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Biomedical subjects

E Wilson

Publications and source records attributed to E Wilson.

At least 37 records · Page 2Linked to original sources
PubMed2000

Synthetic glycopeptide-based delivery systems for systemic gene targeting to hepatocytes.

PURPOSE: To design, synthesize, and test synthetic glycopeptide-based delivery systems for gene targeting to hepatocytes by systemic administration. METHODS: All peptides were synthesized by the solid phase method developed using Fmoc chemistry on a peptide synthesizer. The binding of galactosylated peptides to HepG2 cells and accessibility of the galactose residues on particle surface was demonstrated by a competition assay using 125I-labeled asialoorosomucoid and RCA lectin agglutination assay, respectively. DNA plasmid encoding chloramphenicol acetyl transferase (CAT) gene was complexed with a tri-galactosylated peptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in the presence of an endosomolytic peptide (GM225.1) or endosomolytic lipopeptide (GM227.3) to obtain DNA particles of 100-150 nm in size. The plasmid/peptide complexes were added to HepG2 cell cultures or intravenously administered by tail vein injection into normal mice or rats. Plasmid uptake and expression was quantified by qPCR and ELISA, respectively. RESULTS: Multiple antennary glycopeptides that have the ability to condense and deliver DNA plasmid to hepatocytes were synthesized and complexed with DNA plasmid to obtain colloidally stable DNA/peptide complexes. Addition of DNA/GM245.3/GM225.1 peptide complexes (1:3:1 (-/+/-)) to HepG2 cell cultures yielded CAT expression in transfected cells. The transfection efficiency was significantly reduced in the absence of galactose ligand or removal of endosomolytic peptide. Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5 (-/+)) into the tail vein of normal rats yielded DNA uptake in the liver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3 resulted in more stable DNA particles, and a 10-fold enhancement in liver plasmid uptake. CAT expression was detectable in liver following intravenous administration of DNA/GM246.3 complexes. Addition of endosomolytic lipopeptide GM227.3 into the complexes (DNA/ GM246.3/GM227.3 (1:0.5:1 (-/+/-))) yielded a 5-fold increase in CAT expression. Liver expression was 8-fold and 40-fold higher than lung and spleen, respectively, and localized in the hepatocytes only. The transfection efficiency in liver was enhanced by increasing DNA dose and injection volume. The plasmid uptake and expression in liver using DNA/GM246.3/GM227.3 complexes was 100-200-fold higher than DNA formulated in glucose. Tissue examination and serum biochemistry did not show any adverse effect of the DNA/GM246.3/ GM227.3 (1:0.5:1 (-/+/-)) complexes after intravenous delivery. CONCLUSIONS: Gene targeting to hepatocytes was achieved by systemic administration of a well-tolerated synthetic glycopeptide-based delivery system. The transfection efficiency of this glycopeptide delivery system was dependent on peptide structure, endosomolytic activity, colloidal particle stability, and injection volume.

Amino Acid Sequence↗
PubMed2000

Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration.

The impact of a localized application of ultrasound on gene transfer to primary tumors following systemic administration of cationic lipid based transfection complexes was investigated. We have previously shown that systemic administration of DOTMA (N-[(1-(2-3-dioleyloxy) propyl)]-N-N-N-trimethylammonium chloride):cholesterol-based transfection complexes to tumor-bearing mice resulted in expression in the tumor and other tissues, primarily the lungs. Application of ultrasound to the tumor before or after the injection resulted in a significant increase in gene transfer to the tumor with no increase observed in other tissues. The magnitude of increased expression ranged from three- to 270-fold depending upon the DNA dose. The following parameters were optimized for maximal increase: duration of ultrasound application, the time interval between plasmid injection and sonoporation, and plasmid dose. A combination of plasmid quantitation and fluorescence microscopy showed that ultrasound increased tumor uptake of the plasmid and that uptake was limited to the tumor vasculature. Using an IL- 12 expression plasmid, the combination of a single plasmid dose (10 microg) and ultrasound treatment produced significantly higher levels of IL-12 in tumor. This increased expression was sufficient to inhibit tumor growth compared with the control conditions. These data demonstrate the potential application of sonoporation as an effective method for enhancing the expression of systemically administered genes in tumor endothelium for cancer gene therapy.

Animals↗
PubMed2000

The conditioned response erection (CRE)--a new approach to modelling erectile responses in the rat.

Several animal models are currently used in erectile (dys)function research; these models fail to account for the conditions involving the more spontaneous erections in humans. Recently, we observed an increase in the number of 'spontaneously' occurring erections in rats with previous exposure to apomorphine (APO), a centrally acting drug that initiates penile erections and yawns. Based on this observation, we designed a series of experiments to characterize the development of enhanced, non-apomorphine-induced erections or 'spontaneous' erectile responses to vehicle administration in rats with previous exposure to APO. We further examined the effects of castration on these conditioned erections. Naive (ie never received APO) rats were administered vehicle (1 ml/kg saline) to determine the frequency of baseline erections and yawns. An alternating series of APO (80 microg/kg s.c.) and vehicle administrations were performed over several days and subsequent erectile and yawning responses were recorded. Following 3 sets of 3 APO administrations (with vehicle administered between sets), and the 3rd vehicle administration, these rats were then surgically castrated and allowed 30 days to recover. Following this, APO was administered 3 times to determine erectile and yawning responses post-castration, followed by vehicle administration to determine the effects of castration on conditioned APO responses. The major findings were: (1) that although naive rats had a basal spontaneous erectile response (0.75 +/- 0.88; 4 of 8 rats with at least one erection), repetitive administration (up to 22 treatments) of the central initiator apomorphine significantly increased the number of erections (1.8 +/- 0.7; 7 of 8 rats with at least one erection) and yawning (2.5 +/- 2.47) responses to vehicle administration; and (2) both spontaneous yawning and erectile responses were found to be androgen dependent since castration dramatically lowered the number of erections (0.13 +/- 0.35; 1 of 8 rats with at least one erection) and yawns (0). Therefore, this method of producing erections without a pharmacological manipulation provides an additional animal model which can be used in conjunction with the APO-induced erections in characterizing the physiology and pathophysiology of erectile function in conscious rats.

Androgens↗
PubMed2000

Myosin light chain kinase transference induces myosin light chain activation and endothelial hyperpermeability.

The actomyosin complex is the major cytoskeletal component that controls cell contraction. In this study, we investigated the effects of actomyosin interaction on endothelial barrier function and gap formation. Activated myosin light chain kinase (MLCK) protein was transferred into coronary venular endothelial cell (CVEC) monolayers. Uptake of the activated protein resulted in a significant shift in myosin light chain (MLC) from an unphosphorylated to a diphosphorylated form. In addition, MLCK induced a hyperpermeability response of the monolayer as measured by albumin transendothelial flux. Microscopic examination of MLCK-treated CVECs revealed widespread gap formation in the monolayer, loss of peripheral beta-catenin, and increases in actin stress fibers. Inhibition of all of the above responses by a specific MLCK inhibitor suggests they are the direct result of exogenously added MLCK. These data suggest that activation of MLCK in CVECs causes phosphorylation of MLC and contraction of CVECs, resulting in gap formation and concomitant increases in permeability. This study uses a novel technique to measure the effects of an activated kinase on both its substrate and cellular morphology and function through direct transference into endothelial cells.

Actins↗
PubMed1999

Lifestyle modifications to prevent and control hypertension. 1. Methods and an overview of the Canadian recommendations. Canadian Hypertension Society, Canadian Coalition for High Blood Pressure Prevention and Control, Laboratory Centre for Disease Control at Health Canada, Heart and Stroke Foundation of Canada.

OBJECTIVE: To provide updated, evidence-based recommendations for health care professionals on lifestyle changes to prevent and control hypertension in otherwise healthy adults (except pregnant women). OPTIONS: For people at risk for hypertension, there are a number of lifestyle options that may avert the condition--maintaining a healthy body weight, moderating consumption of alcohol, exercising, reducing sodium intake, altering intake of calcium, magnesium and potassium, and reducing stress. Following these options will maintain or reduce the risk of hypertension. For people who already have hypertension, the options for controlling the condition are lifestyle modification, antihypertensive medications or a combination of these options; with no treatment, these people remain at risk for the complications of hypertension. OUTCOMES: The health outcomes considered were changes in blood pressure and in morbidity and mortality rates. Because of insufficient evidence, no economic outcomes were considered. EVIDENCE: A MEDLINE search was conducted for the period January 1996 to September 1996 for each of the interventions studied. Reference lists were scanned, experts were polled, and the personal files of the authors were used to identify other studies. All relevant articles were reviewed, classified according to study design and graded according to level of evidence. VALUES: A high value was placed on the avoidance of cardiovascular morbidity and premature death caused by untreated hypertension. BENEFITS, HARMS AND COSTS: Lifestyle modification by means of weight loss (or maintenance of healthy body weight), regular exercise and low alcohol consumption will reduce the blood pressure of appropriately selected normotensive and hypertensive people. Sodium restriction and stress management will reduce the blood pressure of appropriately selected hypertensive patients. The side effects of these therapies are few, and the indirect benefits are well known. There are certainly costs associated with lifestyle modification, but they were not measured in the studies reviewed. Supplementing the diet with potassium, calcium and magnesium has not been associated with a clinically important reduction in blood pressure in people consuming a healthy diet. RECOMMENDATIONS: (1) It is recommended that health care professionals determine the body mass index (weight in kilograms/[height in metres]2) and alcohol consumption of all adult patients and assess sodium consumption and stress levels in all hypertensive patients. (2) To reduce blood pressure in the population at large, it is recommended that Canadians attain and maintain a healthy body mass index. For those who choose to drink alcohol intake should be limited to 2 or fewer standard drinks per day (maximum of 14/week for men and 9/week for women). Adults should exercise regularly. (3) To reduce blood pressure in hypertensive patients, individualized therapy is recommended. This therapy should emphasize weight loss for overweight patients, abstinence from or moderation in alcohol intake, regular exercise, restriction of sodium intake and, in appropriate circumstances, individualized cognitive behaviour modification to reduce the negative effects of stress. VALIDATION: The recommendations were reviewed by all of the sponsoring organizations and by participants in a satellite symposium of the fourth international Conference on Preventive Cardiology. They are similar to those of the World Hypertension League and the Joint National committee, with the exception of the recommendations on stress management, which are based on new information. They have not been clinically tested. SPONSORS: The Canadian Hypertension Society, the Canadian Coalition for High Blood Pressure Prevention and Control, the Laboratory Centre for Disease Control at health Canada, and the Heart and Stroke Foundation of Canada.

Adult↗
PubMed1999

A circulating bovine gamma delta T cell subset, which is found in large numbers in the spleen, accumulates inefficiently in an artificial site of inflammation: correlation with lack of expression of E-selectin ligands and L-selectin.

Tissue-specific localization of TCR-defined subsets of gamma delta T cells has been widely reported; however, the mechanisms responsible for this phenomenon are poorly understood. We describe a bovine gamma delta T cell TCR-associated subset that preferentially localizes in the spleen. This subset was characterized by coexpression of CD8, and was found to lack surface expression of E-selectin ligands, GR Ag ligands, as well as low expression of L-selectin. The CD8-positive gamma delta T cell subset did not accumulate at sites of inflammation as efficiently as CD8-negative gamma delta T cells that, in contrast, express E-selectin and GR ligands and high levels of L-selectin. This is the first demonstration of a gamma delta T cell subset, which exhibits a defined tissue tropism, having a unique adhesion molecule expression profile. These results demonstrate that in some cases tissue-specific accumulation of gamma delta T cell subsets can be predicted by expression, or lack of expression, of defined homing molecules.

Animals↗
PubMed1999

Rapid induction and translocation of Egr-1 in response to mechanical strain in vascular smooth muscle cells.

The effect of mechanical strain on transcription and expression of the immediate-early genes, early growth response gene-1 (Egr-1), c-jun, and c-fos, was investigated in neonatal rat aortic vascular smooth muscle (VSM) cells. Cells grown on silicone elastomer plates were subjected to cyclic mechanical strain (1 Hz) at various durations and magnitudes. Egr-1 mRNA increased rapidly in response to cyclic strain, reached a maximum of 10-fold after 30 minutes, and returned to baseline after 4 hours. c-jun exhibited a similar pattern, whereas c-fos mRNA expression was unaffected by strain. Cycloheximide prolonged the increase in Egr-1 and c-jun mRNA and caused superinduction of both. The threshold level of continuous cyclic strain needed to induce expression was 5% for Egr-1 and c-jun. Even a single cycle of mechanical strain that lasted 1 second was sufficient to induce Egr-1 and c-jun mRNA. Strain also increased expression of a transiently transfected Egr-1 promoter-reporter construct. The effect of varying extracellular matrices on strain-induced Egr-1 and c-jun mRNA was examined. In contrast to collagen type 1- and pronectin-coated plates, strain did not significantly alter expression of Egr-1 and c-jun was less induced on laminin-coated plates. On collagen type 1, strain increased Egr-1 protein levels by 2.1-fold at 60 minutes. Immunofluorescence microscopy revealed translocation of Egr-1 to the nucleus in response to strain. These observations indicate that Egr-1 expression and translocation are sensitive to mechanical perturbation of the cell. c-jun is also induced by strain, but c-fos is not. The signal for this induction may involve specific cell-matrix interactions.

Animals↗
PubMed1999

Intratracheal administration of interleukin 12 plasmid-cationic lipid complexes inhibits murine lung metastases.

Administration of plasmid/lipid complexes to the lung airways for the treatment of metastatic pulmonary diseases represents a new strategy of gene therapy. In this study we present evidence that intratracheal administration of a plasmid encoding murine IL-12 complexed with N-[1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride:cholesterol inhibits the growth of lung metastases, using a renal cell carcinoma model. Instillation of pIL-12/lipid complexes resulted in expression of biologically active IL-12 (170-240 pg/ml) and IFN-gamma (100-190 pg/ml) in the bronchoalveolar lavage fluid. A significantly reduced number of lung metastases (26+/-24) was observed in mice instilled with IL-12/lipid complexes 24 hr after tumor challenge, whereas more than 250 metastatic foci were counted in lungs of untreated mice. Moreover, IL-12/lipid inhibited the growth of 3-day-old established metastases when compared with empty plasmid/lipid or IL-12 plasmid in saline. Mice receiving IL-12 gene therapy survived significantly longer (median survival of 43 days) than untreated mice (median survival of 31 days) or mice treated with control plasmid/lipid complexes (median survival of 35 days). These data demonstrate that a nonviral IL-12 gene therapy employing synthetic cationic lipids as a delivery system can be used to inhibit the development of lung metastases. Thus, this method provides support for the use of IL-12/lipid complexes to control the growth of pulmonary metastases and represents a potentially safer alternative to IL-12 protein immunotherapy.

Animals↗
PubMed1999

A case of discordant related abnormal karyotypes from chorionic villi and amniocytes.

A case of three discordant cell lines in prenatal diagnosis is described, of which two were abnormal related structural abnormalities of chromosome 11. One of the abnormal cell lines was seen in all metaphases examined from direct preparations of chorionic villi, the cultured preparations showing an apparently normal male karyotype; the other abnormal cell line was seen in conjunction with a normal cell line in cultured amniocytes. Prenatal diagnosis offered solely on chorionic villus sampling would have yielded a mistakenly normal result on the basis of established criteria for distinguishing true mosaicism from confined placental mosaicism.

Adult↗