Adenovirus-mediated delivery of fas ligand inhibits intimal hyperplasia after balloon injury in immunologically primed animals.

BACKGROUND: Adenoviral constructs have been used for studies of injury-induced vascular hyperplasia in immunologically naive laboratory animals, but their usefulness for intra-arterial gene therapy may be limited by the prevalence of preexisting immunity to adenovirus in the patient population. Here, we explored the efficacy of adenovirus-mediated transfer of Fas ligand, a cytotoxic gene with immunomodulatory properties, in inhibiting injury-induced vascular lesion formation in both naive and immunologically primed animals. METHODS AND RESULTS: Lesion formation was evaluated in balloon-injured carotid arteries of naive and adenovirus-immunized rats that were infected with adenoviral constructs expressing Fas ligand (Ad-FasL), the cyclin-dependent kinase inhibitor p21 (Ad-p21), or beta-galactosidase (Ad-betagal). In naive rats, Ad-FasL induced apoptosis in medial vascular smooth muscle cells and inhibited intimal hyperplasia by 60% relative to Ad-betagal-treated vessels (P<0.05), whereas the cytostatic agent Ad-p21 decreased lesion size by 58% (P<0.05). In animals preimmunized with an adenoviral vector containing no transgene, Ad-FasL significantly inhibited neointima formation (73% reduction, P<0.05), but Ad-p21 failed to inhibit neointima formation relative to controls. Immunologically primed rats displayed robust T-cell infiltration in Ad-p21- and Ad-betagal-treated vessels, but T-cell infiltration was markedly attenuated in Ad-FasL-treated vessels. CONCLUSIONS: Our data demonstrate that adenovirus-mediated Fas ligand delivery can inhibit intimal hyperplasia in both immunologically primed and naive animals, whereas the efficacy of an adenovirus-mediated p21 delivery is limited to immunologically naive animals. This study documents, for the first time, the therapeutic efficacy of intravascular adenoviral gene transfer in animals with preexisting immunity to adenovirus.

Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1alpha/VP16 hybrid transcription factor.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that regulates expression of genes involved in O(2) homeostasis, including vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis. We sought to exploit this native adaptive response to hypoxia as a treatment for chronic ischemia. METHODS AND RESULTS: A hybrid protein consisting of DNA-binding and dimerization domains from the HIF-1alpha subunit and the transactivation domain from herpes simplex virus VP16 protein was constructed to create a strong, constitutive transcriptional activator. After transfection into HeLa, C6, and Hep3B cells, this chimeric transcription factor was shown to activate expression of the endogenous VEGF gene, as well as several other HIF-1 target genes in vitro. The bioactivity of HIF-1alpha/VP16 hybrid gene transfer in vivo was examined in a rabbit model of hindlimb ischemia. Administration of HIF-1alpha/VP16 was associated with significant improvements in calf blood pressure ratio, angiographic score, resting and maximal regional blood flow, and capillary density (all P:<0.01). CONCLUSIONS: The HIF-1alpha/VP16 hybrid transcription factor is able to promote significant improvement in perfusion of an ischemic limb. These results confirm the feasibility of a novel approach for therapeutic angiogenesis in which neovascularization may be achieved indirectly by use of a transcriptional regulatory strategy.

Increased contact time improves adenovirus-mediated CFTR gene transfer to nasal epithelium of CF mice.

Multiple dosing with recombinant adenoviral vectors containing the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the nasal mucosa of cystic fibrosis (CF) transgenic mice reportedly results in only partial correction of the CF defect in chloride (Cl-) secretion without normalizing sodium (Na+) hyperabsorption, perhaps indicating inefficient gene transfer into the nasal airway epithelium in vivo. In this study, we have examined whether optimizing vector administration such as contact time could improve gene transfer efficiency. Changes in basal nasal potential difference (PD), and in PD (delta PD) following addition of amiloride and subsequent removal of Cl- from the luminal perfusate were assayed. As reported previously, the basal nasal PD was significantly more negative in CF mice (-24.9 +/- 2.1 mV) than in normal mice (-6.3 +/- 1.2 mV). Normal mouse nasal mucosa exhibited a large hyperpolarization in response to low Cl- substitution (delta PD of 8.5 +/- 1.9 mV), whereas the nasal mucosa of the CF mouse depolarized in response to this treatment. No correction of either the Cl- or Na+ transport defects were observed when 5 x 10(9) IU of Ad2/CFTR-5 were administered to the nasal passage of CF mice over a period of 5-20 min. However, when CF mice were perfused over a period of 60 min with the same dose of vector, a significant response (delta PD of 5.9 +/- 1.1 mV) to low Cl- substitution was detected 2 days later. In these mice, the basal nasal PD (-10.5 +/- 1.4 mV) and the response to amiloride were also reduced, indicating a partial correction of the Na+ transport defect. Expression of functional CFTR activity was transient with no measurable delta PD signals observed by day 7 post-treatment. These results suggest that prolonging the contact between an adenoviral vector and the respiratory epithelium enhances the efficiency of gene transfer and can result in improved correction of the CF Na+ and Cl- ion transport defects. Therefore, strategies that improve internalization of viral vectors and that prolong their contact time with target cells may result in the improved clinical efficacy of such vectors.

A concentrated and stable aerosol formulation of cationic lipid:DNA complexes giving high-level gene expression in mouse lung.

Advances in gene therapy vectors and techniques hold promise for treatment of many inherited and acquired diseases. For lung indications, especially those involving the epithelium, delivery of the gene therapy vehicle ideally will involve the use of an aerosol. Aerosol delivery of transgenes using cationic lipids is currently limited by the ability to generate highly concentrated formulations of lipid:DNA complexes that are stable and retain their activity following aerosolization. We have examined many of the variables inherent in aerosolizing cationic lipid gene delivery vehicles and have devised a new formulation that incorporates small amounts of a polyethylene glycol-containing lipid. This formulation has allowed the preparation of concentrated dispersions of cationic lipid:plasmid DNA (pDNA) complexes (> 20 mM pDNA) at approximately 10-fold higher concentrations than previously reported. Most of the pDNA in these formulations was bound to the lipid component and thereby protected from nebulizer-induced shearing; the pDNA also maintained full biological activity both in vitro and in vivo. This new formulation thus represents a significant improvement over current methods to prepare concentrated, active cationic lipid gene delivery vectors, and provides a new tool with which to test gene transfer to the lung.

Enhancement of Fas ligand-induced inhibition of neointimal formation in rabbit femoral and iliac arteries by coexpression of p35.

Adenovirus-mediated gene transfer of Fas ligand (FasL) inhibits neointimal formation in balloon-injured rat carotid arteries. Vascular smooth muscle (VSM) cells coexpressing murine FasL and p35, a baculovirus gene that inhibits caspase activity, are not susceptible to FasL-mediated apoptosis in vitro but are capable of inducing apoptosis of VSM cells that do not express p35. We reasoned that coexpression of p35 in FasL-transduced VSM cells in vivo would promote their survival, enhance FasL-induced apoptosis of adjacent VSM cells, and thereby facilitate a greater inhibition of neointimal formation. In balloon-injured rabbit femoral arteries, either Ad2/FasL/p35 or Ad2/FasL was infused into the injured site and withdrawn 20 min later. Both vectors induced a dose-dependent reduction (p < 0.05) of the neointima-to-media ratio when assessed 14 days later. However, Ad2/FasL/p35 exhibited a significantly greater inhibition of neointimal formation than Ad2/FasL. In a more clinically relevant model of restenosis, rabbit iliac arteries were injured with an angioplasty catheter under fluoroscopic guidance. Adenoviral vectors were delivered locally to the injured site over a period of 2 min, using a porous infusion balloon catheter. Twenty-eight days after gene transfer angiographic and histologic assessments indicated a significant (p < 0.05) inhibition of iliac artery lumen stenosis and neointimal formation by Ad2/FasL/p35 (5 x 10(11) particles per artery). The extent of inhibition was comparable to that achieved with Ad2/TK, an adenoviral vector encoding thymidine kinase (5 x 10(11) particles per artery) and coadministration of ganciclovir for 7 days. These data suggest that coexpression of p35 in FasL-transduced VSM cells is more potent at inhibiting neointimal formation and as such represents an improved gene therapy approach for restenosis.

Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung.

Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) that peaked at days 1-2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids.

Comparative susceptibility of a canine cell line and bluetongue virus susceptible cell lines to a bluetongue virus isolate pathogenic for dogs.

Recently, bluetongue virus (BLU) serotype 11 was detected in diseased dogs that had been inoculated with live attenuated vaccine contaminated with this serotype of bluetongue virus (Akita et al., 1994). For various laboratory tests, BLU can be propagated in different cell cultures. No information was found in the literature about the possibility of propagating this virus in canine cells. To determine whether the BLU isolate from the contaminated canine vaccine (BLU-vac) is unique in its ability to replicate in canine cells, this virus was studied in parallel with U.S. prototype strains of BLU (serotypes 2, 10, 11, 13, and 17), in hamster lung (HmLu-1) and canine kidney (MDCK) cell cultures. In HmLu-1 cell cultures, the BLU-vac produced cytopathic effect (CPE) of the same type as the U.S. prototype BLU strains by 4 to 6 d postinoculation. In MDCK cell cultures, all of the BLU strains tested were able to replicate but did not produce CPE. The BLU-inoculated MDCK cells became persistently infected, and these cultures continued to produce infectious BLU even after six serial passages over 2 1/2 mo. In none of these cultures was CPE observed. In mixed cultures containing both HmLu-1 and MDCK cells, CPE first affected the HmLu-1 islands; subsequently, CPE spread also to the areas with MDCK cells. The silent persistent infection of the MDCK cells with BLU indicates that more stringent screening of the cells used in the production of live vaccines for various contaminating viruses is necessary.

Detection of bovine group B rotaviruses in feces by polymerase chain reaction.

A pair of primers designed from the sequence of genome segment 9 of group B rat rotavirus (IDIR) were employed to amplify genome segment 9 of a group B bovine rotavirus in a polymerase chain reaction (PCR) and to sequence the derived PCR products. A new pair of primers were synthesized from the obtained sequence data and used in a PCR detection assay for group B bovine rotavirus in fecal samples. In addition, another pair of primers were designed to produce a PCR-derived internal probe. This probe was used in a chemiluminescent hybridization to confirm the specificity and to increase the sensitivity of the assay. This assay could detect 0.1 fg of target double-stranded RNA. It was specific to group B bovine rotavirus and did not detect group B rat (IDIR) and porcine rotaviruses, group A bovine (NCDV), simian (SA-11), equine (H-2), porcine (OSU), human (DS-1), deer, and avian rotaviruses, coronavirus, or other enteric organisms tested in this study.

PCR detection of group A bovine rotaviruses in feces.

A polymerase chain reaction (PCR) protocol has been developed for identification of bovine group A rotavirus infection in feces. Primers (20mers) complementary to 3' ends of double-stranded RNA genome segment 6 of bovine rotavirus NCDV strain were synthesized and used in PCR. Bovine rotavirus RNA from infected cell culture was employed to optimize the PCR protocol. Rotavirus-negative fecal samples were spiked with known quantities of bovine rotavirus, and the sensitivity of the PCR assay was determined. Fecal samples were extracted with phenol and treated to eliminate unidentified PCR inhibitor(s) in feces, and PCR was performed. PCR products were either visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the assay was 6 x 10(4) viral particles/ml of feces with ethidium bromide-stained agarose gel visualization or 6 x 10(2) viral particles/ml of feces with chemiluminescent hybridization. The PCR assay was applied to 18 fecal specimens from clinical cases. All 16 clinical samples that were positive for rotavirus by enzyme-linked immunosorbent assay (ELISA) or by ELISA and electron microscopy (EM) were positive by PCR. The 2 samples that were rotavirus negative by ELISA or by ELISA and EM were also negative on PCR analysis.

Detection of epizootic hemorrhagic disease virus serotypes 1 and 2 in cell culture and clinical samples using polymerase chain reaction.

The potential for the use of the polymerase chain reaction (PCR) in detecting epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell cultures and clinical samples was studied. Using oligoribonucleotide primers selected from genome segment 6 of EHDV-2 (Alberta strain), the PCR-based assay resulted in a 387-base pair (bp) PCR product. EHDV RNA from US prototype serotypes 1 and 2 and a number of EHDV field isolates, propagated in cell cultures, were detected by this EHDV PCR-based assay. Amplification products were visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the PCR assay was 100 fg of virus RNA (equivalent to 6 x 10(3) virus particles) with ethidium bromide-stained agarose gels. Chemiluminescent hybridization increased the sensitivity of the PCR assay 1,000 times, and specific signals were detected from 0.1 fg of virus RNA (equivalent to 6 virus particles). Amplification product was not detected when the PCR-based assay was applied to RNA from the US bluetongue (BT) virus prototype serotypes 2, 10, 11, 13, and 17 or total nucleic acid extracts from uninfected baby hamster kidney-21 cells, Vero cells, and blood cells from deer that were EHDV seronegative and virus isolation negative. Application of this EHDV PCR-based assay to clinical samples resulted in detection of EHDV RNA from blood and spleen samples from a deer in California with clinical hemorrhagic disease. This EHDV PCR-based assay could provide a rapid, sensitive, and specific assay for detection of EHDV infection in susceptible ruminants.

Detection of bluetongue virus in clinical samples by polymerase chain reaction.

A previously described bluetongue virus (BTV) serogroup polymerase chain reaction (PCR) assay was applied to clinical samples. The sensitivity of the BTV serogroup PCR was increased by the use of non-radioactive chemiluminescent hybridization. Unfractionated whole blood samples from rams experimentally inoculated with cell culture-adapted BTV-11 UC-8 were analyzed by virus isolation (VI) on Vero cells and PCR. VI and PCR were in agreement, with the exception of 3 blood samples that were VI negative and PCR positive. In semen spiked with BTV-11 UC-8, PCR detected as little as 1.6 x 10(2) plaque-forming units of BTV/ml of semen. BTV in the spleen of a sheep submitted for necropsy for suspect BTV infection was detected by both PCR and VI in embryonated chicken eggs. BTV PCR with nonradioactive chemiluminescent hybridization resulted in a level of sensitivity comparable to that of VI and likely more sensitive than VI on Vero cells for blood. This BTV PCR has great promise for rapid, sensitive, and specific detection of active BTV infection in a variety of clinical samples.

Comparison of polymerase chain reaction and virus isolation for detection of epizootic hemorrhagic disease virus in clinical samples from naturally infected deer.

We compared our recently reported reverse transcriptase polymerase chain reaction (PCR)-based assay for detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples with different virus isolation (VI) procedures. Thirty-six blood samples and 1 spleen sample from deer were assessed by the EHDV PCR assay and VI in baby hamster kidney (BHK)-21 cells and embryonated chicken eggs (ECE). The EHDV PCR assay detected EHDV RNA from 6 blood samples obtained from deer during 1988-1989 outbreaks of epizootic hemorrhagic disease and from the spleen and blood samples of a deer with clinical hemorrhagic disease in 1992. The 6 blood samples from the 1988-1989 outbreaks and the spleen sample from the 1992 case were VI positive on BHK-21 cell culture. The blood from the same deer with the PCR- and VI-positive spleen was VI negative in BHK-21 cells and ECE. All EHDV isolates were identified as EHDV serotype 2 by a plaque inhibition test. The results of this study indicate that the sensitivity of the previously described EHDV PCR assay is comparable to or greater than that of the VI method in BHK-21 cell culture or ECE. The EHDV PCR assays could provide a superior diagnostic alternative to the current cumbersome and time-consuming VI procedures.

Detection of bluetongue virus serogroup by polymerase chain reaction.

To facilitate detection of active bluetongue virus (BTV) infection, a polymerase chain reaction (PCR) protocol was developed. The BTV reverse transcriptase PCR (RT-PCR) is a 1-tube reaction and involves chemical denaturation of the double-stranded viral RNA target, a complementary DNA (cDNA) synthesis step, and PCR amplification of the cDNA. BTV RT-PCR using primers derived from highly conserved genome segment 10 results in a 251-base pair (bp) product. BTV RNA from all USA prototype serotypes 2, 10, 11, 13, and 17; a wide spectrum of USA BTV field isolates including serotypes 10, 11, 13, and 17; and a spectrum of Israeli field isolates including serotypes 2, 4, 6, 10, and 16 were detected by BTV RT-PCR. With agarose gels, the 251-bp product was detected from as little as 100 fg-1 pg of BTV RNA, which is equivalent to 5 x 10(3)-5 x 10(4) viral particles or 5 x 10(2)-5 x 10(3) infectious units. With dot blot hybridization, specific PCR product was detected from as little as 1 fg of BTV RNA, which is equivalent to 50 viral particles, or 5 infectious units. This level of sensitivity is comparable to that of virus isolation. The BTV RT-PCR using primers derived from genome segment 10 can detect a wide spectrum of USA and Israeli BTV serotypes and has potential for detection of infection by the BTV serogroup. Application of this BTV PCR to clinical samples is in progress.

PCR detection of acute Ehrlichia canis infection in dogs.

A polymerase chain reaction (PCR)-based detection assay that specifically detected Ehrlichia canis in dogs with acute infections was developed. A region of the 16S ribosomal RNA gene of E. canis was targeted for PCR amplification and chemiluminescent hybridization (CH) with a complementary internal 287-base pair (bp) oligonucleotide probe. The CH improved the PCR assay sensitivity 1,000-fold as compared with visualization on ethidium bromide-stained agarose gels. The PCR assay with CH (PCR/CH) detected as little as 30 fg of E. canis genomic DNA, the equivalent of approximately 150 E. canis organisms. The 495-bp product defined by the specific primers was not detected when genomic DNA from E. platys, E. chaffeensis, E. risticii, and E. equi were used in the PCR/CH assay. The PCR/CH assay was tested with unfractionated blood samples collected from 9 dogs experimentally infected with E. canis. The PCR/CH assay had greater detection sensitivity than did cell culture isolation (CCI) from infected blood. PCR/CH detected E. canis 7 days prior to CCI in 4 of 6 experimentally infected dogs. The results obtained with the PCR/CH assay otherwise consistently matched the results obtained by CCI. This PCR/CH assay is a rapid, sensitive, and specific method for E. canis detection with sensitivity comparable to or exceeding that of CCI. A diagnosis of E. canis using this PCR/CH assay can be made in 2 days as compared with 1-4 weeks for CCI. The PCR/CH assay appears to be an acceptable alternative or complement to current diagnostic techniques.

Cardiovascular effects of a PEGylated apelin.

Several studies have documented cardiovascular effects of apelin, including enhanced inotropy and vasodilation. However, these cardiovascular effects are short lived due to the predicted short circulating half-life of the apelin peptide. To address this limitation of apelin, we pursued N-terminal PEGylation of apelin and examined the cardiovascular effects of the PEGylated apelin. A 40kDa PEG conjugated apelin-36 (PEG-apelin-36) was successfully produced with N-terminal conjugation, high purity (>98%) and minimum reduction of APJ receptor binding affinity. Using an adenylate cyclase inhibition assay, comparable in vitro bioactivity was observed between the PEG-apelin-36 and unmodified apelin-36. In vivo evaluation of the PEG-apelin-36 was performed in normal rats and rats with myocardial infarction (MI). Cardiac function was assessed via echocardiography before, during a 20 min IV infusion and up to 100 min post peptide infusion. Similar increases in cardiac ejection fraction (EF) were observed during the infusion of PEG-apelin-36 and apelin-36 in normal rats. However, animals that received PEG-apelin-36 maintained significantly increased EF over the 100 min post infusion monitoring period compared to the animals that received unmodified apelin-36. Interestingly, EF increases observed with PEG-apelin-36 and apelin-36 were greater in the MI rats. PEG-apelin-36 had a prolonged circulating life compared to apelin-36 in rats. There were no changes in aortic blood pressure when PEG-apelin-36 or apelin-36 was administered. To our knowledge this is the first report of apelin PEGylation and documentation of its cardiovascular effects.

Replication and cytopathic effects of ovine lentivirus strains in alveolar macrophages correlate with in vivo pathogenicity.

Ruminant lentiviruses share genomic sequences and biologic properties with human immunodeficiency viruses. Four ovine lentivirus strains were assessed for cytopathic effects and virus replication. Lentivirus isolate H/24 produced high virus titers and lysis of synovial cells but replicated slowly and caused no fusion of alveolar macrophages. Lentivirus isolates 84/28 and 85/14 produced low virus titers, less syncytia, and limited or no cell lysis in synovial cells and macrophages. In contrast, ovine lentivirus isolate 85/34 produced early peak virus titers and caused rapid fusion and lysis of both macrophages and synovial cells. Ovine lentivirus isolates which were cytopathic for macrophages induced lymphoproliferative disease when inoculated into lambs.

Detection of bluetongue virus in the blood of inoculated calves: comparison of virus isolation, PCR assay, and in vitro feeding of Culicoides variipennis.

The interval after infection when bluetongue virus (BTV) was present in the blood of calves inoculated with BTV serotype 10 (BTV 10) was evaluated by virus isolation (VI) in embryonated chicken eggs (ECE), BTV-specific polymerase chain reaction (PCR), and in vitro blood feeding of vector Culicoides variipennis (C.v.) sonorensis. BTV nucleic acid was detected by PCR in blood cells for 16 to 20 weeks after infection whereas infectious virus was detected by VI in ECE for 2 to 8 weeks. BTV was detected in calf blood by in vitro feeding of C.v. sonorensis for only 0 to 2 weeks after inoculation of calves with BTV 10. Selected bloods which were positive by PCR analysis but not by VI in ECE were not infectious for sheep. The data are consistent with the hypothesis that prolonged viremia in BTV-infected cattle results from association of the virus with blood cells, especially erythrocytes. The fact that calf blood that contained viral nucleic acid as determined by PCR analysis, but not infectious virus as determined by VI in ECE, was not infectious for either the insect vector or sheep suggests that cattle whose blood contains BTV nucleic acid but not infectious virus are unimportant to the epidemiology of BTV infection.