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

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

Values and limits of adenoviral vectors for gene transfer in vivo

Review of the therapeutic use of DNA transfer to treat a certain number of hereditary diseases (such as adenosine deaminase deficiency) or acquired diseases. The strategies (ex vivo manipulation or direct in vivo transfer of the corrective gene), vectors (retrovirus, adenovirus, nonviral vectors), and diseases which can benefit from gene therapy are considered and discussed together with an evaluation of the risk of gene therapy.     

Adenovirus-mediated gene transfer of viral interleukin-10 inhibits the immune response to both alloantigen and adenoviral antigen

Although adenoviral vectors are attractive for gene transfer, their effectiveness is limited by host antiviral immune responses. In this study, we determined if host antiallograft and antiviral immunity could be diminished with an adenoviral vector encoding the immunosuppressive cytokine viral interleukin-10 (vIL-10). AdSV40vIL-10, a vIL-10-expressing adenoviral vector with an SV40 promoter, induced significant prolongation of murine cardiac allograft survival to 32.2 +/- 1.7 days compared to 14.2 +/- 1.0 days for controls (p < 0.01). This effect was specific for vIL-10 encoding vector and could be inhibited by anti-vIL-10 monoclonal antibody (mAb). In vivo administration of adenovirus facilitated the generation of adenovirus-specific cytotoxic T lymphocytes (CTL), whereas treatment with AdSV40vIL-10 prevented CTL priming and generation of virus-specific immunity. AdSV40vIL-10 also induced extended expression of a beta-galactosidase reporter from a co-injected LacZ-encoding adenoviral vector. These results demonstrate that adenovirus-mediated gene transfer and expression of vIL-10 prolong allograft survival and inhibit the immune response to adenoviral antigens, thereby improving the persistence of the vector and extending transgene expression. The efficacy of adenoviral vectors can be improved by incorporating immunosuppressive genes into the vector.     

Antibody to CD40 ligand inhibits both humoral and cellular immune responses to adenoviral vectors and facilitates repeated administration to mouse airway

PURPOSE: Grating acuity values obtained with preferential looking techniques do not correspond to test results obtained with letter charts. The aim of the study was to investigate the agreement between grating acuity and other acuity tests. METHODS: Twenty-eight adult patients with strabismic amblyopia were assessed with the preferential looking-method and other linear and single letter and symbol tests. RESULTS: Assessment with preferential looking regularly led to an overestimation of the acuity values, but between preferential looking and single optotypes there was a much better agreement. This is most likely due to 'crowding' in strabismic amblyopia. A crowding ratio was calculated in the amblyopic eye, and crowding was found to be more prominent for comparisons of preferential looking with optotypes in rows, than for preferential looking and single optotypes. CONCLUSIONS: Strabismic amblyopia cannot be reliably identified with grating acuity. Preferential looking-testing is therefore of limited use as a screening method for the detection of amblyopia, but preferential looking could still be useful in the assessment and treatment.     

Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells

Nonviral gene delivery systems consist predominantly of lipoplexes or receptor-targeting and nontargeting polyplexes. We examined integrin-mediated gene delivery using an Arg-Gly-Asp/oligo-L-lysine ([K]16RGD) cyclic peptide and investigated its gene transfer efficiency when associated with a cationic liposome. We demonstrated that human cystic fibrosis and noncystic fibrosis tracheal epithelial cells in culture express integrins that recognise the RGD integrin-binding motif. We found a 10-fold (P < 0.01) increased expression of a luciferase encoding plasmid in these cells when complexing the plasmid to the [K]16RGD peptide as compared with plasmid alone. This increase was specific to the [K]16RGD peptide since neither a [K]16RGE nor a [K]16 peptide gave a comparable increase. Expression was further enhanced 30-fold (P < 0.01) with lipofectamine and the ratio of DNA/peptide/lipofectamine was critical for specificity and expression. Fluorescence and radioactive labelling of the complex showed that the [K]16RGD peptide increased the endocytic uptake of DNA into cells. The cell association of both DNA and peptide increased even further with lipofectamine. Confocal microscopy showed that the [K]16RGD peptide and the DNA internalised together within 30 min and localised to vesicles in the perinuclear region. These results show that an integrin-binding ligand can deliver genetic material to airway cells and that a cationic liposome can enhance the efficacy of this nonviral vector system.     

Dendritic cells transduced with an adenoviral vector encoding a model tumor-associated antigen for tumor vaccination

Evaluation of the potential role of dendritic cells (DCs) as adjuvants for tumor vaccination has focused primarily on techniques that load DCs with peptide tumor antigens. Our aim has been to optimize the induction of antitumor immunity by enhancing the ability of DCs to present tumor-associated antigens endogenously to the afferent lymphatic system in the appropriate major histocompatibility complex (MHC)-restricted context. We have used replication-defective adenovirus vectors (Ads) to transduce DCs with various genes, including tumor antigen genes. We found that 90% of murine bone marrow derived-DCs could be infected with an Ad vector expressing the beta-galactosidase gene and still retain their physiologic and phenotypic characteristics. Furthermore, we demonstrated that transgene expression was detectable in the spleen for at least 3 days following intravenous injection of Ad-transduced DCs. Using a polyoma middle T (PymT) transgenic murine mammary carcinoma model, we have shown that a single injection of 10(5)-4 x 10(6) DCs transduced with an Ad vector expressing PymT provided complete and specific protection against tumor cell challenge in 100% of vaccinated animals. Immunization against the PymT tumor by injection with the PymT expressing Ad vector alone resulted in varying degrees of effectiveness, was highly dependent upon the route of administration, and led to significant hepatic toxicity that was not seen in mice immunized with DC transduced with the Ad vector. Our results suggest that: (i) DCs can be very efficiently modified by ex vivo Ad transduction to express tumor-specific antigens, (ii) such modified DCs appear nontoxic and stimulate a potent antitumor response.     

Acceleration of widespread adenoviral gene transfer to intact rabbit hearts by coronary perfusion with low calcium and serotonin

Previous attempts at adenoviral gene transfer to the intact heart have been limited by the requirement for prolonged exposure to high virus concentrations. In an ex vivo coronary perfusion model of intact adult rabbit hearts, we previously reported gene transfer to 96% of cardiac myocytes after a 60 min exposure to 1.6 x 10(9) p.f.u./ml Ad beta gal, a recombinant adenovirus encoding beta-galactosidase. Here we sought to decrease the virus exposure time by enhancing microvascular permeability to increase the efficiency of adenoviral gene transfer. Baseline perfusion with 1.0 x 10(8) p.f.u./ml Ad beta gal in normal Krebs solution (1 mM calcium) caused infection of 22% of myocytes at 30 min and 40% at 60 and 120 min. Increasing the virus concentration, decreasing perfusate calcium concentration, or pretreating with serotonin or bradykinin in Krebs solution or L-NAME in heparinized rabbit blood significantly decreased the necessary exposure time. Under optimal conditions of serotonin pretreatment, 50 mumol/l perfusate calcium, and a virus concentration of 1.6 x 10(9) p.f.u./ml, 2 min of coronary perfusion sufficed to produce near-total infection. This profound enhancement of infection parameters has important implications for in vivo myocardial gene transfer, where a similar strategy could facilitate gene therapy for common myocardial disorders.     

Use of the cosmid adenoviral vector cloning system for the in vitro construction of recombinant adenoviral vectors

The large size of the adenoviral genome unfortunately precludes there being many unique, useful restriction sites available for in vitro manipulation. Two methods have been developed for the construction of recombinant adenoviral vectors to date: in vivo homologous recombination or direct ligation in vitro. The efficiency of either the direct ligation method or the homologous recombination method is low because of the large size of the recombinant adenoviral vectors. To circumvent these problems, we have chosen to use the cosmid vector system to facilitate the assembly of recombinant adenoviral vectors. In this paper, we demonstrate for the first time that recombinant adenoviral vectors can be efficiently constructed in vitro by the cosmid vector system. With this method, it is possible to amplify the recombinant adenoviral vector DNA sufficiently to transfect 293 cells. The cosmid adenoviral vector cloning method for in vitro construction of the full-length recombinant adenoviral vectors represented here is simple and efficient and should facilitate the development of recombinant adenoviral vectors for human gene therapy.     

Direct in vivo gene transfer to canine myocardium using a replication-deficient adenovirus vector

Inter-alpha-inhibitor (I alpha I) is a serine protease inhibitor present in human plasma. It has a molecular weight of about 220 kDa which encompasses 3 chains including two heavy chains and one light chain. The light chain, known as bikunin, is responsible for the antitryptic activity of I alpha I in the inhibition of various enzymes, such as trypsin and chymotrypsin. Under physiologic or certain pathologic circumstances, several macromolecules related to I alpha I appear in plasma and urine. However, the physiologic role of I alpha I remains unclear. As far as urolithiasis is concerned, two urinary macromolecules related to I alpha I have been isolated and shown to be potent inhibitors of calcium oxalate formation. One of these inhibitors, uronic-acid-rich protein (UAP), has been identified and well characterized. The sequence of the first 18 amino acid residues of UAP is identical with that of bikunin. Furthermore, the immunoreaction between UAP and I alpha I antibody using immunoblot analysis was positive. UAP isolated from the urine of stone formers exhibited less inhibitory activity towards calcium oxalate crystallization than that derived from the urine of healthy subjects. This suggests a structural abnormality of the inhibitor obtained from stone patients. The organic matrix extracted from kidney stones contained a protein antigenically related to I alpha I. We conclude that UAP is a member of I alpha I family taking part in inhibiting calcium oxalate crystallization, and modulating the formation of stones in the urinary tract.     

Direct gene transfer to mouse melanoma by intratumor injection of free DNA

The evaluation of commercially available test systems of competitive enzyme-linked immunosorbent assay (ELISA), for Aflatoxin M1 (AFM1) was performed in experimental conditions, through repeated analysis, in samples of milk powder contaminated with known concentrations of the toxin. Recoveries of AFM1 added to milk at levels of 0.10, 0.20, 0.50 and 1.00 ng/ml were 83.0%, 87.5%, 103.0% and 111.8% respectively. Relative standard deviations for the above mentioned concentrations were 65.5%, 31.8%, 10.9% and 13.6%, respectively (n = 10, per spiking level). According to these results the ELISA is appropriate for AFM1 research and/or surveying, mainly for concentrations between 0.20-1.00 ng/ml.     

Novel sites of adrenomedullin gene expression in mouse and rat tissues

Adrenomedullin (AM) was originally identified in pheochromocytoma tissue and was characterized as a hypotensive peptide. The tissue distribution and cellular localization of AM messenger RNA (mRNA) were determined in mouse and rat tissues by in situ hybridization. Three probes were used: two nonoverlapping probes to the pro-AM N-terminal 20 peptide (PAMP) and AM peptide regions of mouse pro-AM, and a larger complementary DNA (cDNA) probe spanning both the PAMP- and AM peptide-coding regions. The most intense expression of AM mRNA was in endometrium and epithelial cells lining the uterus and mouse adrenal medulla. Moderate levels of expression were detected in kidney glomerulus and cortical distal tubules, ovarian corpus luteum and follicles, epithelial cells lining the bronchioles, cardiac atrium and ventricle, posterior pituitary (particularly in female rats), stomach, small intestine (microvilli, mucosa and submucosa), spleen, and pancreas. Lower levels were observed in pulmonary alveoli, anterior pituitary, and submandibular gland. No expression was detected in the testis, thymus, skeletal muscle, or liver. The localization of AM mRNA in epithelial cells lining the uterus, bronchioles, and gastrointestinal tract indicates novel roles for AM, possibly as an antimicrobial agent. The strong expression of AM in uterus, ovary, and posterior pituitary suggests that AM plays a role in female reproduction.     

A new yeast artificial chromosome vector designed for gene transfer into mammalian cells

The supercritical fluid extraction (SFE) behavior of cocaine and its major metabolite benzoylecgonine (BZE) was investigated and found to be highly dependent upon the chemical nature of the matrix and the manner in which the target drug analytes are incorporated into or on the matrix. The recovery of cocaine from Teflon wool, filter paper, drug-fortified hair, and drug user hair was studied using a variety of CO2/modifier mixtures. Incorporation of a triethylamine (TEA)/water modifier mixture provided dramatic improvements in the recovery of cocaine from interactive matrixes. The results suggest that the SF extractability of cocaine is not limited by analyte solubility; rather, desorption of cocaine from hair binding sites is a rate-limiting step in the SFE process. A displacement SFE mechanism is hypothesized in which TEA (as the triethylammonium cation) competes with cocaine for negatively charged hair binding sites. The dependence of extractability on hair/drug binding interactions allows the differentiation of cocaine present at different discrete sites in hair based on differences in SFE behavior. These findings suggest the potential for distinguishing exogenous (i.e., environmental) from endogenous (i.e., physiological) sources of drugs in hair. In contrast to the results observed for cocaine, SFE recoveries of BZE were poor from all matrixes and under all conditions studied. Its increased polarity, the presence of an additional binding site, and the possibility of multiple charged states suggest that poor BZE recoveries may be due to both poor analyte solubility and failure to desorb the analyte from hair binding sites under the conditions employed.     

Fluorescent virions: dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells

T cells specific for nucleosomal autoepitopes are selectively expanded in lupus mice and these Th cells drive autoimmune B cells to produce pathogenic antinuclear antibodies. We transfected the TCR-alpha and -beta chain genes of a representative, pathogenic autoantibody-inducing Th clone specific for the nucleosomal core histone peptide H471-94 into TCR-negative recipient cells. Although the autoimmune TCRs were originally derived from SNF1 (I-Ad/q) mice, the transfectants could recognize the nucleosomal autoepitope presented by APC-bearing I-A molecules of all haplotypes tested, as well as human DR molecules. Competition assays indicated that the autoepitopes bound to the MHC class II groove. Most remarkably, MHC-unrestricted recognition of the nucleosomal peptide epitope was conferred by the lupus TCR-alpha chain even when it paired with a TCR-beta chain of irrelevant specificity. Several other disease-relevant Th clones and splenic T cells of lupus mice had similar properties. The TCR-alpha chains of these murine lupus Th clones shared related motifs and charged residues in their CDRs, and similar motifs were apparent even in TCR-alpha chains of human lupus Th clones. The lupus TCR-alpha chains probably contact the nucleosomal peptide complexed with MHC with relatively high affinity/avidity to sustain TCR signaling, because CD4 coreceptor was not required for promiscuous recognition. Indeed, pathogenic autoantibody-inducing, CD4-negative, TCR-alphabeta+ Th cells are expanded in systemic lupus erythematosus. These results have implications regarding thymic selection and peripheral expansion of nucleosome-specific T cells in lupus. They also suggest that universally tolerogenic epitopes could be designed for therapy of lupus patients with diverse HLA alleles. We propose to designate nucleosomes and other antigens bearing universal epitopes "Pantigens" (for promiscuous antigens).     

Behavioral and cellular protection of rat dopaminergic neurons by an adenoviral vector encoding glial cell line-derived neurotrophic factor

Consanguineous marriages are strongly preferred in much of West and South Asia. This paper examines the prevalence and sociodemographic correlates of consanguineous unions in Pakistan using local and national data. Information from 1011 ever-married women living in four multi-ethnic and multi-lingual squatter settlements of Karachi, the main commercial centre of the country, are compared with data from the national 1990/91 Pakistan Demographic and Health Survey (PDHS), based on information provided by 6611 women. Both sets of results indicate that approximately 60% of marriages were consanguineous, over 80% of which were between first cousins. The mean coefficients of inbreeding (F) in the present generation were 0.0316 and 0.0331 for the Karachi and PDHS data respectively. In both surveys the prevalence of consanguineous unions appeared to be unchanged over the past three to four decades. Consanguineous unions were more common among women who were illiterate or had only primary level education, were first or second generation migrants from rural areas of Pakistan or, in the PDHS, lived in rural areas, and whose parents were also consanguineously married.     

Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer

Computer and robot assisted surgery is concerned with the improvements achievable by using computer methods and robotic devices to plan and execute surgical interventions. The registration of different coordinate frames, often achieved through the matching of 3D data sets, represents a crucial step connecting planning and execution. Orthopaedic surgery already features a number of functioning applications which include registration routines relying on presurgically implanted fiducial markers. Replacing such invasive routine with non-fiducial registration procedures is regarded as a necessary step towards a minimisation of surgical invasiveness. A minimally invasive registration technique based on the iterative closest point algorithm is presented and conceived for a specific computer and robot assisted orthopaedic reconstructive intervention, namely total knee arthroplasty. The whole surgical protocol is examined in detail and the experimental results, relative to tests performed on synthetic and animal specimens, are thoroughly reported and discussed. The authors indicate that the proposed registration approach is well-suited for the relevant application and appropriate for in vivo testing.     

Development of a positive method for male stem cell-mediated gene transfer in mouse and pig

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods with low efficiency of transgenic production. A promising approach for producing transgenic animals by using male stem cells was recently reported by Brinster and Zimmermann (1994; Proc Natl Acad Sci 91:11298-11302) and by Brinster and Avarbock (1994: Proc Natl Acad Sci USA 91:11303-11307). However, in order to apply this technique to producing transgenic animals, some difficulties have to be overcome. These include a satisfactory method for short-term in vitro culture for drug selection after transfection with exogenous DNA, and methods for the use of livestock such as pigs. We developed a new method for transferring foreign DNA into male germ cells. Mice and pigs were treated with busulfan, an alkylating agent, to destroy the developing male germ cells, and liposome/bacterial LacZ gene complexes were introduced into each seminiferous tubule by using a microinjection needle. As a control, lipofectin was dissolved in phosphate-buffered saline at a ratio of 1:1, and then injected into seminiferous tubules. In mice, 8.0-14.8% of seminiferous tubule expressed the introduced LacZ gene, and 7-13% of epididymal spermatozoa were confirmed as having foreign DNA by polymerase chain reaction. The liposome-injected testes were all negative for X-gal staining. These results indicate that some spermatozoa were successfully transformed in their early stages by liposome/DNA complexes. In pigs, foreign DNA was also incorporated efficiently into male germ cells, and 15.3-25.1% of the seminiferous tubules containing germ cells expressed the LacZ gene. The data suggest that these techniques can be used as a powerful tool for producing transgenic livestock.