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Biomedical subjects

A E Smith

Publications and source records attributed to A E Smith.

At least 73 records · Page 4Linked to original sources
PubMed1996

Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung.

Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.

Adenoviruses, Human↗
PubMed1996

Antigen-specific tumor vaccines. Development and characterization of recombinant adenoviruses encoding MART1 or gp100 for cancer therapy.

The human melanoma tumor Ags, MART1 and gp100, are specifically recognized by HLA-A2-restricted CD8+ CTLs derived from melanoma patients and appear to be involved in tumor regression. In order to develop immunizing vectors for the treatment of patients with metastatic melanoma, replication-defective recombinant adenoviruses, Ad2CMV-MART1 and Ad2CMV-gp100, which encode these tumor Ags, have been generated. Infection of non-Ag expressing HLA-A2+ cell lines A375 and MDA-231 with the vectors resulted in recognition by Ag-specific CTLs as demonstrated by specific target cell lysis and release of cytokines, including IFN-gamma, TNF-alpha, and granulocyte-macrophage-CSF. Sodium butyrate and TNF-alpha can further augment adenovirus-mediated transgene expression and increase recognition by specific CTLs. Although adenovirus-infected cells expressed the E3/19K protein at detectable levels, significant reduction of surface MHC class I expression was observed in only 3 of 10 tumor cell lines infected with either Ad2CMV-MART1 or Ad2CMV-gp100. Because of the suspected homology between the human MART1 and gp100 genes and their murine counterparts, we immunized C57BL/6 mice with these recombinant adenoviruses and demonstrated that immunization with Ad2CMV-gp100 could protect mice from murine melanoma B16 challenge administered intradermally. Depletion of CD8+ but not CD4+ T cells in vivo from Ad2CMV-gp100-vaccinated mice eliminated the protective effect. The anti-gp100 T cells induced by Ad2CMV-gp100 vaccinated appeared to be responsible for the protection. Thus, these recombinant adenoviruses encoding tumor Ags may be useful as vaccines to induce specific T cell immunity for cancer therapy.

Adenoviridae↗
PubMed1996

Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.

Adenovirus E1 Proteins↗
PubMed1996

Biosynthetic and growth abnormalities are associated with high-level expression of CFTR in heterologous cells.

An inducible gene amplification system was utilized to study the effects of overexpression of cystic fibrosis transmembrane conductance regulator (CFTR) in vitro. BTS, a monkey kidney cell line expressing a temperature-sensitive simian virus 40 (SV-40) large T antigen was stably transfected at the nonpermissive temperature with a plasmid containing an SV-40 origin of replication and the cDNA for either the wild-type CFTR or the mutant G551D-CFTR. Shift of the isolated cell lines to the permissive temperature resulted in induction and accumulation to high levels of the CFTR plasmid, mRNA, and protein. However, high-level expression of CFTR was transient in both BTS-CFTR and BTS-G551D cells due to a decrease in their respective levels of CFTR mRNA. Because G551D-CFTR only exhibits residual Cl channel activity, this suggests that the observed downregulation with BTS-G551D cells may have been induced by either the physical presence of high amounts of CFTR or some low threshold level of Cl- channel activity. Examination of cell growth properties revealed a correlation between high-level expression of wild-type CFTR and growth arrest of the cells at the G2/M phase. However, similar induction of the G551D-CFTR mutant showed only a slight growth inhibition and little enrichment of cells at the G2/M phase. Cytofluorographic analysis further revealed that BTS-CFTR cells were significantly larger than parental BTS or BTS-G551D cells at all stages of the cell cycle. These results indicate that CFTR overexpression is capable of inducing its own downregulation and that high levels of Cl- channel activity can result in increased cell volume and subsequent cell growth abnormalities.

Animals↗
PubMed1996

Ability of adenovirus vectors containing different CFTR transcriptional cassettes to correct ion transport defects in CF cells.

Cystic fibrosis (CF) airway epithelial cells exhibit defective adenosine 3',5'-cyclic monophosphate (cAMP)-mediated chloride (Cl) secretion, abnormal hyperabsorption of sodium (Na+), and aberrant fluid transport. Adenovirus-mediated transduction of cystic fibrosis transmembrane conductance regulator (CFTR) corrects these ion and fluid transport abnormalities in CF cells. However, several challenges remain pertaining to the use of adenovirus vectors for gene delivery, including the efficiency of gene transfer and the host response to the vector. To improve the efficacy of adenovirus-mediated gene transfer, we have constructed a series of recombinant adenoviruses containing different CFTR transcriptional units, and we have evaluated their relative ability to correct electrolyte and fluid transport in polarized CF airway epithelial cells. The ability of the vectors to correct the CF Cl- transport defects was greatest when the human cytomegalovirus promoter was used. The E1a and phosphoglycerate kinase promoters resulted in the greatest persistence of functional CFTR expression. Efficacy of gene expression by recombinant adenoviruses improved as the cells were treated with increasing multiplicities of infection, as the duration of viral contact with the target cells was lengthened, and when the virus concentration was increased. Transduction of functional CFTR Cl- channel activity reversed the abnormal Na+ hyperabsorption observed in CF cells in a dose-dependent manner, suggesting that Na+ channel activity is downregulated by CFTR. Although efficient correction of both cAMP-mediated Cl- transport and fluid secretion could be achieved readily with these vectors, normalization of the Na+ absorption required vector administration at high multiplicities of infection.

Adenoviruses, Human↗
PubMed1996

Advocacy.

Explore the source record for details and available documents.

Child↗
PubMed1996

Biological response of nonhuman primates to long-term repeated lung exposure to Ad2/CFTR-2.

We have assessed the safety and efficacy of repeated adenovirus vector administration by exposing the left caudal lung lobe of rhesus monkeys to as many as 17 exposures of Ad2/CFTR-2. After nine doses of either 3 x 10(9) or 3 x 10(10) infectious units, the monkeys were free of adverse effects as assessed by thoracic radiographs, CBCs, clinical chemistries, arterial blood gases, and physical and clinical signs. In some animals elevated protein levels and increased numbers of cells were recovered in bronchoalveolar lavage (BAL), and in all animals there were increased proportions of lymphocytes in the BAL. After 11 doses, two animals were killed. In the lower dose animal (3 x 10(9) IU), there was little histopathology evident. In the higher dose animal (3 x 10(10) IU), histopathology was largely confined to a focal fibrotic lesion that may have been associated with treatment. At the tenth exposure, the dose was increased to 6 x 10(10) or 3 x 10(11) IU. There was evidence of lung injury by thoracic radiographs after two additional exposures and an increase in protein and number of cells in the BAL. The animals were still free of evidence of adverse effects by other parameters, but histopathologic changes were noted upon death. After 15 or 17 doses, three animals were instilled with Ad2/beta gal-2 and killed 3 days later. These animals had greatly reduced levels of transgene expression when compared with controls.

Adenoviruses, Human↗
PubMed1996

Humoral and cellular immune responses of nonhuman primates to long-term repeated lung exposure to Ad2/CFTR-2.

To evaluate the host immune response to long-term repeat administration of adenovirus vector, rhesus monkeys were treated at intervals of approximately 3 weeks with up to 18 instillations of Ad2/CFTR-2, a second generation vector encoding the cystic fibrosis transmembrane conductance regulator (CFTR). All monkeys instilled with Ad2/CFTR-2 developed a significant humoral immune response against adenovirus but not CFTR. Antibodies with virus neutralizing activity were detected in the serum and bronchoalveolar lavage (BAL) of all vector-treated monkeys and included both IgG and secretory IgA. Virus-specific T cells capable of proliferating in response to stimulation with adenovirus antigen were detected in all vector-treated monkeys. No CFTR-specific proliferation of peripheral blood lymphocytes was detected. An increase in the proportion of CD8+ T cells was noted in the BAL of virus-treated monkeys but cells from the BAL displayed little or no cytolytic activity against infected autologous fibroblasts when tested under a variety of culture conditions. However, MHC-restricted cytolytic activity was detected in the tracheobronchial lymph nodes and spleen of one of three virus-treated monkeys tested. MHC-unrestricted killing of infected fibroblasts was also observed with spleen cells from all animals tested. From these results, it appears that both the humoral and cell-mediated arms of the immune response were stimulated by repeated administration of high doses of Ad2/CFTR-2 suggesting that effective, long-term adenovirus gene therapy may require modification of the vector or treatment of the host to allow the virus to evade host immune defenses.

Adenoviruses, Human↗
PubMed1996

Adenovirus-mediated generation of cAMP-stimulated Cl- transport in cystic fibrosis airway epithelia in vitro: effect of promoter and administration method.

Cystic fibrosis (CF) is a common autosomal recesses disease in which loss of CFTR-Cl- channel function of defective cAMP-stimulated Cl- transport transfer across airway epithelia. Recombinant adenoviruses have shown progress as vectors with which to transfer CFTR cDNA to CF airway epithelia. Here we investigated variables involved in adenovirus-mediated transfer of CFTR by measuring cAMP- stimulated Cl- transport in CF airway epithelia grown as monolayers on permeable filter supports. When we compared the effects of different promoters, we found that persistent correction of Cl- transport was obtained when the vector contained the E1a promoter, or to a lesser extent the PGK promoter. Vector containing the CMV promoter produced a greater initial cAMP-stimulated Cl- current, but the duration of correction was shorter and the infection procedure itself increased CFTR expression, suggesting that high input doses of virus stimulate expression. We compared the level of expression, measured with a beta-galactosidase reporter of CFTR mRNA, with CFTR-mediated Cl- transport. Even low levels of expression generated significant Cl- current and marked increases in expression produced only modest increments in Cl- current. Correction of the CF Cl- transport defect was also improved when the concentration of adenovirus vector was high and when the duration of contact with the epithelium was prolonged. These findings may help optimize the ability of adenovirus vectors encoding CFTR to correct the CF Cl- transport defect.

Adenoviridae↗
PubMed1995

Sequence homologies between nucleotide binding regions of CFTR and G-proteins suggest structural and functional similarities.

Sequence homology between the alpha-subunits of G-proteins and other GTP-binding proteins and certain regions within the nucleotide binding domains (NBDs) of cystic fibrosis transmembrane conductance regulator (CFTR) indicates that these protein structures may be similar. A sequence alignment of the NBDs of CFTR and NBDs from other membrane transporters, forms the basis of a structural model. This model predicts that one of the conserved sequences GGQR, within which a number of CF mutations occur, forms part of the nucleotide binding pocket and serves as an ON/OFF conformational switch as observed in GTP binding proteins. Furthermore, based on subtle sequence differences between the first and second NBDs of CFTR and from structure-activity data, we suggest that the nucleotide binding site environments of the two NBDs are different.

Amino Acid Sequence↗
PubMed1995

Primary and secondary prevention services in clinical practice. Twenty years' experience in development, implementation, and evaluation.

This article reviews lessons from 20 years of experience in development and provision of clinical preventive services at Group Health Cooperative of Puget Sound, a large health maintenance organization. Critical factors for enhancing service include the use of a population-based epidemiologic viewpoint coupled with specific evidence-based criteria to examine issues; involvement of practitioners in the process; a systems approach to implementation focused on predisposing factors of the practitioners and enabling factors in the practice, organizational, and community environments; feedback of program outcomes; and the use of automated clinical information systems. Outcome results from our clinical prevention efforts include a 32% decrease in late-stage breast cancer (1989 to 1990); 89% of 2-year-old children with complete immunizations (1994); decrease in adult smokers from 25% to 17% (1985 to 1994); and an increase in bicycle safety helmet use among children from 4% to 48% along with a 67% decrease in bicycle-related head injuries (1987 to 1992). Systematic population-based approaches to the development and provision of clinical preventive services targeting the one-to-one level of primary care and multiple infrastructure levels of care are forging a synthesis of clinical medicine and public health approaches. This approach will become pervasive as clinical information systems improve, risk information is captured routinely, and practitioners gain skills in the art of patient risk behavior change and population-based care.

Breast Neoplasms↗
PubMed1995

Uncertainty in fitted estimates of apparent in vivo metabolic constants for chloroform.

It has become common practice to rely on fitted estimates of apparent in vivo metabolic constants (e.g., Vmax and KM) in the parameterization of PBPK models. Yet, quantitative estimates of precision in these fitted parameters are not routinely reported. Such information is needed to assess the reliability of model predictions. The purpose of this study was to assess the precision in estimates of Vmax and KM for chloroform, accounting for both the statistical uncertainties in parameter estimates from individual data sets and any additional uncertainty due to differences in the parameter estimates derived from various experiments. Joint confidence regions for Vmax and KM from each experiment, generated using maximum likelihood techniques, were used to evaluate these questions. Three previously published data sets were considered. Estimates of Vmax and KM obtained from these data sets differed more than could be explained as a consequence of a limited number of observations, measurement error, or stochastic error. Issues associated with the use of maximum likelihood techniques to estimate joint confidence regions, the estimation of metabolic constants from individual experiments within a gas uptake study versus the full data set, the degree of overlap in the joint confidence regions for metabolic constants obtained from separate data sets, and the implications for risk assessment are discussed.

Animals↗
PubMed1995

The ability of predicted internal dose measures to reconcile tumor bioassay data for chloroform.

PBPK models are developed in the hope that they will improve our ability to extrapolate from one species to another and from one exposure regime to another. Evidence that a dose measure was successful at reconciling the available animal bioassay data would be encouraging. It would give us some confidence that the dose measure (as evaluated by a PBPK model) might yield reasonable predictions for yet other species (e.g., humans) and other dose routes. We have investigated the ability of a modified version of the Corley et al. (R. A. Corley, A. L. Mendrala, F. A. Smith, D. A. Staats, M. L. Gargas, R. B. Conolly, M. E. Andersen, and R. H. Reitz, 1990, Toxicol. Appl. Pharmacol. 103, 512-527) PBPK model for chloroform to reconcile the available bioassay data. Two rate-dependent dose measures, maximal rate of metabolism in the liver, and percentage of hepatocytes killed per day performed well at reconciling the rodent liver bioassay data, while all rate-independent dose measures performed less well. In contrast, none of the PBPK dose measures were capable of reconciling the rat and mouse kidney tumor response data. Here, administered dose scaled to body surface area performed the best.

Animals↗