Biomedical subjects

B C Trapnell

Publications and source records attributed to B C Trapnell.

At least 19 recordsLinked to original sources

PubMed2001

Alveolar macrophage deficiency in osteopetrotic mice deficient in macrophage colony-stimulating factor is spontaneously corrected with age and associated with matrix metalloproteinase expression and emphysema.

Macrophage colony-stimulating factor (M-CSF) is one of several hematologic growth factors capable of regulating the survival, proliferation, and differentiation of macrophages, but its role in modulation of the accumulation and function of alveolar macrophages (AMs) in vivo is not well defined. Osteopetrotic (Op/Op) mice have no detectable M-CSF and show variable tissue-specific reductions in macrophage numbers. It was hypothesized that AMs would be decreased in number and have altered function in Op/Op mice because of the absence of M-CSF. Lung macrophages identified by Mac-3 staining in lung sections were decreased in number in 20-day-old Op/Op mice (P <.001) but not Op/Op mice older than 4 months (P =.68) compared with findings in age-matched littermate controls. The numbers of AMs recovered by bronchoalveolar lavage (BAL) were also reduced in young but not adult Op/Op mice compared with controls. Expression of interleukin-3 (IL-3) was increased in the lungs of Op/Op mice compared with controls as determined by quantification of IL-3 cytokine levels (P =.04), bioactivity (P =.02), and messenger RNA transcript levels. AMs of Op/Op mice spontaneously released higher levels of matrix metalloproteinases (MMPs) than AMs of controls as determined by immunohistochemical staining of AMs and zymographic assessment of BAL fluid and AM lysates. Consistent with an increased release of MMP, Op/Op mice had abnormal elastin deposition and spontaneously developed emphysema in the absence of molecular or cellular evidence of lung inflammation. These data show that the AM deficiency observed in young Op/Op mice is spontaneously corrected with age and is associated with increased lung levels of IL-3, spontaneous MMP expression by AMs, and destruction of lung tissue.

Age Factors↗

PubMed2001

GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1.

GM-CSF gene targeted (GM(-/-)) mice are susceptible to respiratory infections and develop alveolar proteinosis due to defects in innate immune function and surfactant catabolism in alveolar macrophages (AMs), respectively. Reduced cell adhesion, phagocytosis, pathogen killing, mannose- and Toll-like receptor expression, and LPS- or peptidoglycan-stimulated TNFalpha release were observed in AMs from GM(-/-) mice. The transcription factor PU.1 was markedly reduced in AMs of GM(-/-) mice in vivo and was restored by selective expression of GM-CSF in the lungs of SPC-GM/GM(-/-) transgenic mice. Retrovirus-mediated expression of PU.1 in AMs from GM(-/-) mice rescued host defense functions and surfactant catabolism by AMs. We conclude that PU.1 mediates GM-CSF-dependent effects on terminal differentiation of AMs regulating innate immune functions and surfactant catabolism by AMs.

Animals↗

PubMed2001

Adenovirus infection increases iNOS and peroxynitrite production in the lung.

Host inflammatory and immune responses limit viral gene expression after administration of replication-deficient adenoviruses to the lung. The current study asks whether inducible nitric oxide synthase (iNOS) expression and peroxynitrite generation accompanied the inflammatory response following intratracheal administration of adenovirus. Pulmonary iNOS mRNA and protein were increased 2, 7, and 14 days following administration of 2 x 10(9) plaque-forming units of recombinant adenovirus (Av1Luc1) to BALB/c mice. Adenovirus infection was associated with a marked increase in nitrotyrosine staining. Intense nitrotyrosine staining was observed in alveolar macrophages, respiratory epithelial cells, conducting airways, and alveolar spaces 2 days postinfection. Two weeks after exposure to adenovirus, nitrotyrosine staining was detected within alveolar macrophages, suggesting adenovirus enhanced the nitration of proteins that were subsequently taken up by alveolar macrophages. Western blot analysis using anti-nitrotyrosine antibody did not demonstrate accumulation of nitrated surfactant protein A (SP-A), although a small fraction of aggregated SP-A comigrated with a nitrotyrosine-positive protein. iNOS expression, peroxynitrite, and nitrotyrosine generation accompany and may contribute to inflammatory responses to adenovirus in the lung.

Adenoviridae Infections↗

PubMed2000

Internalization of adenovirus by alveolar macrophages initiates early proinflammatory signaling during acute respiratory tract infection.

Adenovirus is a common respiratory pathogen which causes a broad range of distinct clinical syndromes and has recently received attention for its potential for in vivo gene delivery. Although adenovirus respiratory tract infection (ARTI) results in dose-dependent, local inflammation, the pathogenesis of this remains unclear. We hypothesized that alveolar macrophages (AMphi) rapidly internalize adenovirus following in vivo pulmonary administration and then initiate inflammatory signaling within the lung. To evaluate the role of AMphi in the induction of lung inflammation during ARTI in vivo, we directly assessed adenovirus uptake by murine AMphi and correlated uptake with the initiation of proinflammatory gene expression. Stimulation of cytokine (tumor necrosis factor alpha [TNF-alpha], interleukin-6 [IL-6], macrophage inflammatory protein-2 [MIP-2], and MIP-1alpha) expression in the lung was evaluated at the level of mRNA (by reverse transcription-PCR [RT-PCR]) and protein (by enzyme-linked immunosorbent assay) and by identification of cells expressing TNF-alpha and IL-6 mRNA in lung tissues (by in situ hybridization) and isolated lung lavage cells (by RT-PCR). Adenovirus, labeled with the fluorescent dye (Cy3), was rapidly and widely distributed on epithelial surfaces of airways and alveoli and was very rapidly ( approximately 1 min) localized within AMphi. At 30 min after infection AMphi but not airway epithelial or vascular endothelial cells expressed mRNA for TNF-alpha and IL-6, thus identifying AMphi as the cell source of initial cytokine signaling. IL-6, TNF-alpha, MIP-2, and MIP-1alpha levels progressively increased in bronchoalveolar lavage fluid after pulmonary adenovirus infection, and all were significantly elevated at 6 h (P < 0.05). To begin to define the molecular mechanism(s) by which adenovirus initiates the inflammatory signaling in macrophages, TNF-alpha expression from adenovirus-infected RAW264.7 macrophages was evaluated in vitro. TNF-alpha expression was readily detected in adenovirus-infected RAW cell supernatant with kinetics similar to AMphi during in vivo infection. Blockage of virus uptake at specific cellular sites, including internalization (by wortmannin), endosome acidification and/or lysis (by chloroquine) or by Ca(2+) chelation (by BAPTA) completely blocked TNF-alpha expression. In conclusion, results showed that during ARTI, (i) AMphi rapidly internalized adenovirus, (ii) expression of inflammatory mediators was initiated within AMphi and not airway epithelial or other cells, and (iii) the initiation of inflammatory signaling was linked to virion uptake by macrophages occurring at a point after vesicle acidification. These results have implications for our understanding of the role of the AMphi in the initiation of inflammation following adenovirus infection and adenovirus-mediated gene transfer to the lung.

Acute Disease↗

PubMed1999

Efficient in vivo catheter-based pericardial gene transfer mediated by adenoviral vectors.

Adenoviral vectors are promising agents for a number of in vivo gene therapy applications including diseases of the heart and coronary vessels. Efficient intravascular gene transfer to specific sites has been achieved in occluded vessels, but otherwise is hampered by the effect of blood flow on localized vector uptake in the vessel wall. An alternative delivery approach to coronary arteries is the expression of diffusible gene products into the pericardial space surrounding the heart and coronary arteries. However, in vivo pericardial access is comparatively difficult and has been limited to surgical approaches. We hypothesized that efficient adenovirus-mediated gene expression in pericardial lining mesothelium could be achieved by transmyocardial vector delivery to the pericardium. To evaluate this concept, a hollow, helical-tipped penetrating catheter was used to deliver vector-containing fluid directly into the intrapericardial space. The catheter was introduced percutaneously in anesthetized mongrel dogs, advanced into the right ventricle, and the tip passed through the apical right ventricular myocardium under direct radiographic visualization until the open end of the catheter tip resided in the intrapericardial space. Adenoviral vectors expressing either nuclear-localizing beta-galactosidase, cytoplasmic luciferase, or secreted human alpha 1AT reporters (Av1nBg, Av1Lu, or Av1Aa, respectively) were instilled through the catheter into the intrapericardial space. Three days later the animals were sacrificed and reporter gene expression was evaluated in pericardium, epicardium, and multiple other tissues. In animals receiving Av1nBg, beta-galactosidase activity was evident in most of the pericardial lining endothelium, up to 100% in many areas. In animals receiving Av1Lu, luciferase reporter activity was abundant in pericardial tissues, but near-background levels were observed in other organs. In animals receiving Av1Aa, human alpha 1AT was abundant (16-29 mg/ml) in pericardial fluid, but was undetectable in serum. All animals tolerated the procedure well with no electrocardiographic changes and no clinical sequelae. These observations demonstrate highly efficient adenovirus vector delivery and gene transfer and expression in the pericardium and support the feasibility of localized gene therapy via catheter-based pericardial approaches. We suggest that the pericardial sac may serve as a sustained-release protein delivery system for the generation of desired gene products or their metabolites for diffusion into the epicardial region.

Adenoviridae↗

PubMed1999

Pulmonary inflammation associated with repeated, prenatal exposure to an E1, E3-deleted adenoviral vector in sheep.

Fetal gene therapy may prove useful in treating diseases that manifest in the perinatal or early postnatal period. Adenoviruses effectively transfer gene expression to a variety of tissues but also stimulate inflammatory and immune responses. The purpose of this study was to test the hypothesis that exposure of fetal sheep to a first generation adenovirus vector encoding bacterial beta-galactosidase, Av1nBg, before the development of the immune system, is safe, minimizes inflammatory and immune responses and induces tolerance. A total of 22 fetal sheep was studied; of these, two were born with respiratory distress, seven were electively killed and 13 died in utero. The incidence of mortality was higher than the < or = 10% we have experienced with other fetal sheep studies and was not likely related to complications arising from surgical or anesthetic procedures. Inflammatory and fibrotic responses were observed in the lungs and may represent untoward long-term consequences of in utero adenoviral gene therapy. Tolerance to Av1nBg was not established, and repeated exposure to Av1nBg before birth was associated with significant pathology and mortality.

Adenoviridae↗

PubMed1999

SP-A enhances viral clearance and inhibits inflammation after pulmonary adenoviral infection.

Surfactant protein A (SP-A) is a member of the collectin family of host defense molecules expressed primarily in the epithelial cells of the lung. To determine the role of SP-A in pulmonary adenoviral infection, SP-A-deficient (SP-A -/-) mice were intratracheally infected with a replication-deficient recombinant adenovirus, Av1Luc1. Lung inflammation was markedly increased in SP-A -/- compared with SP-A +/+ mice and was associated with increased hemorrhage and epithelial cell injury. Polymorphonuclear cells in bronchoalveolar lavage fluid (BALF) were increased in SP-A -/- mice after administration of adenovirus. Coadministration of adenovirus and purified human SP-A ameliorated adenoviral-induced lung inflammation in SP-A -/- mice. Concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-1beta were increased in BALF of SP-A -/- mice. Likewise, TNF-alpha, IL-6, macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic protein-1, and MIP-2 mRNAs were increased in lung homogenates from SP-A -/- mice 6 and 24 h after viral administration. Clearance of adenoviral DNA from the lung and uptake of fluorescent-labeled adenovirus by alveolar macrophages were decreased in SP-A -/- mice. SP-A enhances viral clearance and inhibits lung inflammation during pulmonary adenoviral infection, providing support for the importance of SP-A in antiviral host defense.

Adenoviridae↗

PubMed1999

Interleukin-1 receptor antagonist inhibits interleukin-8 expression in A549 respiratory epithelial cells infected in vitro with a replication-deficient recombinant adenovirus vector.

In an earlier study, we showed that a recombinant adenovirus vector with deletions in the E1 and E3 regions of the viral genome (AV1LacZ4) induces expression of interleukin (IL)-8 in A549 cells (a human respiratory cell line). IL-8 can be induced through several pathways, including activation by IL-1. We tested the hypothesis that the induction of IL-8 by the AV1LacZ4 adenovirus is accomplished by means of the IL-1/IL-8 activation pathway, which could be blocked by IL-1 receptor antagonist (IRAP). Viral infections of A549 cells were performed at a multiplicity of infection (MOI) of 50 in the presence and absence of IRAP (50 ng/ml). A549 cells were also stimulated with tumor necrosis factor (TNF)-alpha (100 ng/ml), a known stimulant of IL-8, in the presence and absence of IRAP. IL-8 expression was evaluated by Northern blot analysis and enzyme-linked immunosorbent assay. Levels of IL-8 protein and messenger RNA (mRNA) were greater in the infected cells than in the uninfected ones at 24, 48, and 96 h (P < 0.01). Virus-infected cells treated with IRAP expressed 75% less IL-8 mRNA and protein (P < 0.01) than did untreated cells, whereas IRAP pretreatment of TNF-alpha-stimulated cells did not affect IL-8 production. IL-1 production by the virus-infected cells was detectable by concentration of the supernatants and reverse transcription-polymerase chain reaction. We conclude that IL-8 is produced by virus vector-infected cells, partly through IL-1 activation that can be downregulated by IRAP.

Adenoviridae↗

PubMed1998

Nonspecific inflammation inhibits adenovirus-mediated pulmonary gene transfer and expression independent of specific acquired immune responses.

Replication-deficient adenovirus vectors (Avs) have shown high-efficiency gene transfer in a variety of animal models, but demonstrated lower than expected efficiency in the intensely inflammatory milieu of the respiratory tract of individuals with cystic fibrosis (CF). Specific acquired immune responses directed at adenovirus capsid proteins are known to limit the duration of transgene expression and the effectiveness of vector readministration. In these models, however, nonspecific inflammation is also frequently noted to accompany specific immune responses. Because inflammation can occur early after Av administration, we hypothesized that inflammation may block Av-mediated gene transfer in the lung independent of specific immune responses. To evaluate this hypothesis, we measured pulmonary gene transfer and expression in the absence or presence of the potent antiinflammatory agent dexamethasone. To address and eliminate concerns over the potentially confounding effects of systemic, vector-specific acquired immune responses, evaluations were confined to a 3-day period following Av administration and were carried out, in parallel, in normal and immunodeficient (athymic) mice. Dexamethasone significantly reduced Av-associated inflammation in all animals as measured by a significant reduction of blinded, quantitative lung histopathology scores and by reduced proinflammatory cytokine release. Concomitant with reduced inflammation, gene transfer efficiency was significantly increased in both normal and immunodeficient animals as measured by transgene product activity (beta-galactosidase) in total lung homogenates 3 days after vector administration. This finding could not be explained by a direct effect of dexamethasone on transgene specific activity. To begin to understand the molecular mechanisms of Av-induced inflammatory responses, lung levels of the chemoattractive chemokines MIP-2, MIP-1alpha, and MCP-1 were quantified. All were elevated significantly in Av-exposed animals. Dexamethasone reduced levels of MCP-1 and MIP-1alpha, but not MIP-2, consistent with the observed pattern of inflammatory cell changes. Expression of several proinflammatory cytokines including TNF-alpha, IL-6, IL-1beta, and IFN-gamma were also elevated in Av-exposed animals and modulated by dexamethasone. These observations demonstrate that nonspecific inflammation is an important determinant of the efficiency of in vivo pulmonary gene transfer and expression independent of specific immune responses and may have important implications for human gene therapy for diseases of the lung.

Adenoviridae↗

PubMed1998

Lung-specific expression of adenovirus E3-14.7K in transgenic mice attenuates adenoviral vector-mediated lung inflammation and enhances transgene expression.

Herein, we report that the adenovirus E3-14.7K protein inhibits the inflammatory response to adenovirus in transgenic mice in which the E3-14.7K gene was selectively expressed in the respiratory epithelium, using the human surfactant protein C (SP-C) promoter. E3-14.7K mRNA and protein were detected specifically in the lungs of SPC/E3-14.7K transgenic mice. Responses of the transgenic mice to Av1Luc1, an E1-E3-deleted Ad vector encoding the luciferase reporter gene, were examined, including vector transgene expression and lung inflammation. In wild-type mice, luciferase activity declined rapidly and was lost 14 days following Av1Luc1 administration. The loss of luciferase activity was associated with pulmonary infiltration by macrophages and lymphocytes. In heterozygous SPC/E3-14.7K mice, luciferase activity was increased by 7 days compared with control littermates, and pulmonary infiltration by macrophages was decreased. In homozygous (+/+) SPC/E3-14.7K mice, luciferase activity was increased 7, 14, and 21 days following administration compared with wild-type mice, and lung inflammation was markedly reduced. After Av1Luc1 administration, PCNA staining of bronchiolar and alveolar respiratory epithelial cells was decreased in SPC/E3-14.7K transgenic mice, indicating decreased epithelial cell proliferation, a finding consistent with the observed reduction in inflammation. CD4 and CD8 lymphocyte populations were only mildly altered, while humoral responses to adenoviral vectors were unchanged in the SPC/E3-14.7K mice. The E3-14.7K protein expressed selectively in respiratory epithelial cells suppresses Ad-induced pulmonary epithelial cell cytotoxicity and lung inflammation in vivo and prolongs reporter gene expression.

Adenoviridae↗

PubMed1998

Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy.

Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy.

Adenoviridae↗

PubMed1997

Recombinant adenoviral vector disrupts surfactant homeostasis in mouse lung.

Although replication-deficient adenoviruses efficiently transfer genes into epithelial cells of the lung, host immune responses limit the extent and duration of gene expression. To define further the role of inflammatory responses to first-generation, recombinant, deltaE1, deltaE3 adenovirus in lung pathology and surfactant protein homeostasis, expression of the surfactant proteins SP-A, SP-B, and proSP-C was determined by immunohistochemistry 2, 7, and 14 days following intratracheal administration of 2 x 10(9) pfu of a recombinant adenovirus, Av1Luc1, to BALB/c nu/nu and BALB/c wild-type mice. Two to 7 days after virus administration, an acute inflammatory response was observed in both mouse strains. Respiratory epithelial cells were sloughed, and extracellular accumulation of SP-A and SP-B was detected in the airways. Diminished immunostaining for SP-A and SP-B was noted in type II cells, and SP-A and SP-B mRNA expression was decreased in focal regions of the lungs from both mouse strains. One week after virus administration, immunostaining for proSP-C was markedly increased in cells lining the regenerating alveolar epithelial surfaces. Two weeks after Av1Luc1 treatment of nu/nu mice, immunostaining for SP-A, SP-B, and proSP-C was similar to those patterns observed prior to adenoviral administration. In immunocompetent wild-type mice, however, immunostaining for surfactant proteins was absent in areas associated with chronic lymphocytic infiltration. The recombinant adenoviral vector, Av1Luc1, caused acute inflammatory responses in the respiratory epithelium with disruption of surfactant protein homeostasis in both wild-type and nu/nu mice. Alterations in surfactant homeostasis persisted in wild-type mice. Thus, both acute and thymic-dependent immune responses limit transgene expression and disrupt surfactant protein gene expression and homeostasis. Because surfactant proteins are critical to host defense and to the maintenance of alveolar stability following injury, these findings raise concerns regarding both acute and chronic toxicity of first-generation recombinant adenoviral vectors for gene transfer.

Adenoviridae↗

PubMed1997

Anti-T cell receptor antibody prolongs transgene expression and reduces lung inflammation after adenovirus-mediated gene transfer.

Replication-deficient delta E1a-E3 adenovirus mediates efficient gene transfer to the mouse lung; however it induces a host immune response mediated, in part, by T cells. This immune response is associated with loss of transgene expression. Monoclonal antibodies (mAb) against the T cell receptor (TCR) complex can inhibit both CD4+ and CD8+ T cell responses in vivo and are the most potent anti-T cell agents in clinical use. To determine whether such mAbs can be used to prolong adenovirus-mediated transgene expression, the vector Av1Luc1 (delta E1a-E3 recombinant adenovirus encoding the firefly luciferase gene) was administered intratracheally to C57BL/6 mice on day 0. Three days prior to adenovirus administration (day -3), mice were treated with a single i.p. injection of the anti-TCR mAb H57. Controls received phosphate-buffered saline. Animals were sacrificed on days 3, 14, 28, and 56 and lungs were assessed for transgene expression and histopathology. Luciferase activity decreased markedly in the controls by day 14, but was maintained at high levels in animals receiving anti-TCR mAb. A mild, focal, predominantly neutrophilic inflammation was observed in the alveoli of all mice 3 days after virus administration. In PBS-treated controls, this inflammation progressed to a moderate to severe multifocal, perivascular and peribronchiolar lymphoid infiltration at 14 days. B cells and T cells were present in approximately equal numbers, with CD4+ T cells predominating over CD8+ T cells by 3- to 28-fold. Treatment with H57 resulted in near-complete prevention of the lymphocytic inflammatory infiltrate and increased luciferase activity throughout the 56-day study period, in association with TCR modulation and T cell depletion. Thus, anti-TCR mAb decreases inflammation and prolongs gene expression following adenovirus-mediated gene transfer.

Adenoviridae↗

PubMed1997

Adenovirus-mediated transduction of ribozymes abrogates HER-2/neu and pleiotrophin expression and inhibits tumor cell proliferation.

The combination of specific gene targeting technologies with efficient gene delivery systems could provide the means to evaluate the concept of anticancer strategies designed to block expression of potentially rate-limiting tumor promoting factors. Here, we constructed adenoviruses expressing hammerhead-ribozymes targeted to two of these factors, the tyrosine kinase receptor HER-2/neu or the growth factor pleiotrophin (PTN). Adenovirus-mediated transduction of either HER-2/neu- or PTN-targeted ribozymes depleted the respective RNAs and inhibited protein expression significantly in three different human cancer cell lines. This resulted in almost complete abrogation of HER-2/neu- or PTN-dependent cancer-cell proliferation, thus demonstrating the feasibility of this approach as a future cancer gene therapy.

Adenoviridae↗

PubMed1996

Infection of A549 cells with a recombinant adenovirus vector induces ICAM-1 expression and increased CD-18-dependent adhesion of activated neutrophils.

A significant number of pulmonary exacerbations in patients with cystic fibrosis (CF) and asthma are associated with respiratory virus infections. The molecular mediators of this process are beginning to be understood. Viral infection of respiratory epithelial cultures in vitro leads to the production of intercellular adhesion molecule-1 (ICAM-1) (a ligand for inflammatory cell adhesion and activation) and a number of proinflammatory cytokines. Human gene therapy vectors derived from human adenoviruses (AV) are currently under evaluation for CF transmembrane regulator (CFTR) gene delivery to the airway epithelium of CF patients. However, studies in animal models using these AV vectors demonstrate pulmonary inflammation following AV exposure. Using an in vitro model, we examined the hypothesis that exposure of respiratory epithelial cells to AV vectors results in upregulation of ICAM-1 gene expression. Infections were performed using a replication-deficient, first-generation AV vector. A549 cells (a human pulmonary adenocarcinoma cell line) were exposed to AV at multiplicity of infection of 50-150 plaque-forming units/cell (resulting in > 90% of cells expressing the reporter gene by 48 hr following exposure). Measurements of ICAM-1 expression were made at time intervals following virus exposure using enzyme immunoassay, flow cytometry, and Northern blot analysis. Cell-bound ICAM-1 was significantly increased 96 hr following vector exposure, two to four times control, p < 0.001). The AV-exposed A549 cells also supported increased levels of adhesion of activated neutrophils 96 hr following AV exposure (four times control, p < 0.001) that was blocked by antibody to CD18. AV exposure of A549 monolayers increases expression of biologically active ICAM-1. Strategies to minimize host cellular proinflammatory responses to the replication-deficient AV vectors may improve their safety for gene therapy.

Adenocarcinoma↗

PubMed1996

Safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lungs of nonhuman primates.

To define the toxicity of cystic fibrosis transmembrane conductance regulator gene (CFTR) gene therapy with a replication-deficient recombinant adenovirus (Av1Cf2) in a nonhuman primate model, 10(10) plaque forming units (pfu) were instilled directly through a bronchoscope into the right lung of 5 macaques, and a lower dose of 4 x 10(6) pfu was administered to the right lung of 1 macaque. One sham-treated control received phosphate-buffered saline (PBS). The macaques were evaluated sequentially by clinical examination, vital signs, weight, hematology, blood chemistry, chest radiography, pulse oximetry, and bronchoalveolar lavage (BAL) at baseline and 3-28 days post-treatment. After the period of observation, macaques were sacrificed for autopsy and histological examination. The macaques tolerated the experimental therapy clinically with no changes in body temperature, oxygen saturation, heart rate, body weight, or blood pressure. However, 1 macaque with visible evidence of aspiration at the time of initial bronchoscopy developed tachypnea with right lower lobe (RLL) pneumonia on chest radiograph and by histology. There were no changes in Hgb, Wbc, BUN, plasma electrolytes, bilirubin, or hepatic transaminases. In the macaques that received 10(10) pfu, there was a progressive increase in the number of CD8+ lymphocytes in BAL that was maximal at 28 days. Histological examination of the treated lungs of the high-dose macaques at 3 days showed marked peribronchial and perivascular cuffing by inflammatory cells and alveolar accumulation of neutrophils and macrophages. The alveolitis appeared to be resolving at 28 days, although the perivascular and peribronchial aggregates of mononuclear cells were still present. In the high-dose macaques, BAL interleukin-8 (IL-8) was increased at all time points (256-388 pg/ml versus 1-84 pg/ml at baseline and in control), whereas IL-1 beta was increased only at days 21 and 28 (341-852 pg/ml versus 30-92 pg/ml at baseline and in control). There were no increases in BAL cell counts, IL-1 beta or IL-8, and histological changes were mild in the macaque that received 4 x 10(6) pfu. Evaluation for Av1Cf2-derived human CFTR expression using RS-PCR demonstrated expression at 3, 10, and 21, but not 28 days in macaques treated with 10(10) pfu of Av1Cf2. In situ hybridization analysis demonstrated human CFTR mRNA in the alveolar regions of the lobes that received the vector at 10 and 21 days. There was no evidence of expression after treatment with 4 x 10(6) pfu. This study showed that high-dose adenoviral vector administration to the lung achieved CFTR gene transfer and expression but was associated with increased concentrations of cytokines in BAL and alveolar inflammation. A low dose, equivalent to the maximum clinical dose currently proposed for phase I trials in human subjects, was not associated with cellular or cytokine evidence of inflammation, and histological abnormalities were mild.

Adenoviridae↗

PubMed1996

Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in comparison of data from different preclinical and clinical studies and may also have important implications for how adenovirus vectors can optimally be used in human gene therapy.

Adenoviridae↗

PubMed1996

Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy.

Adenoviridae↗