leading to enhanced smooth muscle cell proliferation and migration and ultimately intimal hyperplasia. The aim of the present study was to directly compare the effects of adenoviral-mediated gene delivery of two nitric oxide synthase (NOS) isoforms, eNOS and iNOS on endothelial regeneration and intimal hyperplasia following endothelial injury in the rabbit carotid artery. The right carotid arteries of male New Zealand white rabbits were denuded by passing a 3French Fogarty balloon catheter along the artery three times. In all, 1 x 10(9) PFU of adenoviral(Ad)eNOS, AdiNOS or Adbeta-galactosidase (Adbeta-Gal) was then delivered intraluminally and allowed to dwell for 20 min. Transgene expression was sought after 3 days by immunohistochemistry and at 7 days by quantitative reverse transcriptase PCR. The effect on intimal hyperplasia was sought using histological staining after 14 days. Evans blue staining was used to determine the effect on endothelial regeneration. eNOS and iNOS expression was detected in transduced arteries. Neointima/media ratios were significantly reduced in eNOS (0.07+/-0.044) and iNOS (0.087+/-0.086) transduced arteries compared with Adbeta-Gal (0.332+/-0.14) transduced arteries (n=7). In addition, AdeNOS treatment (4.21+/-3.12% de-endothelialized area) enhanced endothelial regeneration compared to Adbeta-Gal treatment (10.05+/-4.98), while treatment with AdiNOS (25.17+/-11.92) inhibited endothelial regeneration in the injured rabbit carotid artery (n=7-8). These results highlight the potential of NOS gene therapy, in particular, eNOS gene therapy as a potential therapeutic strategy for the prevention of restenosis after vascular injury.

The Mitochondrial Citrate/Isocitrate Carrier Plays a Regulatory Role in Glucose-stimulated Insulin Secretion

Glucose-stimulated insulin secretion (GSIS) is mediated in part by glucose metabolism-driven increases in ATP/ADP ratio, but by-products of mitochondrial glucose metabolism also play an important role. Here we investigate the role of the mitochondrial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhibition of CIC activity in INS-1-derived 832/13 cells or primary rat islets by the substrate analogue 1,2,3-benzenetricarboxylate (BTC) resulted in potent inhibition of GSIS, involving both first and second phase secretion. A recombinant adenovirus containing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced CIC expression in 832/13 cells and caused parallel inhibitory effects on citrate accumulation in the cytosol. Ad-siCIC treatment did not affect glucose utilization, glucose oxidation, or ATP/ADP ratio but did inhibit glucose incorporation into fatty acids and glucose-induced increases in NADPH/NADP+ ratio relative to cells treated with a control siRNA virus (Ad-siControl). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas overexpression of CIC enhanced GSIS and raised cytosolic citrate levels. In normal rat islets, Ad-siCIC treatment also suppressed CIC mRNA levels and inhibited GSIS. We conclude that export of citrate and/or isocitrate from the mitochondria to the cytosol is an important step in control of GSIS.

Abstract 583: Identification of Target Proteins for Neuronal Calcium Sensor-1 (NCS-1) in the Heart

Introduction: Changes in intracellular Ca 2+ concentration regulate numerous cellular functions that are mediated by Ca 2+ binding to a large family of EF-hand Ca 2+ sensor proteins. We previously reported that one of them, neuronal calcium sensor-1 (NCS-1) was present in both cytosol and membrane fractions of rat heart. Adenovirus-mediated gene delivery of NCS-1 to isolated ventricular myocytes results in an increase in myocardial contractile function, with a slight alteration of sarcoplasmic reticular function. Aims and Methods: To further investigate the role of NCS-1 in the regulation of cardiac contractility, we used the yeast two-hybrid system to identify novel protein interactions among cardiac E-C coupling-associated proteins and NCS-1. Human NCS-1 was used as a bait to screen a human heart cDNA library by yeast mating. Results: 120 positive colonies were initially obtained under high stringency mating selection. DNA inserts of 47 true positive clones were sequenced; 27 clones contained unique DNA sequences, including 4 unknown genes. The remaining 23 identified proteins are primarily expressed in the cytoplasm, with fewer proteins localized at the plasma membrane. Cellular functions of these proteins include membrane transport, metabolism energy pathways, cell growth/maintenance, signal transduction/cell communication and structural/cytoskeletal-associated proteins. Interestingly, the yeast two hybrid studies show that NCS-1 interacts with Troponin I (cTnI), a key inhibitory protein involved in the regulation of E-C coupling in cardiac muscle. cTnI inhibits sarcomere contraction by interfering with actin-myosin interaction. Co-immunoprecipitation studies in cardiomyocytes also suggest an interaction between NCS-1 and Troponin I. Conclusion: Our results deonstrate that NCS-1 is involved in multiple aspects of Ca 2+ signaling in the heart. Furthermore, identification of a possible protein-protein interaction between Troponin I and NCS-1 provides novel insights into a cellular mechanism by which NCS-1 can augment cardiac contractile function.

Gene Therapy Using Adenovirus-Mediated Full-length Anti-HER-2 Antibody for HER-2 Overexpression Cancers

Therapeutic monoclonal antibody is increasingly applied in many clinical applications, although complicated technologies and high cost still limit their wide applications. To obtain the sustained serum antibody concentration with one single injection and lower the cost of antibody protein therapy, an adenovirus-mediated full-length antibody gene therapy was developed. Full-length antibody light-chain and heavy-chain sequences were linked with internal ribosome entry site and constructed into adenoviral vector under the control of cytomegalovirus promoter. Antibody expression in vitro and in vivo were tested with ELISA, and its antitumor efficacy was evaluated in SKOV-3-inoculated nude mice. Ad5-TAb-generated anti-HER-2 antibody presented the similar binding specificity with commercial trastuzumab. A single i.v. injection of 2 x 10(9) plaque-forming units of Ad5-TAb per mouse resulted in not only a sustained over 40 microg/mL serum antibody level for at least 4 weeks but also significant tumor elimination in the ovarian cancer SKOV-3-inoculated nude mice. An in vivo full-length antibody gene delivery system allows continuous production of a full-length antibody at high concentration after a single administration. Bioactive antibody macromolecules can be generated via gene transfer in vivo. All the data suggest that this novel adenovirus-mediated antibody gene delivery can be used for the exploitation of antibodies, without being hampered by the sophisticated antibody manufacture techniques and high cost, and, furthermore, can shorten the duration and reduce the expense of antibody developments.

Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes

AbstractUpon local delivery, adenovirus (Ad) serotype 5 viruses use the coxsackie and Ad receptor (CAR) for cell binding and αv integrins for internalization. When administered systemically, however, their role in liver tropism is limited because CAR-permissive and mutated viruses show similar biodistribution, a finding recently attributed to blood coagulation factor (F) IX or complement protein C4BP binding to the adenovirus fiber and “bridging” to either low-density lipoprotein receptor-related protein or heparan sulfate proteoglycans. Here, we show that hepatocyte transduction in vitro can be enhanced by the vitamin K-dependent factors FX, protein C, and FVII in addition to FIX but not by prothrombin (FII), FXI, and FXII. This phenomenon was not dependent on proteolytic activation or cell signaling activity and for FX was mediated by direct virus-factor binding. Human FX substantially enhanced hepatocyte transduction by CAR-permissive and mutated viruses in an ex vivo liver perfusion model. In vivo, global down-regulation of vitamin K-dependent zymogens by warfarin significantly diminished liver uptake of CAR-deleted Ads; however, this phenomenon was fully rescued by acute infusion of human FX. Our results indicate a common and pivotal role for distinct vitamin K-dependent coagulation factors in mediating hepatocyte transduction by adenoviruses in vitro and in vivo.

Effects of adenovirus‐expressing IL‐10 in alleviating airway inflammation in asthma

Allergic asthma strongly correlates with airway inflammation caused by cytokines secreted by allergen-specific type-2 T helper (Th2) cells, but the immunologic regulation of cell function is yet to be acquired. Further, IL-10 has been found to exert both antiinflammatory and immunoregulatory activities. This study aimed to elucidate the therapeutic effects of IL-10 administration via adenovirus-mediated gene delivery on airway inflammation in the ovalbumin (OVA)-induced murine model of asthma. BALB/c mice were sensitized by intraperitoneal injections with OVA and challenged by nebulized OVA. The sensitized mice were given an intratracheal delivery of adenoviral vector expressing the murine IL-10 gene (AdIL-10), or mock adenoviral vector 4 days before the inhalation challenge of the OVA. Inflammatory parameters, such as the development of airway hyper-responsiveness (AHR), bronchial lavage fluid eosinophils, and chemokines were assayed. Intratracheal administration of AdIL-10 could efficiently inhibit antigen-induced AHR and significantly decrease the number of eosinophils and neutrophils in the bronchoalveolar lavage fluid of OVA-sensitized and challenged mice during the effector phase. Our data showed that the intratracheal transfer of the IL-10 gene could affect the recruitment of inflammatory cells during the challenge phase in a way that would result in the inhibition of airway inflammation. These findings suggest that the development of an immunoregulatory strategy based on IL-10 might shed light on more effective treatment.

Role of cyclooxygenase‐2 induction by transcription factor Sp1 and Sp3 in neuronal oxidative and DNA damage response

Cyclooxygenase-2 (COX-2) has been implicated in neuronal survival and death. However, the precise regulatory mechanisms involved in COX-2 function are unclear. In the present study we found that COX-2 is induced in response to glutathione depletion-induced oxidative stress in primary cortical neurons. Two proximal specific Sp1 and Sp3 binding sites are responsible for the COX-2 promoter activity under normal as well as oxidative stress conditions through enhanced Sp1 and Sp3 DNA binding activity. Site-directed mutagenesis confirmed that -268/-267 positions serve as specific Sp1 and Sp3 recognition sites under oxidative stress. Enforced expression of Sp1 and Sp3 using HSV vectors increased the promoter activity, transcription, and protein level of COX-2 in cortical neurons. The dominant negative form of Sp1 abrogated the oxidative stress-induced promoter activity and expression of COX-2. We also demonstrated that adenovirus-mediated COX-2 gene delivery protected neurons from DNA damage induced by oxidative, genotoxic, and excitotoxic stresses and by ischemic injury. Moreover, COX-2(-/-) cortical neurons were more susceptible to DNA damage-induced cell death. These results indicate that in primary neurons Sp1 and Sp3 play an essential role in the modulation of COX-2 transcription, which mediates neuronal homeostasis and survival by preventing DNA damage in response to neuronal stress.