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Biomedical subjects

J M Wilson

Publications and source records attributed to J M Wilson.

At least 163 records · Page 9Linked to original sources
PubMed1998

Long-term effect of perfluorocarbon distension on the lung.

BACKGROUND/PURPOSE: We have demonstrated previously that postnatal lung growth can be accelerated by continuous intrapulmonary distension with perfluorocarbon (PFC). The purpose of this study was to examine the effect of PFC distension on long-term lung function and to determine if shorter periods of lung distension would be sufficient to stimulate growth. METHODS: Eight neonatal lambs underwent a right thoracotomy. The superior segment of the right upper lobe of the experimental group (n = 5) was isolated and distended with PFC to an intrabronchial pressure of 7 to 10 mm Hg. The inferior segment was removed. After a 7-day distension period (the maximum period of exposure currently allowed for humans), the experimental animals underwent removal of the intrabronchial catheter and surgical closure of the bronchial stump. Control animals (n = 3) underwent right upper lobe inferior segmentectomy alone. Animals were allowed to breathe spontaneously and reach 3 to 6 months of age. Before death, all animals were evaluated by chest radiographs and ventilation-perfusion scans. Pulmonary venous blood gas levels were obtained. Lungs were harvested and airway fixed for morphometric analysis. RESULTS: Chest radiographs of the experimental group showed variable amounts of intrapleural and interstitial PFC but were otherwise normal. Results of ventilation/perfusion (VQ) scans of all experimental animals were normal. On retrieval, the right upper lobe of experimental animals appeared slightly hyperinflated compared with controls. Right upper lobe pulmonary vein PaO2 to left pulmonary vein PaO2 ratio was comparable in experimental and control animals (1.2 +/- .41 v 0.92 +/- .15). DNA to protein ratios were slightly higher in the right upper lobes of experimental animals, however, the difference was not statistically significant (.64 +/- .11 v .42 +/- .03). On histological evaluation, the airway epithelium and alveoli of the right upper lobe of experimental animals appeared normal. Lung morphometry results showed no statistically significant differences in alveolar number between experimental and control animals. CONCLUSIONS: From these preliminary data we conclude that (1) lung architecture is preserved in juvenile animals subjected to intrapulmonary PFC distension as neonates, (2) lung function is preserved in the lobar segment after PFC distension, and (3) intrapulmonary PFC distension appears to be safe at 3 to 6 months follow-up. However, 7 days of PFC distension is insufficient to promote lung growth.

Animals↗
PubMed1998

Insulin-like growth factor-I gene expression in three models of accelerated lung growth.

BACKGROUND/PURPOSE: We have learned previously that in utero tracheal ligation reverses the structural and physiological effects of surgically created congenital diaphragmatic hernia. In addition, we have discovered that postnatal lung growth similarly can be accelerated using liquid-based airway distension with perfluorocarbon. Another model of accelerated lung growth is that of compensatory growth seen after neonatal pneumonectomy. In all of these models, growth has occurred because of an increase in alveolar number rather than enlargement of preexisting alveoli. However, the molecular mechanisms underlying these processes remain unknown. The purpose of this study was to determine if gene expression could be altered by changes in physical forces in the prenatal and postnatal lung. METHODS: The three models of accelerated lung growth studied were the following: (1) The prenatal group, consisted of fetal lambs (n = 12) that underwent the surgical creation of a left diaphragmatic hernia at 90 days' gestation. Six of these animals also underwent simultaneous tracheal ligation. (2) The PFC group consisted of five neonatal animals that underwent isolation of the superior segment of the right upper lobe, with intrabronchial distension with perfluorocarbon to 7 to 10 mm Hg pressure for a 3-week period. (3) The postpneumonectomy group consisted of four neonatal animals that underwent left pneumonectomy. In the fetal study, lungs were retrieved at term (130 days), and in the postnatal study, lungs were retrieved 3 weeks after initial intervention. In all cases, RNA was extracted from snap-frozen lung samples and Northern blot analysis performed. RESULTS: Insulinlike growth factor-I, insulinlike growth factor-II, and vascular endothelial growth factor gene expression were analyzed by densitometry. Insulinlike growth factor-I gene expression was found to be decreased in association with experimental diaphragmatic hernia (P = .005), but restored to normal with tracheal ligation. Insulinlike growth factor-I gene expression was significantly increased in both postnatal models of accelerated lung growth (P = .022, P = .016). No significant differences were found in insulinlike growth factor-II or vascular endothelial growth factor gene expression. CONCLUSIONS: The authors conclude from these preliminary data that (1) insulin like growth factor-I gene expression is reduced in experimental fetal diaphragmatic hernia and restored to normal by tracheal ligation, and (2) insulinlike growth factor-I gene expression is increased in both the liquid-based airway distension and postpneumonectomy models of accelerated postnatal lung growth. The authors speculate that all of these manipulations exploit a natural pathway essential for normal lung growth.

Animals↗
PubMed1998

Efficient induction of protective anti-malaria immunity by recombinant adenovirus.

The immunogenicity of a previously constructed replication-defective recombinant adenovirus expressing the CS protein of Plasmodium yoelii was compared with that of irradiated sporozoites. We found that immunization of BALB/c mice with a single dose of this recombinant adenovirus induced a much greater CS-specific T-cell response compared with immunization with irradiated sporozoites. More importantly, we found that this recombinant adenovirus induces similar or higher levels of protective immunity than those induced by irradiated sporozoites, eliciting an appreciable resistance to malaria infection.

Adenoviridae↗
PubMed1998

Methods of gene delivery.

Human gene therapy continues to be an exciting concept for the treatment of disease. This field of research remains in its early stages, but already a number of studies have provided "proof-of-principle." Although there is no unequivocal evidence of efficacy, there have been demonstrated physiologic changes that are relevant to the disease process. One of the major challenges still confronting the field is the design of more efficient vectors. The gene delivery systems being used today will undoubtedly be seen as crude when compared with future developments. It is unlikely that there will ever be a universal vector, but rather there will be multiple vectors specifically designed for certain organ sites and certain disease. It is reasonable to assume that, in the future, there will be synthetic vectors that co-opt the needed properties from both cells and viruses. It will be necessary to do much more fundamental research in cell biology, virology, immunology, and pathophysiology before vectors can be significantly improved. Many of the tools for this research are in place, and the driving force will be provided by the imaginativeness of the committed investigators.

Adenoviridae↗
PubMed1998

In vivo measurement of ureagenesis with stable isotopes.

We have utilized stable isotopes to measure in vivo rates of ureagenesis. In one testing procedure, 15NH4Cl was administered orally to controls and to heterozygotes for ornithine transcarbamylase deficiency. Controls produced [15N]urea at a rate that was greater than that of symptomatic carriers, but indistinguishable from that of asymptomatic carriers. In contrast, both symptomatic and asymptomatic heterozygotes produced [5-15N]glutamine more rapidly than the controls. Ureagenesis could also be measured by administering sodium [1-13C]acetate to a healthy adult and measuring subsequent formation of [13C]urea. The latter approach involves the use of isotope ratio mass spectrometry to determine isotopic abundance. This technique is much more sensitive than gas chromatography-mass spectrometry for the measurement of isotopic label, a consideration that makes the method more suitable for the study of subjects in whom ureagenesis is severely compromised, for example the human male neonate with a near complete deficiency of ornithine transcarbamylase.

Adult↗
PubMed1998

Developing adenoviral-mediated in vivo gene therapy for ornithine transcarbamylase deficiency.

There are a number of reasons for choosing ornithine transcarbamylase (OTC) deficiency as a candidate for gene therapy: the gene has been cloned; the disorder is relatively common; the current clinical outcome is poor; and there are authentic animal models. In considering the development of gene therapy for OTC deficiency, we focused on the use of in vivo gene therapy with an adenoviral vector. Using the partially OTC-deficient sparse fur mouse we found transduction and expression could be achieved using an intravenous infusion of a recombinant adenovirus containing the OTC cDNA. The results were transient as a result of immune activation in response to the vector and vector-transduced cells. By modifying the adenoviral construct, creating an E1 deletion-E2 temperature-sensitive mutation, we blunted the cytotoxic T lymphocyte immune response and achieved correction of biochemical abnormalities for 2-3 months. We also found that transduction and expression following gene transfer occurred sufficiently rapidly to protect against acute hyperammonaemia within 24 h. Subsequent preclinical studies in mice and non-human primates demonstrated that E1-E4-deleted vectors had a substantially improved safety profile and similar efficacy. With this evidence of efficacy and safety of adenoviral vectors, we are embarking on a phase I trial of intravascular gene transfer using an E1-E4-deleted vector in adults with partial OTC deficiency.

Adenoviruses, Human↗
PubMed1998

Blunting of immune responses to adenoviral vectors in mouse liver and lung with CTLA4Ig.

Adenoviral vectors deleted of E1 are attractive vehicles for in vivo gene therapy because efficient gene transfer can be achieved. Immune responses to the vector and vector-transduced cells lead to destruction of target cells, inflammation and difficulties with vector readministration. Immune effectors have been identified as CD8+ cytotoxic T lymphocytes, which destroy vector-transduced cells, as well as B cells which secrete neutralizing antibodies and block repeated gene transfer. The central role of CD4+ T cells in the activation of both of these effector functions has focused immunosuppressive strategies towards blockade of costimulatory molecules. We describe in this study a strategy which aims to inhibit CD4+ T cell activation by transiently administering an inhibitor of the CD28/B7 pathway, ie CTLA4Lg, at the time an E1-deleted adenovirus is administered to liver or lung. In lung, CTLA4Ig treatment significantly blocked the formation of neutralizing antibodies, allowing efficient readministration of virus, whereas transgene expression was only moderately prolonged. In contrast, CTLA4Ig did not suppress neutralizing antibody formation in the context of liver gene therapy, but resulted in more stable gene expression. In vitro assays revealed suppression of T cell activation in either organ. This observation suggests that transient inhibition of the CD28/B7 pathway at the time of virus instillation can partially interfere with both arms of the immune response to adenovirus-mediated gene transfer circumventing the need for chronic immune suppression.

Abatacept↗
PubMed1998

High throughput method for creating and screening recombinant adenoviruses.

Replication defective adenoviruses are being considered as vectors in therapeutic applications of gene therapy, as well as research tools in studying gene function. Important to their successful utilization is the development of techniques to isolate new recombinants quickly, which are not contaminated with wild-type virus. We describe a modification of the traditional technique to create recombinant adenoviruses in which a 5' plasmid containing vector sequence is cotransfected into 293 cells with viral DNA. In our protocol, the viral DNA is derived from the 3' portion of an E1-deleted recombinant that expresses the green fluorescent protein. Visualization of the cotransfection by fluorescent microscopy distinguishes recombinant plaques (nonfluorescent or 'white plaque') from background plaques (green fluorescent or 'green plaque'). Using this approach we have been able to increase substantially the success and throughput for creating new recombinants while minimizing contamination. This has been used to isolate adenoviral vectors deleted in a number of essential genes.

Adenoviridae↗
PubMed1998

Mouse beta-defensin 1 is a salt-sensitive antimicrobial peptide present in epithelia of the lung and urogenital tract.

One component of host defense at mucosal surfaces appears to be epithelium-derived peptides with antimicrobial activity called defensins. Human beta-defensin 1 (hBD-1) represents the first member of the beta-defensin family isolated from humans and has been implicated in the pathogenesis of cystic fibrosis. We describe in this report the isolation and characterization of a murine homolog of hBD-1 called mouse beta-defensin 1 (mBD-1). The predicted amino acid sequence shows the hallmark features of other known epithelial beta-defensins, including the ordered array of six cysteine residues. Analysis of a genomic clone of mBD-1 revealed two exons separated by a 15-kb intron. By use of fluorescence in situ hybridization, the mBD-1 gene was localized at the proximal portion of chromosome 8, the site where mouse alpha-defensins are found. Lysates from cells transfected with the mBD-1 cDNA showed antibacterial activity against gram-positive and gram-negative bacteria. mBD-1 transcripts were found in kidney, liver, and female reproductive organ tissues. In the airways, mBD-1 is expressed diffusely throughout the epithelial cells of the large proximal airways with less expression in the small distal airways and no expression in alveolar cells. The present study demonstrates that a beta-defensin potentially homologous to human beta-defensin 1 is present in the respiratory system and other mucosal surfaces in mice.

Amino Acid Sequence↗
PubMed1998

Adeno-associated virus as a vector for liver-directed gene therapy.

Factors relevant to the successful application of adeno-associated virus (AAV) vectors for liver-directed gene therapy were evaluated. Vectors with different promoters driving expression of human alpha-1-antitrypsin (alpha-1AT) were injected into the portal circulation of immunodeficient mice. alpha-1AT expression was stable but dependent on the promoter. Southern analysis of liver DNA revealed approximately 0.1 to 2.0 provirus copies/diploid genome in presumed head-to-tail concatamers. In situ hybridization and immunohistochemical analysis revealed expression in approximately 5% of hepatocytes clustered in the pericentral region. These results support the use of AAV as a vector for diseases treatable by targeting of hepatocytes.

Animals↗
PubMed1998

Cytotoxic T-lymphocyte target proteins and their major histocompatibility complex class I restriction in response to adenovirus vectors delivered to mouse liver.

The activation of cytotoxic T lymphocytes (CTLs) to cells infected with adenovirus vectors contributes to problems of inflammation and transient gene expression that attend their use in gene therapy. The goal of this study was to identify in a murine model of liver gene therapy the proteins that provide targets to CTLs and to characterize the major histocompatibility complex (MHC) class I restricting elements. Mice of different MHC haplotypes were infected with an E1-deleted adenovirus expressing human alkaline phosphatase (ALP) or beta-galactosidase as a reporter protein, and splenocytes were harvested for in vitro CTL assays to aid in the characterization of CTL epitopes. A library of vaccinia viruses was created to express individual viral open reading frames, as well as the ALP and lacZ transgenes. The MHC haplotype had a dramatic impact on the distribution of CTL targets: in C57BL/6 mice, the hexon protein presented by both H-2Kb and H2Db was dominant, and in C3H mice, H-2Dk-restricted presentation of ALP was dominant. Adoptive transfer of CTLs specific for various adenovirus proteins or transgene products into either Rag-I or C3H-scid mice infected previously with an E1-deleted adenovirus verified the in vivo relevance of the adenovirus-specific CTL targets identified in vitro. The results of these experiments illustrate the impact of lr gene control on the response to gene therapy with adenovirus vectors and suggest that the efficacy of therapy with adenovirus vectors may exhibit considerable heterogeneity when applied in human populations.

Adenoviridae↗
PubMed1998

Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers.

Immune responses to vector-corrected cells have limited the application of gene therapy for treatment of chronic disorders such as inherited deficiency states. We have found that recombinant adeno-associated virus (AAV) efficiently transduces muscle fibers in vivo without activation of cellular and humoral immunity to neoantigenic transgene products such as beta-galactosidase, which differs from the experience with recombinant adenovirus, where vibrant T-cell responses to the transgene product destroy the targeted muscle fibers. T cells activated following intramuscular administration of adenovirus expressing lacZ (AdlacZ) can destroy AAVlacZ-transduced muscle fibers, indicating a prior state of immunologic nonresponsiveness in the context of AAV gene therapy. Adoptive transfer of dendritic cells infected with AdlacZ leads to immune mediated elimination of AAVlacZ-transduced muscle fibers. AAVlacZ-transduced antigen-presenting cells fail to demonstrate beta-galactosidase activity and are unable to elicit transgene immunity in adoptive transfer experiments. These studies indicate that vector-mediated transduction of dendritic cells is necessary for cellular immune responses to muscle gene therapy, a step which AAV avoids, providing a useful biological niche for its use in gene therapy.

Animals↗
PubMed1998

Role of E4 in eliciting CD4 T-cell and B-cell responses to adenovirus vectors delivered to murine and nonhuman primate lungs.

Adenovirus vectors delivered to lung are being considered in the treatment of cystic fibrosis (CF). Vectors from which E1 has been deleted elicit T- and B-cell responses which confound their use in the treatment of chronic diseases such as CF. In this study, we directly compare the biology of an adenovirus vector from which E1 has been deleted to that of one from which E1 and E4 have been deleted, following intratracheal instillation into mouse and nonhuman primate lung. Evaluation of the E1 deletion vector in C57BL/6 mice demonstrated dose-dependent activation of both CD4 T cells (i.e., TH1 and TH2 subsets) and neutralizing antibodies to viral capsid proteins. Deletion of E4 and E1 had little impact on the CD4 T-cell proliferative response and cytolytic activity of CD8 T cells against target cells expressing viral antigens. Analysis of T-cell subsets from mice exposed to the vector from which E1 and E4 had been deleted demonstrated preservation of TH1 responses with markedly diminished TH2 responses compared to the vector with the deletion of E1. This effect was associated with reduced TH2-dependent immunoglobulin isotypes and markedly diminished neutralizing antibodies. Similar results were obtained in nonhuman primates. These studies indicate that the vector genotype can modify B-cell responses by differential activation of TH1 subsets. Diminished humoral immunity, as was observed with the E1 and E4 deletion vectors in lung, is indeed desired in applications of gene therapy where readministration of the vector is necessary.

Adenoviridae↗
PubMed1998

beta-Naphthoflavone abolishes interrenal sensitivity to ACTH stimulation in rainbow trout.

We report for the first time that beta-naphthoflavone (BNF) abolishes ACTH stimulation of cortisol production in rainbow trout (Oncorhynchus mykiss). There was significantly higher hepatic cytochrome P450 content and ethoxyresorufin O-de-ethylase and uridine-5'-diphosphoglucuronic acid transferase activities in BNF-treated fish than in sham-treated controls. BNF did not significantly affect either plasma turnover or tissue distribution of [3H]cortisol-derived radioactivity. Hepatic membrane fluidity and hepatocyte capacity for cortisol uptake were not altered by BNF as compared with the sham-treated fish. These results taken together suggest that BNF does not affect cortisol-clearance mechanisms in trout. A 3 min handling disturbance period elicited a plasma cortisol response in the sham-treated fish; however, the response in the BNF-treated fish was muted and significantly lower than in the sham fish. This in vivo response corroborates the lack of interrenal sensitivity to ACTH in vitro in the BNF-treated fish, suggesting that BNF affects the ACTH pathway in trout. Our results suggest the possibility that cytochrome P450-inducing compounds may affect cortisol dynamics by decreasing interrenal responsiveness to ACTH stimulation in fish, thereby impairing the physiological responses that are necessary for the animal to cope with the stressor.

Adrenocorticotropic Hormone↗
PubMed1998

The inhibition of bovine carbonic anhydrase by saccharin and 2- and 4-carbobenzoxybenzene sulfonamide.

The present work demonstrates that the high-activity zinc metalloenzyme, carbonic anhydrase (CA II) from bovine erythrocytes is inhibited by the cyclic sulfimide, saccharin, and 2- and 4-carbobenzoxybenzene sulfonamide. A spectrophotometric method was employed to monitor the enzymatically catalyzed hydrolysis of p-nitrophenyl acetate by following the increase in absorbance at 410 nm which accompanies p-nitrophenoxide/p-nitrophenol formation. The more rapid enzymatic hydration of CO2 was monitored by using a stopped-flow spectrophotometer as well as by a modified colorimetric method of Wilbur and Anderson. The studies show that, at a given molar ratio of inhibitor to enzyme, the degree of inhibition of the enzymaic hydration of CO2 and hydrolysis of p-nitrophenyl acetate by the inhibitory compounds is essentially the same. Kinetic analyses were made at 25.0 degrees at pH 6.5 (MES buffers), pH 6.9 (HEPES buffers) and pH 7.9 (HEPES buffers) with ionic strength regulated by the addition of appropriate quantities of sodium sulfate. Lineweaver-Burk plots were used to evaluate apparent inhibition constants for each of the three inhibitors. For all the inhibitors studied, inhibition appears to be mixed (competitive/noncompetitive). For saccharin in the presence of sodium sulfate, the extent of inhibition is considerably decreased. It was found for the three inhibitors that the inhibitory potency decreases with increasing pH, and that the inhibitory potency is extremely sensitive to the shape of these rather closely related molecules. For example, apparent inhibition constants for the enzymatic hydrolysis of p-nitrophenyl acetate at pH 6.9 were Ki (saccharin) = 0.20 mM, Ki (2-carbobenzoxybenzene sulfonamide) = 0.54 mM and Ki (4-carbobenzoxybenzene sulfonamide) = 1.6 microM. For the enzymatic hydration of CO2 at pH 6.9, 0.10 mM saccharin caused 50% inhibition while 7.0 nM 4-carbobenzoxybenzene sulfonamide resulted in 50% inhibition. The results suggest that sulfonamide inhibition is caused by formation of a monodentate ligand at the zinc ion of the enzyme active site and that the more linear 4-carbobenzoxybenzene sulfonamide is better able to enter a conical enzyme active site than is 2-carbobenzoxybenzene sulfonamide or saccharin.

Algorithms↗
PubMed1998

Congenital diaphragmatic hernia survival and use of extracorporeal life support at selected level III nurseries with multimodality support.

BACKGROUND: Congenital diaphragmatic hernia (CDH) has been cited to have a mortality rate of 50%. There have been multiple studies at individual institutions demonstrating potential benefits from various strategies including extracorporeal life support (ECLS), delayed repair, and lower levels of ventilator support. There has been no multicenter survey of institutions offering these modalities to describe the current use of ECLS and survival of these infants. In addition, the relationship between the number of patients with CDH managed at an individual institution and outcome has not been evaluated. METHODS: We queried 16 level III neonatal intensive care centers on the use of ECLS and survival of infants with CDH who were treated during 2 consecutive years (1993 to 1995). Data are presented as mean +/- SEM, median, and range. RESULTS: Data were collected on 411 patients. Of these, 71% +/- 8% were outborn and 8% +/- 3% were considered nonviable. Overall survival of CDH infants was 69% +/- 4% (range, 39% to 95%). The survival rate of infants on ECLS was 55% +/- 4%, whereas survival of infants not requiring ECLS was significantly increased at 81% +/- 5% (p = 0.005). The mean rate of ECLS use was 46% +/- 2%. There was no correlation between the number of cases per year at an individual institution and overall survival, ECLS survival, or ECLS use (r = 0.341, 0.305, and 0.287, respectively). There was also no correlation between case volume at an individual institution and ECLS survival (r = 0.271). CONCLUSIONS: The current survival rate and rate of ECLS use in infants with CDH at level III neonatal intensive care units in the United States are 69% +/- 4% and 46% +/- 2%, respectively. There is no correlation between the yearly individual center experience with managing CDH and rate of ECLS use or outcome.

Extracorporeal Circulation↗
PubMed1998

Fundamentals of coagulation and glycoprotein IIb/IIIa receptor inhibition.

An understanding of the coagulation process and the role of platelets is essential to recognizing the shortcomings of older anticoagulant therapies and appreciating the clinical potential of newer forms of antiplatelet and anticoagulant therapy for acute coronary syndromes. The anticoagulant actions of heparin are severely limited by dependence on antithrombin III, neutralization by platelet factor 4, and the resistance of clot-bound thrombin and platelet membrane-bound factor Xa to the heparin-antithrombin III complex. Unlike heparin, the direct thrombin inhibitors (such as hirudin) are active against both circulating and clot-bound thrombin. However, in recent clinical trials they have not resulted in major improvements in patient outcome. Another new class of drugs, the glycoprotein IIb/IIIa receptor antagonists, blocks the final common pathway of platelet aggregation and is capable of preventing platelet accumulation at sites of injury. The net effect is a dramatic reduction in the amount of platelet membrane available to support the process of coagulation. Clinical trials with the glycoprotein IIb/IIIa inhibitors have suggested that this class of agents may be particularly effective in reducing the thrombotic complications associated with coronary interventional procedures and may be useful in the treatment of acute coronary syndromes.

Blood Coagulation↗