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T O'Brien

Publications and source records attributed to T O'Brien.

At least 91 records · Page 5Linked to original sources
PubMed1998

Adipocyte macrophage colony-stimulating factor is a mediator of adipose tissue growth.

Adipose tissue growth results from de novo adipocyte recruitment (hyperplasia) and increased size of preexisting adipocytes. Adipocyte hyperplasia accounts for the severalfold increase in adipose tissue mass that occurs throughout life, yet the mechanism of adipocyte hyperplasia is unknown. We studied the potential of macrophage colony-stimulating factor (MCSF) to mediate adipocyte hyperplasia because of the profound effects MCSF exerts on pluripotent cell recruitment and differentiation in other tissues. We found that MCSF mRNA and protein were expressed by human adipocytes and that adipocyte MCSF expression was upregulated in rapidly growing adipose tissue that encircled acutely inflamed bowel and in adipose tissue from humans gaining weight (4-7 kg) with overfeeding. Localized overexpression of adipocyte MCSF was then induced in rabbit subcutaneous adipose tissue in vivo using adenoviral-mediated gene transfer. Successful overexpression of MCSF was associated with 16-fold increases in adipose tissue growth compared with a control adenovirus expressing beta-galactosidase. This occurred in the absence of increased cell size and in the presence of increased nuclear staining for MIB-1, a marker of proliferation. We conclude that MCSF participates in adipocyte hyperplasia and the physiological regulation of adipose tissue growth.

Adenoviridae↗
PubMed1998

Direct cloning of DNA that interacts in vivo with a specific protein: application to RNA polymerase II and sites of pausing in Drosophila.

A new method is described for cloning DNA sequences occupied by a specific protein on chromatin in vivo . The approach uses UV cross-linking to couple proteins covalently to DNA and the resulting complexes are then purified under stringent conditions. Particular adducts are immunoprocipitated with antibody to the protein of interest. The resulting DNA (iDNA) is amplified by PCR, cloned and characterized. The model system used was RNA polymerase II (Pol II), whose density on particular DNAs under various conditions is well documented. Pol II can exist in several states on DNA. While Pol II can simply be bound to DNA, the bulk of DNA-associated Pol II is transcriptionally engaged in either the transcribing or paused states. Paused Pol IIs that have previously been characterized are found at promoters and have the distinctive property that their transcription in isolated nuclei is stimulated by sarkosyl or high salt. Here we isolate and sequence DNAs that cross-link to Pol II molecules. We identify by nuclear run-on assays those DNAs that have Pol II engaged in transcription. Twenty one percent of the iDNA clones that have detectable transcriptionally engaged Pol II appear to be paused, in that they display sarkosyl-stimulated trancription in a nuclear run-on transcription assay. At least some of these map to the 5'-ends of genes. These results suggest that transcriptional pausing of Pol II is a general phenomenon in vivo.

Animals↗
PubMed1998

Systematic evaluation of distribution of transgene expression after adenovirus-mediated gene transfer to the transplanted heart.

In the transplantation setting, the study and potential treatment of acute and chronic rejection by means of gene therapy will require widespread transgene expression in the donor organ. The distribution of transgene expression after adenovirus-mediated gene transfer via the coronary vasculature in a model of abdominal heterotopic heart transplantation in syngeneic rats (n = 6) was evaluated at 1 week. Reporter gene expression was evenly distributed in the base, the midventricle, and the apex of the transplanted hearts. This study demonstrates that intracoronary administration of the adenoviral vector to the donor heart results in widespread transgene expression.

Adenoviridae↗
PubMed1998

Transbronchial gene transfer of endothelial nitric oxide synthase to transplanted lungs.

BACKGROUND: Experiments were designed to study the efficiency, distribution, and toxicity of transbronchial adenoviral-mediated transfer of endothelial constitutive nitric oxide synthase (ecNOS) gene to transplanted lungs. METHODS: Syngeneic orthotopic single-lung transplantation in the rat was performed after airway administration (300 microL, 1 x 10(9) pfu/mL) of either the ecNOS gene or the marker gene beta-Gal (control group) to donor lungs (n=4 each). After 4 days, transgene expression, inflammation, and the presence of apoptosis in the transplanted lungs were assessed by molecular, immunohistochemical, and histologic techniques. RESULTS: Gene transfer was confirmed by a positive polymerase chain reaction signal for the recombinant ecNOS gene, and recombinant messenger RNA by reverse transcription polymerase chain reaction. Positive immunohistochemical staining for ecNOS was present in more than 75% of pneumocytes only in ecNOS transduced lungs. Calcium-dependent nitric oxide synthase activity was increased in ecNOS- compared with betaGal-transduced lungs (2,139+/-756 versus 47+/-28 pmol x mg protein(-1) x h(-1); p < 0.05). Minimal to mild inflammation was observed in all transplanted lungs; fewer than 0.5% of cells in both groups were apoptotic. CONCLUSIONS: Transbronchial transfer of ecNOS gene to the transplanted lung results in transduction of pneumocytes with expression of a functionally active transgene product.

Adenoviridae↗
PubMed1998

Ex vivo gene transfer of endothelial nitric oxide synthase to atherosclerotic rabbit aortic rings improves relaxations to acetylcholine.

Cholesterol feeding results in impaired endothelium dependent vasorelaxation. The role of nitric oxide in this process is unclear. The aim of this study was to evaluate the role of nitric oxide in cholesterol-induced vasomotor dysfunction by examining the effect of overexpression of eNOS in the hypercholesterolemic rabbit aorta on vascular reactivity. Vascular rings from the thoracic aorta of hypercholesterolemic rabbits were exposed ex vivo either to an adenoviral vector encoding endothelial nitric oxide synthase (AdeNOS) or Escherichia coli beta Galactosidase (AdbetaGal). Transgene expression was examined by histochemistry for beta galactosidase, immunohistochemistry for eNOS and cyclic GMP measurements and vasomotor studies were performed. Transgene expression was found to localize to the endothelium and adventitia. cGMP levels were significantly greater in AdeNOS compared to AdbetaGal transduced rings. Acetylcholine mediated relaxation was significantly impaired in cholesterol fed rabbits and was markedly improved by overexpression of eNOS. These results suggest that reduced NO bioavailability observed in cholesterol-induced vascular dysfunction can be partially overcome by eNOS gene transfer.

Acetylcholine↗
PubMed1998

Gene therapy in lung transplantation: effective gene transfer via the airways.

OBJECTIVES: Gene therapy may provide a means of modifying factors that contribute to the development of pathologic processes in transplanted lungs. Experiments were designed to study the feasibility of adenovirus-mediated gene transfer by way of the airways to the transplanted lung. METHODS: Orthotopic left lung transplantation (Lewis to Lewis rats) was performed on four groups of animals. 300 microl of adenovirus solution encoding for beta-galactosidase was infused into the left bronchus of donor rats at viral concentrations of 10(8) pfu/ml (n = 5), 10(9) pfu/ml (n = 6), and 10(10) pfu/ml (n = 6), and the lung was ventilated for 5 minutes. Controls (n = 6) received medium only. Seven days after transplantation, native and transduced, transplanted lungs were harvested. Sections of lung were fixed and stained with a solution of X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) and staining was evaluated for distribution by cell type and intensity. RESULTS: Beta-galactosidase expression was absent in the control group and in the native lungs. Two of five lungs in the 10(8) group expressed beta-galactosidase, but in a limited distribution and intensity. All six lungs in the 10(9) group and five of six lungs in the 10(10) group expressed beta-galactosidase. The distribution and intensity of beta-galactosidase expression ranged from only a few cells staining per slide to up to 75%. Pneumocytes were the most frequently stained cell type followed by alveolar macrophages. CONCLUSIONS: Gene transfer to the transplanted lung via the bronchial route is feasible and offers a novel technique to modify pathologic processes in the transplanted lung.

Adenoviridae↗
PubMed1998

Transfer and expression of recombinant nitric oxide synthase genes in the cardiovascular system.

Gene therapy involves the transfer of a functional gene into host cells to correct the malfunction of a specific gene or to alleviate the symptoms of a disease. For gene transfer to the cardiovascular system, adenoviral vectors are the most efficient means of transfer. Recently, transfer and functional expression of recombinant nitrio oxide synthase (NOS) genes to cerebral and cardiovascular beds have been demonstrated both ex vivo and in vivo. Here, Alex Chen and colleagues review current progress in the field of vascular NOS gene transfer and the potential use of NOS gene therapy for a number of cardiovascular diseases. Although the feasibility of the NOS gene transfer approach has been demonstrated in animal models, currently available vectors have a number of technical and safety limitations that have to be solved before human NOS gene therapy for cardiovascular disease can be attempted.

Animals↗
PubMed1998

Influence of temperature on adenovirus-mediated gene transfer.

OBJECTIVE: The transfer of recombinant genes to donor organs may allow for novel therapeutic approaches to the challenges of acute and chronic rejection. Adenoviral vectors are capable of efficient gene transfer, but use of these vectors during donor organ preservation may be less efficient due to the low temperature. This study was designed to examine the effect of temperature on the efficiency of adenovirus-mediated gene transfer. METHODS: Gene transfer to human endothelial cells, porcine vascular smooth muscle cells and cultured rat thoracic aortas was examined. Incubation with an adenoviral vector encoding for E. coli beta-galactosidase was performed for 1 h at three different temperatures: 4 degrees C, 10 degrees C and 37 degrees C. Transgene expression was assessed by histochemical staining for beta-galactosidase in transduced cells and by evaluation of beta-galactosidase activity in organ cultures. RESULTS: Both in human endothelial cells and vascular smooth muscle cells the percentage of positively staining cells following transduction at 37 degrees C was significantly greater than at 4 degrees C and at 10 degrees C (30.55 +/- 7.26% vs. 14.29 +/- 3.79% and 12.43 +/- 2.47%, respectively for endothelial cells, P < 0.01 vs. 4 degrees C and 10 degrees C; and 28.25 +/- 4.52% vs. 17.91 +/- 3.76% and 16.63 +/- 3.92%, respectively for smooth muscle cells, P < 0.05 vs. 4 degrees C, P < 0.01 vs. 10 degrees C). Beta-galactosidase activity was significantly greater in aortas transduced at 37 degrees C than in vessels transduced at 4 degrees C and 10 degrees C (289,700 +/- 113,300 vs. 149,600 +/- 54,390 and 108,800 +/- 23,140 relative chemiluminesce units/mg of total protein, respectively; P < 0.05 vs. 4 degrees C, P < 0.001 vs. 10 degrees C). CONCLUSIONS: The present study demonstrates that the efficiency of adenovirus-mediated gene transfer is significantly reduced at lower temperatures. The need for cold preservation of donor organs may render efficient adenovirus-mediated gene transfer more difficult in the transplantation setting.

Adenoviridae↗
PubMed1998

Functional analysis of the human TAFII250 N-terminal kinase domain.

The largest subunit of the human transcription factor TFIID, TAFII250, was previously reported to contain serine/threonine kinase domains that can autophosphorylate and transphosphorylate the large subunit of the basal factor TFIIF. Here, we identify the regions of the N-terminal kinase domain (amino acids 1-414) necessary for kinase activity and examine its function in vivo. Point mutations within two patches of amino acids in the kinase domain decrease both autophosphorylation and transphosphorylation activities. Importantly, we find that TAFII250-bearing mutations within the N-terminal kinase domain exhibit a significantly reduced ability to rescue ts13 cells that express a temperature-sensitive TAFII250. Moreover, transcription from the cyclin A and cdc2 promoters becomes impaired when cotransfected with hTAFII250 containing inactive forms of the N-terminal kinase domain. Our results suggest that the TAFII250 kinase activity is required to direct transcription of at least some genes in vivo.

Cell Line↗
PubMed1998

High expression of Wnt7b in human superficial bladder cancer vs invasive bladder cancer.

Aberrant Wnt gene expression is involved in the development of breast cancer, but its role in other tumours is unknown. Wnts regulate cadherin function, previously shown to be more commonly deregulated in invasive bladder cancer. This study investigated whether factors upstream of cadherins were aberrantly expressed in superficial bladder cancer. The expression of one transforming (Wnt7b) and one non-transforming (Wnt5a) Wnt gene in four human bladder carcinoma cell lines, and in normal human bladder tissues (n = 8) and bladder cancers (n = 48) were analysed by ribonuclease protection analysis. All cell lines expressed an approximately equal level of Wnt7b mRNA. Wnt5a and Wnt7b mRNAs were both expressed in normal bladder tissues and bladder tumours. The median expression of Wnt7b was fourfold higher in superficial tumours (n = 29) than in normal tissues (n = 8, P = 0.002) and five fold higher than in invasive tumours (n = 17, P = 0.003). There was no significant difference between normal tissues and invasive tumours (P = 0.3). The expression of Wnt5a did not vary significantly between normal tissues and superficial tumours (P = 0.4), normal tissues and invasive tumours (P = 0.3) or superficial tumours and invasive tumours (P = 0.2). The differential expression of Wnt7b suggests a role in the early events of superficial bladder tumorigenesis involving cell adhesion and provides further evidence of different pathways of evolution of superficial and invasive cancer.

Carcinoma↗
PubMed1998

Medical-resource use for suspected tuberculosis in a New York City hospital.

OBJECTIVE: To compare resource use by diagnostic outcome among hospital admissions during which tuberculosis (TB) was suspected. DESIGN: Retrospective study based on chart review and microbiology laboratory data. SETTING: The department of medicine in a municipal hospital serving central Brooklyn, New York. PARTICIPANTS: We identified all adult admissions in 1993 during which TB was suspected. We assigned each admission to one of four mutually exclusive groups defined by the results of microbiological tests (acid-fast bacilli [AFB] smear and culture): culture-positive and smear-positive (C+S+); culture-positive and smear-negative (C+S-); culture-negative and smear-positive (C-S+); or culture-negative and smear-negative (C-S-). Each admission was divided into two separate periods to which the utilization of medical resources was assigned: the diagnostic and the postdiagnostic periods, which were separated by the date of receipt of the first definitive culture report. RESULTS: Data on 519 admissions (93 C+S+; 57 C+S-; 30 C-S+; and 339 C-S-) were analyzed. Although C+S+ were more likely than other groups to have an admitting diagnosis of TB, approximately one quarter of the admissions without TB (C-S+, C-S-) were admitted with the principal diagnosis of TB. For the four groups, C+S+, C+S-, C-S+, and C-S-, the respective rates of TB isolation and anti-TB treatment, and median lengths of isolation were 98%, 87%, and 34 days; 74%, 74%, and 7 days; 83%, 83%, and 15 days; and 44%, 29%, and 0 days. During the diagnostic period, the rate and length of isolation were similar in the AFB-smear-positive groups (C+S+ and C-S+). We estimated that admissions without culture-proven TB (C-S+ and C-S-) accounted for 3,174 (36%) of the 8,712 days of TB isolation expended and for 65% of the 16,671 days of anti-TB treatment. The vast majority of this resource consumption (2,737 [86%] of 3,174 days of isolation) occurred during the diagnostic period before a definitive culture result was known. CONCLUSIONS: Our results suggest that prolonged diagnostic uncertainty and misclassification of cases due to false-positive and false-negative smears are associated with substantial medical-resource consumption. New diagnostic modalities that reduce the period of diagnostic uncertainty could reduce the utilization of resources later found to be unnecessary.

Adult↗
PubMed1998

In vitro evaluation of a new lithium dilution method of measuring cardiac output and shunt fraction in patients undergoing venovenous extracorporeal membrane oxygenation.

OBJECTIVE: To evaluate, in vitro, a method of measuring cardiac output and shunt fraction during venovenous extracorporeal membrane oxygenation (ECMO). DESIGN: Experimental study using an in vitro model. SETTING: A teaching hospital. MODEL: An ECMO circuit was set up in parallel with a patient circuit consisting of tubing through which saline was circulated from a 50-L reservoir by a pump which was set at 3 L/min to represent cardiac output. A second pump in the ECMO circuit drew saline from the patient circuit and passed it through a membrane oxygenator. The flow from the membrane oxygenator either returned directly to the patient circuit or was diverted, via a third pump, back into the ECMO circuit, thereby producing a shunt. INTERVENTIONS: By adjusting the flow rates of the second (ECMO) and third (shunt) pumps, three shunt fractions of 12%, 25%, and 50% were produced at three different ECMO flow rates. Lithium chloride (0.15 mmol) was injected just downstream of the membrane oxygenator; the lithium ion concentration-time curves were recorded simultaneously in the flow returning to the saline reservoir and in the flow just upstream of the membrane oxygenator using lithium selective electrodes. MEASUREMENTS AND MAIN RESULTS: Nine pairs of curves were recorded, one pair for each combination of ECMO and shunt flow rates. Analysis of these curves allowed shunt flow and "cardiac output" to be calculated and compared with the flow rates delivered by the pumps. Mean "cardiac output" derived from the lithium dilution curves was 2.98 +/- 0.18 (SD) L/min, compared with a delivered pump flow of 3 L/min. Measured shunt flow = 0.008 + 1.09 x actual shunt flow (R = 0.997). CONCLUSIONS: This method would allow cardiac output and shunt flow to be measured in patients undergoing venovenous ECMO. It could result in better patient management and improved cannula design.

Blood Flow Velocity↗
PubMed1998

Adenovirus-mediated gene transfer of macrophage colony stimulating factor to the arterial wall in vivo.

Macrophage colony stimulating factor (MCSF) is believed to play a key role in one of the earliest events in atherosclerosis, ie, monocyte to macrophage differentiation in the arterial intima. The aim of this study was to examine the biological effects of vascular wall expression of MCSF. A recombinant adenovirus vector encoding human MCSF (AdMCSF) was generated by standard techniques of homologous recombination in 293 cells. The rabbit carotid artery was transduced with AdMCSF. As negative controls, carotid arteries were transduced with either an adenoviral vector encoding beta-galactosidase, an adenoviral vector encoding apolipoprotein E, or diluent alone. Intima-media thickness ratio was calculated 5 and 21 days after transduction. The cell type present in intimal infiltrates was analyzed by immunohistochemistry. MCSF expression was demonstrated in the vessel wall of AdMCSF-transduced vessels by reverse transcription-polymerase chain reaction and immunofluorescence. In contrast to control vessels, adenovirus-mediated MCSF expression was associated with an intimal cellular infiltrate consisting of smooth muscle cells and small numbers of macrophages. Whereas the intima-media thickness ratio was greater in AdMCSF-transduced vessels at 5 days, this difference was no longer statistically significant at 21 days. These results suggest that MCSF may play a role in recruitment of monocytes and macrophages to the vessel wall and may contribute to smooth muscle cell proliferation and migration.

Adenoviridae↗
PubMed1998

Adventitial expression of recombinant eNOS gene restores NO production in arteries without endothelium.

The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVbeta-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3 x 10(-11) to 10(-9) mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVbeta-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3 x 10(-11) to 10(-9) mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor, NG-nitro-L-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.

Adenoviridae↗
PubMed1998

Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery after experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Gene transfer with recombinant viral vectors encoding vasodilator proteins may be useful in therapy of cerebral vasospasm after subarachnoid hemorrhage (SAH). Relaxations mediated by nitric oxide are impaired in cerebral arteries affected by SAH. The present study was designed to determine the effect of SAH on the efficiency of ex vivo adenovirus-mediated gene transfer to canine basilar arteries and to examine whether expression of recombinant endothelial nitric oxide synthase (eNOS) gene may have functional effects on vasomotor reactivity of spastic arteries affected by SAH. METHODS: Replication-deficient recombinant adenovirus vectors encoding bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVbeta-Gal) genes were used for ex vivo gene transfer. Rings of basilar arteries obtained from control dogs and dogs exposed to SAH were incubated with the vectors in minimum essential medium. Twenty-four hours after gene transfer, expression and function of the recombinant genes were evaluated by (1) histochemical or immunohistochemical staining, (2) beta-galactosidase protein measurement, and (3) isometric tension recording. RESULTS: Transduction with AdCMVbeta-Gal and AdCMVeNOS resulted in the expression of recombinant beta-galactosidase and eNOS proteins mostly in the vascular adventitia. The expression of beta-galactosidase protein was approximately 2-fold higher in SAH arteries than in normal arteries. Endothelium-dependent relaxations caused by bradykinin and substance P were suppressed in SAH arteries. The relaxations to bradykinin were significantly augmented in both normal and SAH arteries after AdCMVeNOS transduction but not after AdCMVbeta-Gal transduction. The relaxations to substance P were augmented by AdCMVeNOS transduction only in normal arteries. Bradykinin and substance P caused relaxations even in endothelium-denuded arteries, when the vessels were transduced with AdCMVeNOS. These endothelium-independent (adventitia-dependent) relaxations to bradykinin observed after AdCMVeNOS transduction were similar between normal and SAH arteries, whereas those to substance P were significantly reduced in SAH arteries compared with normal arteries. CONCLUSIONS: These results suggest that expression of recombinant proteins after adenovirus-mediated gene transfer may be enhanced in cerebral arteries affected by SAH and that successful eNOS gene transfer to spastic arteries can at least partly restore the impaired nitric oxide-mediated relaxations through local (adventitial) production of nitric oxide.

Adenoviridae↗
PubMed1998

Primary and secondary reinforcers in performance of a 1.0-mile walk/jog by adolescents with moderate mental retardation.

Based on visual inspection of data, reinforcement procedures, namely, use of verbal praise and verbal praise plus token exchange, were at least mildly influential in improving performance of 5 youth with moderate mental retardation compared to their initial performance without reinforcers. These findings suggest specific reinforcement may improve the time for aerobic activity by adolescents with moderate mental retardation.

Adolescent↗
PubMed1998

Adenoviral-mediated gene transfer to human adipocytes in vitro, and human adipose tissue ex vivo and rabbit femoral adipose tissue in vivo.

Adenoviral-mediated gene transfer has proven useful in several organ systems to understand gene action and to provide a potential therapeutic modality for localized, organ-specific gene overexpression. However, the application of adenoviral-mediated gene transfer to adipocytes and adipose tissue has not been evaluated. We evaluated the feasibility of in vitro and ex vivo transfer of the beta-galactosidase gene to human adipocytes and adipose tissue by means of adenoviral vectors. The efficiency (percentage of cells transduced) of adenoviral-mediated gene transfer of the beta-galactosidase gene to human adipocytes in vitro and to human adipose tissue ex vivo was 21 +/- 3% and 14 +/- 3%, respectively. Adenoviral-mediated gene transfer in a rabbit femoral adipose tissue was also demonstrated in vivo. Adenoviral-mediated gene transfer may facilitate studies on understanding the biology of adipocytes and provide a potential tool for the modulation of adipocyte function in vivo and thereby for the treatment of obesity.

Adenoviridae↗