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Biomedical subjects

J M Wilson

Publications and source records attributed to J M Wilson.

At least 289 records · Page 16Linked to original sources
PubMed1994

Biochemical and functional analysis of an adenovirus-based ligand complex for gene transfer.

Ligand-mediated approaches to gene transfer offer an alternative to viral vectors for both in vivo and in vitro applications. Although a significant percentage of the plasmid-based DNA complex is lost to lysosomal degradation following receptor-mediated endocytosis, simultaneous infection with adenovirus has been shown to increase the level of transgene expression [Curiel, Agarwal, Wagner and Cotten (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8850-8854; Wagner, Zatloukal, Cotten, Kirlappos, Mechtler, Curiel and Birnstiel (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6099-6103]. In this study we describe an adenovirus-based ligand complex where the plasmid DNA, polycation-ligand conjugate and adenovirus are contained within a single particle structure. At the core of the transfection particle is a replication-defective recombinant adenovirus encoding a cDNA minigene for human placenta alkaline phosphatase that was chemically modified with poly(L-lysine) (Ad-pLys). Electron microscopy of an adenovirus-based ligand complex formed by successively adding plasmid DNA and an asialo-orosomucoid-poly(L-lysine) conjugate to Ad-pLys revealed structures that appeared as intact viral particles coated with a dense biomolecular layer. Adenovirus-based ligand complexes containing either a luciferase or beta-galactosidase reporter plasmid were shown to efficiently deliver the plasmid transgene to cells that express the hepatic asialoglycoprotein receptor. Furthermore, the poly(L-lysine) modification greatly reduced the infectivity potential of the virus without causing a concomitant loss of augmented gene transfer. As an alternative to infectious virions, incomplete products of viral assembly were also considered as a source for endosomalytic activity. However, these defective virions were unable to significantly enhance plasmid transgene delivery.

3T3 Cells↗
PubMed1994

Effects of DNA adduct structure and distribution on the mutagenicity and genotoxicity of two platinum anticancer drugs.

cis-Diamminedichloroplatinum(II) (cis-DDP) and cis,trans,cis-ammine(cyclohexylamine)-dibutyratodichloroplatinu m(IV) (ACDDP) are anticancer drugs that bind to DNA, forming replication blocking adducts. ACDDP, probably manifests its cytotoxicity through the metabolite cis-ammine(cyclohexylamine)dichloroplatinum(II) (ACDP). The biological effects of ACDP and cis-DDP were compared by studying polymerase inhibition in vitro and mutagenesis and genotoxicity in vivo in the duplex genome of bacteriophage M13mp18 replicated in Escherichia coli. cis-DDP and ACDP adducts were equally genotoxic within the statistical error limits of the analysis. Survival of genomes platinated by either drug, increased by threefold in cells pretreated with u.v. irradiation to induce the SOS functions of the host. In the M13mp18 lacZ' gene fragment, the mutagenicity of ACDP was lower than that of cis-DDP; the difference was approximately twofold at a dose of two adducts per 370 base-pair mutational target. Mutagenesis by both compounds was SOS-dependent. The structural basis for lower mutagenicity of ACDP is proposed to be its reduced reactivity at d(ApG) sites. This effect is attributed to an orientational isomerism that precludes the formation of one of two possible DNA adducts at d(ApG) residues. The types of mutations induced for both drugs were similar, but they occurred with different distributions. Both compounds induced primarily G-->T transversions at d(GpG) sites whereas G-->A transitions and A-->T transversions, many at d(ApG), d(GpNpG), and d(GpG) sites, were also well represented. The mapping of DNA adducts by DNA synthesis inhibition revealed excellent correlation between the location of DNA lesions and the sites of mutations. Analysis of the distribution of mutations and the distribution of potential platination sites revealed no sequence-dependent mutation hotspots; i.e. mutagenesis was random throughout the lacZ' region of the M13mp18 bacteriophage genome. These results offer insights into the molecular mechanism of mutagenicity of platinum anticancer drugs.

Antineoplastic Agents↗
PubMed1994

Successful adenovirus-mediated gene transfer in an in vivo model of human malignant mesothelioma.

Malignant mesothelioma remains a frustrating clinical problem with uniformly poor responses to current therapeutic regimens. However, the localized nature of the disease, the potential accessibility of the tumor, and the relative lack of distant metastases make it a particularly attractive candidate for somatic gene therapy. The purpose of this study was to evaluate the ability of an adenoviral vector system to transfer genetic material to human mesothelioma cells in vitro and in vivo. Using a replication-deficient recombinant adenovirus carrying the Escherichia coli lacZ marker gene, we found that human mesothelioma cell lines were susceptible to adenovirus infection. Furthermore, surprisingly effective gene transfer was accomplished within tumor implants of human mesothelioma growing within the peritoneal cavity of immunodeficient mice after intraperitoneal administration of virus. These studies demonstrate that adenoviral vectors hold promise as vehicles to deliver gene therapy in human malignant mesothelioma.

Abdomen↗
PubMed1994

Evolution of the technique of congenital diaphragmatic hernia repair on ECMO.

With the increase in popularity of delayed repair of congenital diaphragmatic hernia (CDH), many institutions are using extracorporeal membrane oxygenation (ECMO) to stabilize patients preoperatively. This practice has led to controversy regarding whether the repair should be performed while the patient is on ECMO or after decannulation. This report details the authors' experience with repair of CDH on ECMO. Of the 154 high-risk CDH patients treated at Children's Hospital, Boston, MA, since ECMO became available (1984), 97 received ECMO, including 31 who had repair performed while on ECMO. In group I (nine patients), repair was carried out only if the patients were unweanable from ECMO after 7 days. Activated clotting times (ACT) were maintained at 200 to 220 seconds. In group II (22 patients), repair was performed on ECMO electively, before decannulation. ACT were maintained at 180 to 200 seconds. Additionally, all patients in group II received aminocaproic acid before surgery. This was administered continuously for 72 hours postoperatively or until decannulation. Patients in group II had significantly less overall blood loss (P = .02), and lower transfusion requirement (P = .0003) than those in group I. Additionally, four of the nine patients in group I required reexploration because of hemorrhage; this was not required for any patient in group II (P = .005). Although the survival rates differed, this may have been because of a bias in patient selection between the two groups. From these preliminary data, the authors conclude that repair of congenital diaphragmatic hernia on ECMO can be performed safely, with a minimum of hemorrhagic complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminocaproates↗
PubMed1994

Congenital diaphragmatic hernia and associated anomalies: their incidence, identification, and impact on prognosis.

The general concept of the association of congenital diaphragmatic hernia (CDH) with other anomalies has been well described. This study is aimed at assessing the distribution of the associated anomalies (AA) by organ system, their influence on prognosis, and the practical signs that should prompt a diagnostic search. One hundred and sixty-six high-risk patients with CDH (symptomatic within the first 6 hours of life) were treated in this institution in the past decade. Sixty-five patients (39.2%) were found to have one or more AA, and 101 had isolated CDH. Of patients with anomalies, cardiac (excluding patent foramen ovale and patent ductus arteriosus) was the most frequent type of AA (63%). Hypoplastic heart syndrome was the most common defect. Many patients had multiple AA. For purposes of analysis, the patients were divided into three groups: isolated CDH, cardiac anomalies, and all other anomalies. The groups were compared with respect to several common clinical and laboratory variables, as well as survival. The frequency and timing of antenatal diagnosis were also noted. The analysis led to the following conclusions. (1) AA are present in more than one third of high-risk patients with CDH; in this group, cardiac lesions predominate. (2) High-risk CDH infants with AA have significantly lower APGAR scores and a lower BPDPO2 (best postductal PO2 before ECMO or surgery) than those with isolated CDH. This is even more evident in the group with cardiac AA. In such patients, a careful search for an undetected AA, especially cardiac, is warranted.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Multiple↗
PubMed1994

Experimental fetal tracheal ligation reverses the structural and physiological effects of pulmonary hypoplasia in congenital diaphragmatic hernia.

Infants with congenital diaphragmatic hernia (DH) and profound pulmonary hypoplasia are currently unsalvageable. The authors previously demonstrated that tracheal ligation (TL) accelerates fetal lung growth and reverses the pulmonary hypoplasia of fetal nephrectomy. The purpose of this study was to determine if the pulmonary hypoplasia of experimental DH could be similarly reversed and, if so, whether the resulting lungs would show better function than those of their DH counterparts. Eighteen fetal lambs were divided into three experimental groups of six animals each. In group 1, DH was created at 90 days' gestation. In group 2, DH was created at 90 days' gestation and TL performed during the same operation. Group 3 consisted of sham-operated controls. These animals were delivered near full-term, and their lungs analyzed by standard morphometric techniques. Ten additional fetal lambs were divided into two experimental groups of five animals each. In group 4, DH was created at 90 days' gestation. In group 5, DH was created at 90 days' gestation and TL performed 20 days later, at 110 days' gestation. These animals were pressure-ventilated via tracheostomy over a 2-hour period in which PaO2, PaCO2, and compliance were measured. Intratracheal pressure (ITP) was measured at the time of delivery in all groups. Upon retrieval, DH animals had abdominal viscera in the chest and small lungs; in contrast, DH/TL animals had the herniated viscera reduced from the chest by enlarged lungs. DH/TL lungs showed markedly increased growth, with significant increases in lung volume:body weight ratio (LV:BW; P = .0001), alveolar surface area (ALV.SA; P = .0001), and alveolar number (ALV#) (P = .0001) when compared with those of the DH or control group. This growth was associated with a normal maturation pattern based on histological appearance, normal airspace fraction, and normal alveolar numerical density. ITP in the DH/TL group was increased when compared with that of DH and control animals (P = .0001). Total lung DNA and protein were both elevated in the DH/TL animals (P = .0001). However, the DNA:protein ratio remained normal, suggesting lung growth had occurred through cell proliferation, not by hypertrophy. When ventilated over a range of settings, DH/TL lungs were more compliant (P = .0001) and achieved higher PaO2s (P < .003) and lower PaCO2s (P = .0001) than their DH counterparts. From these data, the authors conclude: (1) Experimental fetal DH produces hypoplastic lungs that are not capable of adequate gas exchange with conventional ventilation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Congenital diaphragmatic hernia: the hidden morbidity.

It is often thought that survivors of congenital diaphragmatic hernia (CDH) have an isolated problem related to lung hypoplasia, and little data exist regarding the extrapulmonary problems of high-risk CDH patients who do survive. In 1990, the authors began a multidisciplinary follow-up clinic for CDH patients. Members of the program include representatives from the departments of surgery, pulmonary medicine, development, nursing, and nutrition. Since this program began, the authors have followed up on 33 infants who survived after treatment of high-risk CDH, ie, those who were symptomatic within 6 hours of birth. Twenty patients were treated with extracorporeal membrane oxygenation (ECMO). Neurological problems were common in these patients: seven children (21%) required hearing aids, and seven others had abnormal results with brain-stem auditory evoked response (BAER) testing. Extraaxial fluid collections or enlarged ventricles were present on head computed tomography scans of 10 children, and four children had clinical seizure activity. Fifteen patients had developmental delays, which improved rapidly once the children began to thrive. Six patients required eyeglasses or had strabismus, and one patient is congenitally blind. There were a variety of problems related to growth and nutrition, with six patients needing fundoplications, and 13 patients below the fifth percentile for weight. Of 10 patients with patch repairs, two had recurrent hernias. Six others required surgery for bowel obstruction. Eleven patients had pectus excavatum, usually mild, and four had mild to moderate degrees of scoliosis. There were undescended testicles in five boys, vesicoureteral reflux in two patients, and kidney stones in two patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Electroencephalography↗
PubMed1994

Antenatal diagnosis of isolated congenital diaphragmatic hernia is not an indicator of outcome.

It has been reported previously that infants diagnosed with congenital diaphragmatic hernia (CDH) antenatally have a much poorer prognosis than those diagnosed postnatally. The authors identified 173 high-risk (symptomatic within the first 6 hours of life) infants with CDH treated in the past decade. Seventy-seven cases were diagnosed antenatally and 96 were diagnosed postnatally. The survival rate was slightly worse for the antenatal group (34% v 48% for the postnatal group; P = .04). However, 59 of the 173 patients (34%) had other life-threatening congenital anomalies. Among the 114 patients with isolated CDH, the survival rate increased to 59% in the antenatal group and 63% in the postnatal group--a difference that was not significant. The timing of antenatal diagnosis (> or < 25 weeks) had no impact on the statistics (P = .08). The only parameter consistently different between all groups and subgroups was the Apgar score at 5 minutes, which was always better in the antenatal group (P < .02), possibly reflecting more efficient resuscitation. Of the 59 patients with other life-threatening anomalies (42 cardiac), there was one survivor among the 34 in the antenatal group and only two among the 25 in the postnatal group, despite the fact that 39 patients had undergone resuscitation, which included ECMO in 25.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Multiple↗
PubMed1994

Regional thyroid hormone levels in rat brain.

Levels of T4 and T3 were measured in half-brain and seven brain regions. Although thyroid hormones were distributed throughout the brain, T4 levels were highest in cerebellum and thalamus. The highest T3 levels were observed in the thalamus and lowest levels were found in olfactory bulb, hypothalamus, and amygdala.

Animals↗
PubMed1994

MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses.

The use of E1-deleted recombinant adenoviruses in gene therapy has consistently been associated with transient gene expression and inflammation due to immune-based destruction of the infected cells. We have used murine models of adenovirus-mediated gene transfer to liver to investigate these immunologic mechanisms. Adoptive transfer experiments, as well as studies involving genetic knockout mice, confirmed the original hypothesis that cell-mediated immunity induced by E1-deleted adenovirus destroyed trans-gene-expressing hepatocytes and defined MHC class I-restricted CD8+ cytolytic lymphocytes as the primary immune effectors for hepatocyte destruction. Responses mediated by CD4+ cells per se were insufficient to mediate destruction of hepatocytes in vivo, despite the activation of virus-specific T helper cells of Th1 subsets. A better understanding of the response of the host to in vivo gene therapy is important in evaluating its usefulness in humans.

Adenoviridae Infections↗
PubMed1994

Successful ex vivo gene therapy directed to liver in a patient with familial hypercholesterolaemia.

An ex vivo approach to gene therapy for familial hypercholesterolaemia (FH) has been developed in which the recipient is transplanted with autologous hepatocytes that are genetically corrected with recombinant retroviruses carrying the LDL receptor. We describe the treatment of a 29 year old woman with homozygous FH by ex vivo gene therapy directed to liver. She tolerated the procedures well and in situ hybridization of liver tissue four months after therapy revealed evidence for engraftment of transgene expressing cells. The patient's LDL/HDL ratio declined from 10-13 before gene therapy to 5-8 following gene therapy, improvements which have remained stable for the duration of the treatment (18 months). This represents the first report of human gene therapy in which stable correction of a therapeutic endpoint has been achieved.

Adult↗
PubMed1994

Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis.

Although first generation recombinant adenoviruses, deleted of sequences spanning E1a and E1b, have been useful for in vivo applications of gene therapy, expression of the recombinant gene has been transient and often associated with the development of inflammation. We show that with first generation adenovirus-mediated gene transfer to the mouse lung, viral proteins are expressed leading to destructive cellular immune responses and repopulation of the lung with nontransgene containing cells. Second generation E1 deleted viruses further crippled by a temperature sensitive mutation in the E2a gene were associated with substantially longer recombinant gene expression and less inflammation. Stable expression of human CF transmembrane conductance regulator has been achieved in lungs of CF mice instilled with a second generation virus.

Adenoviridae Infections↗
PubMed1994

Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2a.

Recombinant adenoviruses have tremendous potential for gene therapy of cystic fibrosis (CF) lung disease. First-generation recombinant viruses, rendered replication defective by deleting E1, have been associated with high-level recombinant gene expression in airway epithelial cells when administered directly to the lung. Experience in mice and non-human primates indicates that transgene expression is transient (i.e., lasting less than 21 days) and associated with the development of inflammation. We suggest an hypothesis to explain these findings that is based on expression of viral proteins in genetically modified cells that leads to destructive cellular immune responses and repopulation of lung epithelia with non-transgene-containing cells. This hypothesis has been evaluated in the current study using the cotton rat model. Instillation of the first-generation lacZ virus, H5.010CBlacZ, into cotton rat airway led to high-level gene expression in conducting and respiratory airway that was transient and associated with a substantial mononuclear, CD8-dominated, infiltrates. Treatment of the animals with cyclosporine blunted the inflammatory response and prolonged recombinant gene expression in both conducting and respiratory airways. Expression of viral early and late genes was detected in a subpopulation of lacZ-expressing epithelial cells of conducting airway and alveoli. Instillation of virus into cotton rat tracheal xenografts grown in athymic nu/nu mice led to efficient and stable transgene expression in the absence of pathology, underscoring the importance of T cell-mediated immunity. A recombinant adenovirus was constructed that is disabled in its capacity to replicate by the introduction of a temperature-sensitive mutation in the E2a gene as well deletion of E1 sequences. Instillation of this virus into cotton rat airway led to high-level transgene expression that was more stable than that achieved with the first-generation virus and was associated with less early and late gene expression as well as a diminished infiltration of CD8+ T cells in conducting airway epithelium. Interestingly, the introduction of the E2a mutation had no effect on the persistence of transgene expression, the pattern of late viral gene expression, nor the CD8+ T cell response within alveolar cells. These data suggest that cell-specific variation in the cell biology of recombinant adenoviruses exists in the lung. The present studies in cotton rats confirm the role of cellular immunity in the biology of adenovirus-mediated gene therapy to the lung and suggest that modifications in the design of recombinant adenoviruses to minimize or ablate transgene expression will be useful in improving the potential of this technology for gene therapy of CF.

Adenovirus E2 Proteins↗
PubMed1994

Gene therapy for cystic fibrosis using E1-deleted adenovirus: a phase I trial in the nasal cavity. The University of North Carolina at Chapel Hill.

Cystic fibrosis (CF) is an autosomal recessive disease that reflects mutations in the CFTR gene. Multiple mutations in this gene have been detected that lead to a protein (CFTR) that is abnormally metabolized, dysfunction, or both. The full spectrum of the activities of the gene product have not been defined, but it is clear that CFTR can act as a cAMP-regulated Cl- channel. This type of defect is consistent with the physiologic characterization of CF epithelia, which has revealed abnormalities in salt and water transport. In the lung, abnormalities in epithelial salt and water metabolism lead to abnormal mucociliary clearance. This defect in clerance represents a major failure of lung defense and leads ultimately to infection of the lung with Staphylococcus aureus, Pseudomonas aeruginosa, and other bacterial organisms. The chronic inflammatory response to this persistent intraluminal bacterial infection leads to protease-induced destruction of airway walls and finally, lung failure. More than 95% of CF patients die of lung disease. The clinical therapy of CF lung disease is limited to agents designed to promote clearance of secretions from the lung and antibiotics to treat the chronic bacterial infection. Recent laboratory demonstrations that introduction of the normal CFTR cDNA into CF cells corrects the ion transport defects of these cells has led to the hypothesis that gene therapy in the lung can be an effective, novel mode of therapy for this lung disease. The classic gene transfer vectors, e.g., retroviruses, appear to be not well suited for therapy of lung disease because of the low proliferation rate of airway epithelia in vivo. Recently, adenoviruses, which have a natural tropism for airway epithelia, have been genetically modified (E1-deleted) in an attempt to reduce potential toxicity of this virus and provide space for the CFTR cDNA. A series of in vitro studies have shown that this vector is highly efficient for transferring CFTR into airway epithelial cells in culture and correcting the CF defect. Further, studies in whole animals appear to indicate that this mode of gene transfer is associated with a low degree of toxicity. The present study is a dose-effect study designed to test for the safety and efficacy of E1-deleted recombinant adenovirus containing the CFTR cDNA under a CMV-beta-actin promoter in CF nasal epithelia.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviridae↗
PubMed1994

Noninvasive imaging of the cardiovascular system with electron beam computed tomography and magnetic resonance imaging.

Two new noninvasive imaging modalities have emerged in recent years that provide images of the heart and great vessels with high temporal and spatial resolution: electron beam (or ultrafast) computed tomography and magnetic resonance imaging. Because both modalities acquire images in three dimensions, they can provide unique diagnostic information above and beyond that of more traditional noninvasive modalities such as two-dimensional echocardiography and radionuclide techniques. This review discusses the technical evolution and clinical application of these two imaging techniques, with particular emphasis on progress made over the past year. Specific areas addressed for each include technical aspects and clinical applications from both the imaging and physiology perspective.

Cardiomyopathies↗
PubMed1994

Coupled secretion of chloride and mucus in skin of Xenopus laevis: possible role for CFTR.

We used the isolated skin of Xenopus laevis to investigate the relationship between the secretion of salt, water, and mucus by submucosal glands expressing the cystic fibrosis transmembrane conductance regulator (CFTR). In situ hybridization and immunofluorescence provided evidence for specific expression of CFTR in the mucus-secreting cells of the subepidermal glands. Stimulation of isolated sheets of skin with 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate produced active Cl secretion and a marked increase in tissue conductance that was correlated with mucous cell degranulation and the distention of the glandular ducts. This coordinated increase in active secretion of salt and mucus was abolished by pretreatment of skins with bumetanide or by removing Cl from the bathing solutions. These results provide evidence for an intimate coupling between electrolyte transport and mucus secretion that may illuminate the pathophysiology of mucus-producing glands in cystic fibrosis lung disease.

Animals↗