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Timothy O'Brien

Publications and source records attributed to Timothy O'Brien.

30 records · Page 2Linked to original sources
PubMed2002

Gene transfer to coronary artery bypass conduits.

BACKGROUND: Gene therapy is a rational approach to prevention of stenosis in saphenous vein grafts used as conduits for coronary artery bypass grafting. To explore this possibility we developed methods for adenoviral-mediated gene transfer to canine saphenous veins. METHODS: During a single procedure, autogenous canine saphenous vein segments were transduced ex vivo and used as coronary artery bypass grafts. The proximal end of each vein was ligated, adenovirus containing the Escherichia coli beta-galactosidase gene (Ad.CMVLacZ) was delivered at titers of 2.5 x 10(9) or 5 x 10(9) plaque-forming units (pfu)/mL to the lumen through a distal heparin lock, and the segment was immersed in the viral solution for 1 hour at 37 degrees C. Control segments were exposed to diluent alone in an identical manner. Aortocoronary anastomoses were made using cardiopulmonary bypass. Transgene expression was assessed qualitatively and quantitatively after 3 days. RESULTS: Beta-galactosidase levels showed a dose-dependent increase: 0.00 +/- 0.00 ng/mg total protein for controls; 5.60 +/- 2.27 ng/mg total protein for a viral titer of 2.5 x 10(9) pfu/mL and 11.97 +/- 6.14 ng/mg for 5 x 10(9) pfu/mL. The two dosage groups differed significantly from each other (p = 0.035) and from controls (p = 0.003). X-gal staining demonstrated mostly endothelial and scattered adventitial transgene expression. CONCLUSIONS: Transgene expression after ex vivo gene transfer into saphenous vein grafts in a canine coronary artery bypass model indicates that this method may be useful for delivery of therapeutic genes to prevent or retard vein graft arteriosclerosis.

Adenoviridae↗
PubMed2002

Dietary antioxidants preserve endothelium dependent vasorelaxation in overfed rats.

OBJECTIVE: A high fat diet contributes to obesity and acutely impairs endothelium dependent vasorelaxation. While a high cholesterol diet chronically impairs endothelium dependent vasorelaxation in rabbits, this model is associated with severe hypercholesterolemia. The effect of chronic high fat feeding on endothelial function in the setting of more normal lipid levels has not been studied. Our aim was to study vascular function in rats overfed for 6 months and to determine the role of oxidative stress in the alteration of vascular function associated with this diet. METHODS: Forty-five male Sprague-Dawley rats were placed on the following diets for 6 months, control diet, high fat diet or high fat diet supplemented with vitamins A, E and selenium. Six months later blood samples were collected and vascular function was assessed in the aorta. RESULTS: The rats fed a high fat diet were heavier than controls (608.4 +/- 41.8 vs. 700.3 +/- 50.1 vs. 699.5 +/- 52.6 g, P<0.05 for control vs. high fat and high fat plus antioxidant groups) but lipid levels were similar in each group (cholesterol, 145.9 +/- 53.4 vs. 140.5 +/- 44.0 vs. 152.7 +/- 36.1 mg/dl and triglycerides, 173.2 +/- 106.7 vs. 197.4 +/- 131.3 vs. 166.1 +/- 65.3 mg/dl, P, NS). Relaxations to acetylcholine and calcium were significantly impaired in the high fat diet group compared with controls (EC(50), 6.90 +/- 0.22, 7.12 +/- 0.32; AUC, 96.9 +/- 51.6, 155.5 +/ - 73.7) but were not different between the antioxidant supplemented group and controls (EC(50), 7.06 +/- 0.37; AUC, 151.9 +/- 67.4). Relaxations to DEA NONOate were similar in each group. CONCLUSIONS: Dietary antioxidants preserved endothelium dependent vasorelaxation in rats fed a high fat diet for 6 months.

Analysis of Variance↗
PubMed2002

Adenoviral-mediated overexpression of catalase inhibits endothelial cell proliferation.

Although hydrogen peroxide (H(2)O(2)) induces proliferation of vascular smooth muscle cells, its role in endothelial cell proliferation is unclear. Our aim was to study the role of hydrogen peroxide in endothelial cell proliferation by overexpressing catalase. Human aortic endothelial cells were transduced with adenoviral vectors encoding beta-galactosidase (Adbetagal) or catalase (AdCat) or were exposed to diluent alone (control). Transgene expression was demonstrated by beta-galactosidase staining, Western analysis, and significantly increased enzyme activity in AdCat-transduced cells. Overexpression of catalase decreased DNA synthesis in AdCat compared with control and Adbetagal-transduced cells (536.8 +/- 31 vs. 1,875.1 +/- 132.9 vs. 1,347.5 +/- 93.7 dpm/well, respectively; P < 0.05 vs. control and Adbetagal). Six days after transduction with AdCat (multiplicity of infection = 50), cell numbers were significantly reduced (AdCat: 38 +/- 1.8% of cell counts in control, P < 0.05; and 45 +/- 2% of cell count in Adbetagal, P < 0.05). Incubation with aminotriazole 10 mmol/l, an inhibitor of catalase, prevented this effect. The number of apoptotic cells was increased one- and threefold 2 and 4 days, respectively, after transduction with AdCat. Exogenous administration of low concentrations of H(2)O(2) (50 microM) significantly increased cell proliferation, whereas it was inhibited by higher concentrations. These results suggest that H(2)O(2) is an important modulator of endothelial cell proliferation.

Adenoviridae↗
PubMed2002

Protective vasomotor effects of in vivo recombinant endothelial nitric oxide synthase gene expression in a canine model of cerebral vasospasm.

BACKGROUND AND PURPOSE: Post-subarachnoid hemorrhage (SAH) cerebral vasospasm is a potentially devastating condition whose pathogenesis involves impaired nitric oxide (NO) bioavailability. We aimed to determine whether recombinant endothelial NO synthase (eNOS) gene expression may protect vasomotor function and prevent vasospasm in a canine experimental SAH model. METHODS: Recombinant adenoviral vectors (5x10(9) plaque-forming units/animal) encoding genes for eNOS (AdeNOS) and beta-galactosidase (AdLacZ) or vehicle were injected into the cerebrospinal fluid (CSF) of dogs on day -1 (ie, 24 hours before the first intra-CSF injection of blood on day 0). Cerebral angiography was performed at day 0 (baseline) and day 7 (immediately before death), and tissues were harvested for additional studies. RESULTS: Western analysis and immunohistochemistry detected recombinant eNOS exclusively in cerebral arteries isolated from AdeNOS-transduced dogs, and in this group of animals CSF NO concentrations were significantly elevated by day 2. Analysis of day 7 versus day 0 cerebral angiograms for each group revealed significant spasm at the basilar artery midpoint in AdLacZ-transduced and nontransduced dogs but not in AdeNOS-transduced dogs. Isometric force recording of basilar arteries isolated from AdeNOS-transduced dogs showed significantly augmented relaxations to bradykinin and reduced contractions to endothelin-1. CONCLUSIONS: Our results suggest that expression of recombinant eNOS in the adventitia of cerebral arteries may contribute toward protection against post-SAH vasospasm.

Animals↗
PubMed2002

Expression and function of recombinant S1179D endothelial nitric oxide synthase in canine cerebral arteries.

BACKGROUND AND PURPOSE: Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179DeNOS) increases nitric oxide (NO) production constitutively in the absence of agonist stimulus. The present study was designed to determine the effect of mutant S1179DeNOS gene expression on vasomotor function of canine cerebral arteries. METHODS: Isolated basilar and middle cerebral arteries were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector (10(10) plaque-forming units per milliliter) encoding the S1179DeNOS gene (AdCMVS1179DeNOS), the wild-type eNOS gene (AdCMVeNOS), or the green fluorescent protein (GFP) reporter gene (AdCMVGFP). Twenty-four hours after transduction, arteries were suspended in an organ chamber for isometric force recording, and levels of cGMP were measured by radioimmunoassay. RESULTS: Transgene protein expression was detected mainly in the vascular adventitia. In AdCMVS1179DeNOS-transduced arteries, basal levels of cGMP were significantly elevated compared with those in control (nontransduced), AdCMVGFP-, or AdCMVeNOS-transduced vessels (n=8; P<0.01). The elevation of cGMP was abolished by a NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), or by incubation in the calcium-free medium in the presence of calcium chelators. In AdCMVS1179DeNOS-transduced arteries, contractions to endothelin-1 (10(-10) to 10(-8) mol/L) were significantly reduced compared with those in control and AdCMVGFP-transduced arteries (n=7; P<0.05). The vasoconstrictor effect of endothelin-1 was restored in the presence of the NOS inhibitor L-NAME. CONCLUSIONS: Our results suggest that in cerebral arteries, expression of recombinant S1179DeNOS increases basal production of NO and inhibits the vasoconstrictor effect of endothelin-1. This effect may have therapeutic application in prevention and treatment of cerebrovascular diseases.

Amino Acid Substitution↗
PubMed2002

"Diabetic mastopathy," or sclerosing lymphocytic lobulitis, is strongly associated with type 1 diabetes.

OBJECTIVE: To demonstrate the strong association of diabetic mastopathy or sclerosing lymphocytic lobulitis with type 1 diabetes mellitus by studying appropriate control groups and to describe risk factors and natural history of the disorder. RESEARCH DESIGN AND METHODS: This was a retrospective cross-sectional study of four groups of patients conducted at a setting tertiary care medical center. We examined benign breast biopsies (investigator masked to identity) from age-matched patients with types 1 and 2 diabetes, autoimmune thyroid disease, or none of the above disorders for sclerosing lymphocytic lobulitis. Several risk factors proposed for the disorder (age at diagnosis of benign breast disease, duration of diabetes, age at onset of diabetes, prevalence of retinopathy, neuropathy, nephropathy and cheiroarthropathy, glycemic control, parity, oral contraceptive use, menopausal status, or number of breast biopsies) were evaluated, and patients were contacted to describe the natural history of the disorder. RESULTS: Sclerosing lymphocytic lobulitis was identified in 69.7% of the subjects with type 1 diabetes and 1.8% of those with autoimmune thyroid disease diagnosed with benign breast disease at surgery. It did not occur in patients with type 2 diabetes with or without insulin treatment or in control subjects. Only retinopathy and peripheral neuropathy were associated with sclerosing lymphocytic lobulitis. Breast carcinoma or lymphoma did not occur subsequently in any type 1 diabetes patient with or without sclerosing lymphocytic lobulitis. CONCLUSIONS: Sclerosing lymphocytic lobulitis is strongly associated with type 1 diabetes. Retinopathy and neuropathy are associated with the disorder. The risk of malignancy is not increased.

Adult↗
PubMed2002

Clinical trials in vascular gene therapy: the missing piece of the puzzle.

OBJECTIVE: Vascular gene transfer provides unique opportunities for novel therapies for cardiovascular disease. To define the limitations and expectations of translating preclinical strategies to clinical therapy, the kinetics and extent of vascular transgene expression must be determined. DATA SOURCES: Published clinical and preclinical studies of vascular gene transfer. DATA EXTRACTION: Kinetics and extent of vascular transgene expression. DATA SYNTHESIS: In spite of ongoing clinical studies in vascular gene transfer, data regarding the kinetics and extent of transgene expression are very limited. CONCLUSIONS: For the efficient translation of preclinical to clinical vascular gene transfer studies, the kinetics and extent of transgene expression should be determined. Novel imaging technologies and unique clinical scenarios for obtaining tissue should be considered.

Cardiovascular Diseases↗
PubMed2002

Overexpression of HER-2/neu in uterine serous papillary cancer.

PURPOSE: Uterine serous papillary carcinoma (USPC) is a highly aggressive variant of endometrial cancer and histologically similar to high-grade ovarian cancer. HER-2/neu, the transmembrane receptor encoded by the c-erbB2 gene, is overexpressed by immunohistology in approximately 25% of ovarian cancers. In this study, we have evaluated the expression of HER-2/neu in several fresh, established, paraffin-embedded, fixed USPCs. In addition, we have tested the sensitivity of USPC cells to Herceptin treatment. EXPERIMENTAL DESIGN: Ten consecutive USPC specimens were assessed by immunohistochemistry for the intensity of expression of HER-2/neu. In addition, three USPC cell lines were analyzed for expression of HER-2/neu by flow cytometry as well as for sensitivity to Herceptin-mediated, complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), and inhibition of cell proliferation. RESULTS: Eight of 10 (80%) of the USPCs assessed immunohistochemically for the intensity of expression of HER-2/neu stained heavily for HER-2/neu (2+ to 3+). Fresh and established primary USPC cell lines were found to express significantly more HER-2/neu receptor by flow cytometry (on the average, 10-fold greater) when compared with HER-2/neu-positive primary or established breast and ovarian cancer cell lines (P < 0.001). Importantly, although these USPC cell lines were resistant to chemotherapy in vivo and to natural killer- and complement-mediated cytotoxicity in vitro, they were found to be highly sensitive to Herceptin-mediated ADCC. USPC cell proliferation was also inhibited by Herceptin. A significant enhancement of ADCC was demonstrated when effector cells were exposed to low doses of IL-2 in vitro. Physiological concentrations of human serum IgG did not inhibit Herceptin-mediated ADCC against USPC. CONCLUSIONS: On the basis of these findings and previous reports showing a positive in vivo correlation between efficacy of Herceptin therapy and the level of HER-2/neu overexpression by tumor cells, we propose that Herceptin might be a novel and attractive therapeutic strategy in patients harboring chemotherapy-resistant, recurrent, or metastatic USPC.

Aged↗
PubMedDate not supplied

Cytoprotection of beta cells: rational gene transfer strategies.

Gene transfer to pancreatic islets may prove useful in preventing islet cell destruction and prolonging islet graft survival after transplantation in patients with type 1 diabetes mellitus (T1DM). Potentially, a host of therapeutically relevant transgenes may be incorporated into an appropriate gene delivery vehicle and used for islet modification. An increasing understanding of the molecular pathogenesis of immune-mediated beta cell death has served to highlight molecules which have become suitable candidates for promoting islet cell survival in the face of oxidative stress. This review aims to give an overview of some conventional gene transfer strategies aimed at promoting islet cell survival in the face of cytokine onslaught. These strategies target three aspects of islet cell physiology: redox status and antioxidant defence, anti-apoptotic gene expression and mediators of cytokine signal transduction pathways.

Diabetes Mellitus, Type 1↗
PubMedDate not supplied

Don't lose heart--therapeutic value of apoptosis prevention in the treatment of cardiovascular disease.

Cardiovascular disease is a leading cause of death worldwide. Loss of function or death of cardiomyocytes is a major contributing factor to these diseases. Cell death in conditions such as heart failure and myocardial infarction is associated with apoptosis. Apoptotic pathways have been well studied in non-myocytes and it is thought that similar pathways exist in cardiomyocytes. These pathways include death initiated by ligation of membrane-bound death receptors, release of pro-apoptotic factors from mitochondria or stress at the endoplasmic reticulum. The key regulators of apoptosis include inhibitors of caspases (IAPs), the Bcl-2 family of proteins, growth factors, stress proteins, calcium and oxidants. The highly organized and predictive nature of apoptotic signaling means it is amenable to manipulation. A thorough understanding of the apoptotic process would facilitate intervention at the most suitable points, alleviating myocardium decline and dysfunction. This review summarizes the mechanisms underlying apoptosis and the mediators/regulators involved in these signaling pathways. We also discuss how the potential therapeutic value of these molecules could be harnessed.

Animals↗
PubMedDate not supplied

Giant mediastinal parathyroid adenoma with hypercalcemia.

Parathyroid disease is uncommon, but when found it is always of interest to the physician. Most patients with parathyroid disease also have hypercalcemia. Benign adenomatous disease is the most frequent surgical parathyroid lesion seen. Most of these lesions occur in the neck and are usually <2 cm in diameter. We report the case of a 50-year-old patient with familial neurofibromatosis, a serum calcium of 19 mg/dl, nephrocalcinosis, and renal failure. Evaluation revealed a large (5.7 x 4.5 x 2.9-cm) mediastinal adenoma. At resection, the tumor weighed 39.5 g. Symptomatology rapidly improved postoperatively.

Adenoma↗