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T O'Brien

Publications and source records attributed to T O'Brien.

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

Improved efficiency of gene transfer to the transplanted lung by retrograde vascular gene delivery.

Experiments were designed to evaluate the efficiency of antegrade compared to retrograde vascular gene transfection of donor lungs used for transplantation. Rat donor lungs (n = 5/group) were transduced with an adenoviral vector encoding for beta-galactosidase (AdbetaGal), either antegrade in the pulmonary artery (Group A, 3 x 10(8) pfu, Group B, 3 x 10(9) pfu) or retrograde into the pulmonary vein (Group C, 3 x 10(8) pfu), immediately after pneumoplegia. After storage at 4 degrees C for 1 h, the transduced lungs were transplanted orthotopically in syngeneic animals. The lungs were assessed for transgene expression by ELISA and X-Gal-staining at day 7 after operation. Inflammation was graded based on the extent of inflammatory cell infiltration. Transgene expression was similar between Groups A (1.7 +/- 0.7 ng/mg protein) and B (2.1 +/- 1.0 ng/mg protein). With retrograde delivery, there was a four-fold (8.3 +/- 2.6 ng/ mg protein) increase (P < 0.05) in transgene expression compared to either group A or B. In all groups, pneumocytes were transduced most frequently. The degree of inflammation correlated positively with the extent of transgene expression (r = 0.75, P < 0.01). The efficiency of vascular gene delivery to transplanted lungs can be improved by retrograde delivery of the vector via the pulmonary vein. Transgene expression predominates in pneumocytes following both antegrade and retrograde delivery. The severity of inflammation in the transplanted lung appears to correlate with the extent of transgene expression.

Adenoviridae↗
PubMed2000

A feline model of experimentally induced islet amyloidosis.

The study of the pathogenesis of islet amyloidosis and its relationship to the development and progression of type 2 diabetes mellitus has been hampered by the lack of an experimentally inducible animal model. The domestic cat, by virtue of the fact that it is one of the few species that spontaneously develop a form of diabetes mellitus that closely resembles human type 2 diabetes, including the formation of amyloid deposits derived from islet amyloid polypeptide (IAPP), was considered to be an excellent candidate species in which to attempt to develop a nontransgenic animal model for this disease process. To develop the model, 8 healthy domestic cats were given a 50% pancreatectomy, which was followed by treatment with growth hormone and dexamethasone. Once a stable diabetic state was established, cats were randomly assigned to groups treated with either glipizide or insulin at doses appropriate to control hyperglycemia. Cats were maintained on this treatment regimen for 18 months and then euthanized. Based on light microscopic examination of Congo red-stained sections of pancreas, all cats were negative for the presence of islet amyloid at the time of pancreatectomy. At the end of the study all 4 glipizide-treated cats had islet amyloid deposits, whereas only 1 of 4 insulin-treated cats had detectable amyloid. In addition, the glipizide treated cats had threefold higher basal and fivefold higher glucose-stimulated plasma IAPP concentrations than insulin-treated cats, suggesting an association between elevated IAPP secretion and islet amyloidosis. Blood-glycosylated hemoglobin concentrations were not significantly different between the two treatment groups. This study documents for the first time an inducible model of islet amyloidosis in a nontransgenic animal.

Amyloid↗
PubMed2000

eNOS gene transfer to vascular smooth muscle cells inhibits cell proliferation via upregulation of p27 and p21 and not apoptosis.

OBJECTIVE: Smooth muscle cell (SMC) proliferation is a critical component of vascular diseases such as atherosclerosis and restenosis. Nitric oxide (NO) donors and gene transfer of endothelial nitric oxide synthase (eNOS) have been shown to inhibit SMC proliferation. NO may cause this effect by delaying cell cycle progression and/or induction of apoptosis. The aim of the current study was to examine the mechanism of eNOS-mediated inhibition of SMC proliferation. In addition, the effect of eNOS expression in vascular SMCs on the expression of the cyclin dependent kinase inhibitors, p27 and p21 was examined. METHODS: SMCs were transduced with an adenoviral vector encoding eNOS (AdeNOS) or beta-galactosidase (Ad beta Gal) at a multiplicity of infection of 100. Non-transduced cells served as additional controls. Transgene expression was sought by NADPH diaphorase staining, immunohistochemistry and Western Blotting. Functionality of the recombinant protein was assessed by measurement of cGMP. Cell cycle analysis was performed by flow cytometry and p27 and p21 expression were studied by western blot analysis. Apoptosis was sought by Annexin V staining and DNA laddering. RESULTS: eNOS expression was detected in transduced SMCs. cGMP levels were increased in eNOS-transduced compared to control cells. Expression of eNOS in SMCs resulted in a delay in cell cycle progression and upregulation of p27 and p21. There was no increase in apoptosis detected in eNOS transduced cells after 24 or 72 h. CONCLUSION: eNOS gene transfer to vascular SMCs inhibits cell proliferation via upregulation of p27 and p21 resulting in a delay in cell cycle progression.

Animals↗
PubMed2000

Highly efficient ex vivo gene transfer to the transplanted heart by means of hypothermic perfusion with a low dose of adenoviral vector.

BACKGROUND: Hypothermic conditions required for donor heart preservation may reduce gene-transfer efficiency. Experiments were designed to determine whether a perfusion technique could improve the efficiency of gene transfer to donor hearts. METHODS: An adenoviral vector encoding beta-galactosidase (3.5 x 10(8) plaque-forming units) was infused into explanted rat hearts under 4 conditions (each n = 6): (1) the virus was diluted in 350 microL of University of Wisconsin solution and infused as a high-pressure bolus into the coronary arteries of donor hearts through the aortic root; (2) the virus was diluted in 5 mL of University of Wisconsin solution and circulated by means of a peristaltic pump (flow, 0.75 mL/min) through the vasculature of the donor heart for 30 minutes; (3) 5 mL of viral solution was circulated as for group 2 for 15 minutes; and (4) 5 mL of viral solution was circulated for 5 minutes at a flow rate of 2.4 mL/min. Transduced hearts were transplanted into the abdomen of syngeneic rats, and transgene expression was assessed by means of immunoassay 4 days later. RESULTS: The median beta-galactosidase content was (1) 45.0 ng/mg protein (25th-75th percentile, 33-73 ng/mg), (2) 640 ng/mg protein (25th-75th percentile, 614-878 ng/mg), (3) 493.8 ng/mg protein (25th-75th percentile, 456-527 ng/mg), and (4) 503.3 ng/mg protein (25th-75th percentile, 475-562 ng/mg; P <.01 for group 2 vs group 1, and P <.05 for groups 3 and 4 vs group 1). Transgene expression was predominantly in myocytes and favored the subepicardial region of the right ventricle. CONCLUSION: Hypothermic perfusion of the donor heart with an adenoviral vector resulted in efficient transgene expression compared with that induced by a single bolus injection.

Adenoviridae↗
PubMed2000

The development of immunoassays to four biological threat agents in a bidiffractive grating biosensor.

A critical need exists for a field deployable biosensor to detect environmental infectious agents in collected air samples rapidly, with sensitivity and specificity approaching that of standard laboratory procedures. The ideal sensor would analyze unknown samples in minutes, have programmable operation for unattended sample analysis, and be capable of multiple agent analysis for a number of agents. The goal of this project was to further the development of the bidiffractive grating biosensor (BDG) created through collaboration between Battelle Memorial Institute (BMI), Hoffman LaRoche (HLR), and the Naval Medical Research Command (NMRC). This manuscript details the development, optimization, and evaluation of this device as a potential field deployable biosensor. Well-characterized immunochemical reagents developed by the Biological Defense Research Department (BDRD) at NMRI were employed to develop assays in the BDG. These results were compared to those obtained with antigen capture enzyme linked immunosorbent assays (ELISAs). Four separate antigens were evaluated: Staphylococcus aureus enterotoxin B (SEB), ricin (RIC), Francisella tularensis (FT), and Clostridium botulinum toxin (BOT).

Biological Warfare↗
PubMed2000

Leg length discrepancy--an experimental study of compensatory changes in three dimensions using gait analysis.

Patients with leg length discrepancy (LLD) develop compensatory mechanisms, which result in kinematic alterations in the lower limbs and pelvis. We investigated these compensatory mechanisms. Seven normal subjects underwent three-dimensional (3-D) gait analysis using a CODA MPX 30(R) analyser. The subjects were fitted with raises of varying heights to one lower limb and then analysed in static and dynamic modes. Pelvic obliquity was the most common mechanism used to compensate for small degrees of leg length discrepancy up to 2.2 cm, particularly in static standing. With larger degrees of discrepancy, the subjects developed flexion of the knee of the longer leg. Compensatory mechanisms during walking were more complex and included a combination of kinematic changes at the pelvis, knee and ankle. We developed mathematical models for the compensatory mechanisms at all the three levels. We conclude that 3-D gait analysis is a useful tool in the assessment of patients with functional and structural leg length discrepancies.

Adaptation, Physiological↗
PubMed2000

Early diagnosis of bubonic plague using F1 antigen capture ELISA assay and rapid immunogold dipstick.

Plague is still prevalent in more than 20 countries. Two F1 antigen diagnostic assays (an immunocapture ELISA and an immunogold chromatography dipstick) were evaluated using bubo aspirates, serum and urine specimens from patients suspected with plague. The specificity of the two F1 assays was found 100%. Using bacteriology as a gold reference diagnostic assay, 52 patients were Yersinia pestis culture positive and 141 negative. The sensitivity of the F1 ELISA test was 100% in bubo, 52% in serum and 58% in urine specimens. In culture negative patients, the F1 antigen could be found in 10% bubo aspirates, 5% serum and 7% urine specimens of culture negative patients for whom a seroconversion for anti-F1 antibodies was also observed. The sensitivity of the dipstick assay was 98% on bubo aspirates specimens. Compared to the ELISA test, the agreement rate was 97.5% and the correlation coefficient tau = 0.90 (p < 10(-3)). In conclusion, the diagnosis of bubonic plague has to be performed on bubo fluid rather than on serum or urine specimens. Both the F1 ELISA and the dipstick assays are valuable tools for an early diagnosis and for the surveillance of plague.

Antibodies, Monoclonal↗
PubMed2000

Effect of interferon-alpha on CD20 antigen expression of B-cell chronic lymphocytic leukemia.

Chimeric CD20 monoclonal antibody as alternative therapy in relapsed low-grade non-Hodgkin's lymphoma (NHL) has produced responses in nearly 50% of patients. Augmenting CD20 expression on tumor cells and/or inducing its expression may increase the cell kill and effectiveness of antibody therapy. Peripheral blood lymphocytes from 19 patients with B-cell chronic lymphocytic leukemia (B-CLL) were incubated in vitro in the presence of interferon-alpha (IFN-alpha) (500 U/ml and 1,000 U/ml) for 24 and 72 hours. The effect on CD20 expression was studied by flow cytometry. The differences in the percentage positivity, the mean fluorescence intensity (MFI), and the product of percentage positivity and MFI were used to assess upregulation. There was a significant upregulation of CD20 expression on B cells seen at both concentrations after 24-hour priming (p < 0.01). B-CLL cells cultured for 72 hours in the presence of IFN-alpha also showed upregulation of CD20 expression; however, the degree of upregulation was much lower than that seen at 24 hours. There was no statistically significant increase in CD20 antigen expression on normal lymphocytes following cytokine exposure. These results suggest that IFN-alpha priming may augment the effectiveness of antibody therapy by directly upregulating CD20 antigen expression in addition to its indirect action through effector cells of the host.

Antigens, CD↗
PubMed2000

Adenovirus-mediated gene transfer to human cerebral arteries.

Gene therapy is being investigated as a putative treatment option for cardiovascular diseases, including cerebral vasospasm. Because there is presently no information regarding gene transfer to human cerebral arteries, the principal objective of this study was to characterize adenovirus-mediated expression and function of recombinant endothelial nitric oxide synthase (eNOS) gene in human pial arteries. Pial arteries (outer diameter 500 to 1,000 microm) were isolated from 30 patients undergoing temporal lobectomy for intractable seizures and were studied using histologic staining, histochemistry, electron microscopy, and isometric force recording. Gene transfer experiments were performed ex vivo using adenoviral vectors encoding genes for bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVLacZ). In transduced arteries, studied 24 hours after exposure to vectors, expression of recombinant beta-galactosidase and eNOS was detected by histochemistry, localizing mainly to the adventitia (n = 4). Immunoelectron microscopy localized recombinant eNOS in adventitial fibroblasts. During contractions to U46619, bradykinin-induced relaxations were significantly augmented in AdCMVeNOS-transduced rings compared with control and AdCMVLacZ-transduced rings (P < 0.01; n = 6). The NOS inhibitor L-nitroarginine methylester (L-NAME) caused significantly greater contraction in AdCMVeNOS-transduced rings (P < 0.001; n = 4) and inhibited bradykinin-induced relaxations in control and transduced rings (P < 0.001; n = 6). The current findings suggest that in AdCMVeNOS-transduced human pial arteries, expression of recombinant eNOS occurs mainly in adventitial fibroblasts where it augments relaxations to NO-dependent agonists such as bradykinin. Findings from the current study might be beneficial in future clinical applications of gene therapy for the treatment or prevention of cerebral vasospasm.

Adenoviridae↗
PubMed2000

Detection of Francisella tularensis in biological specimens using a capture enzyme-linked immunosorbent assay, an immunochromatographic handheld assay, and a PCR.

The early detection of Francisella tularensis, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease. Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of Francisella tularensis subsp. holarctica and Francisella tularensis subsp. tularensis. No cross-reactivity with Francisella tularensis subsp. novicida, Francisella philomiragia, and a panel of other possibly related bacteria, including Brucella spp., Yersinia spp., Escherichia coli, and Burkholderia spp., was observed. The detection limit of the assay was 10(3) to 10(4) bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA. In addition, a novel immunochromatographic membrane-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of F. tularensis, were used in this study. Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that of the PCR was about 10 times higher. All three techniques were successfully applied to detect F. tularensis in tissue samples of European brown hares (Lepus europaeus). Whereas all infected samples were recognized by the cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with F. tularensis, especially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.

Antibodies, Monoclonal↗
PubMed2000

B(1) and B(2) bradykinin receptors on adventitial fibroblasts of cerebral arteries are coupled to recombinant eNOS.

Our previous ex vivo and in vivo studies reported that expression of the recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts recovers NO production in arteries without endothelium in response to bradykinin. The present study was designed to characterize subtypes of bradykinin receptors on adventitial fibroblasts coupled to the activation of recombinant eNOS. Endothelium-denuded segments of canine basilar arteries were transduced with beta-galactosidase (beta-Gal) gene or eNOS gene ex vivo, using a replication-defective adenoviral vector (10(10) plaque-forming units/ml) for 30 min at 37 degrees C. Twenty-four hours later, isometric force recording or cGMP measurement was carried out. B(1) bradykinin receptor agonist (des-Arg(9)-bradykinin, 10(-10)-10(-8) mol/l) did not significantly affect vascular tone in control or beta-Gal gene-transduced canine basilar arteries without endothelium. In contrast, this agonist caused concentration-dependent relaxations in recombinant eNOS gene-transduced arteries without endothelium. Relaxations to B(1) receptor agonist in the eNOS arteries were abolished by B(1) receptor antagonist (des-Arg(9)-[Leu(8)]bradykinin, 6 x 10(-9) mol/l) but not by B(2) receptor antagonist (Hoe-140, 5 x 10(-8) mol/l). Bradykinin did not significantly alter vascular tone in control or beta-gal arteries without endothelium, whereas this peptide (10(-11)-10(-8) mol/l) induced concentration-dependent relaxations, as well as an increase in cGMP formation in endothelium-denuded eNOS-transduced arteries. Stimulatory effects of bradykinin were prevented in the presence of a B(2) receptor antagonist but not in the presence of a B(1) receptor antagonist. B(1) and B(2) receptor antagonists had no effect on relaxations to substance P, confirming the selectivity of the compounds. Our results suggest that B(1) and B(2) bradykinin receptors are coupled to activation of recombinant eNOS expressed in adventitial fibroblasts.

Animals↗
PubMed2000

Effects of recombinant eNOS gene expression on reactivity of small cerebral arteries.

Resistance arteries are an important target for vascular gene therapy because they play a key role in the regulation of tissue blood flow. The present study was designed to determine the effects of recombinant endothelial (e) nitric oxide synthase (NOS) gene expression on vasomotor reactivity of small brain stem arteries (internal diameter, 253 +/- 2.5 microm). Arterial rings were exposed ex vivo to an adenoviral vector (10(9) and 10(10) plaque-forming units/ml) encoding eNOS gene or beta-galactosidase gene. Twenty-four hours after transduction, vascular function was examined by isometric force studies. Transgene expression was evident mainly in adventitia. In arteries with endothelium transduced with eNOS gene but not with control beta-galactosidase gene, relaxations to bradykinin and substance P were significantly augmented. Removal of endothelium abolished relaxations to bradykinin and substance P in control and beta-galactosidase arteries. However, in endothelium-denuded arteries transduced with recombinant eNOS, bradykinin and substance P caused relaxations that were abolished in the presence of the NOS inhibitor N(G)-nitro-L-arginine methyl ester. In control arteries, endothelium removal augmented relaxations to the nitric oxide donors sodium nitroprusside and diethylamine NONOate. This augmentation was absent in eNOS gene-transduced arteries without endothelium. Our results suggest that, in small brain stem arteries, expression of recombinant eNOS increases biosynthesis of nitric oxide. Adventitia of small arteries is a good target for expression of recombinant eNOS. Genetically engineered adventitial cells may serve as a substitute source of nitric oxide in cerebral arteries with dysfunctional endothelium.

Animals↗
PubMed2000

Inhibition of adrenal cell aldosterone synthesis by endogenous nitric oxide release.

Adrenal zona glomerulosa (ZG) cells do not contain nitric oxide (NO) synthase (NOS). We conferred endothelial NOS activity onto adrenal ZG cells through transduction with a recombinant adenovirus encoding the endothelial NOS gene (AdeNOS) to determine the effect of endogenous NO on aldosterone synthesis. A 135-kDa protein band immunoreactive to anti-endothelial NOS antibody was observed in Western blots of AdeNOS-transduced ZG cells but not in control cells or cells transduced with adenovirus encoding the beta-galactosidase gene (AdbetaGal). Nitrate/nitrite production in AdeNOS-transduced ZG cells increased from 0.15+/-0.01 to 0.27+/-0.01 micromol/L after stimulation with 1 nmol/L angiotensin II. The treatment of AdeNOS-transduced cells with 30 micromol/L L-nitro-arginine decreased angiotensin II-stimulated nitrite production from 0.27+/-0. 01 to 0.17+/-0.01 micromol/L. Basal and angiotensin II-stimulated nitrite production was not increased in AdbetaGal-transduced or control cells. AdeNOS-transduced cells demonstrated diaminofluorescein-2 diacetate fluorescence, which was blocked by pretreatment with L-nitro-arginine. Angiotensin II-stimulated aldosterone synthesis decreased from 5123+/-177 pg/mL in AdbetaGal-transduced ZG cells to 72+/-27 pg/mL in AdeNOS-transduced cells. Treatment with the NOS inhibitor thiocitrulline (30 micromol/L) increased angiotensin II-stimulated aldosterone synthesis to 2158+/-45 pg/mL after AdeNOS transduction. These data demonstrate that adenovirus-mediated gene transfer of eNOS in ZG cells results in the expression of active endothelial NOS enzyme and that this endogenous NO production by ZG cells decreases aldosterone synthesis.

Adenoviridae↗
PubMed2000

In vivo gene transfer of endothelial nitric oxide synthase to carotid arteries from hypercholesterolemic rabbits enhances endothelium-dependent relaxations.

BACKGROUND AND PURPOSE: Hypercholesterolemia is associated with abnormal endothelium-dependent vasorelaxation due to decreased nitric oxide bioavailability. Our aim was to examine the effect of adenovirus-mediated gene transfer of endothelial nitric oxide synthase (eNOS) to the hypercholesterolemic rabbit carotid artery in vivo. In addition, we examined whether adenovirus-mediated gene transfer was associated with vascular dysfunction. METHODS: Rabbits were fed a 1% cholesterol diet for 4 weeks followed by a 0.5% cholesterol diet for 6 weeks. Vascular reactivity was assessed in nontransduced carotid arteries from chow- and cholesterol-fed animals. In addition, carotid arteries were surgically isolated, and 2 separate doses of adenoviral vectors encoding eNOS or beta-galactosidase (AdbetaGal) on the contralateral side were delivered to the lumen (1x10(10) and 5x10(10) pfu/mL). RESULTS: Abnormal acetylcholine-mediated endothelium-dependent vasorelaxation was detected in the carotid artery from cholesterol-fed animals, whereas responses to calcium ionophore A23187 and diethylamine NONOate were normal. Vascular reactivity was similar in nontransduced and AdbetaGal-transduced hypercholesterolemic vessels. In vessels transduced with eNOS, transgene expression was demonstrated by immunostaining in both the endothelium and the adventitia and by Western blot analysis. High-dose but not low-dose eNOS gene transfer enhanced endothelium-dependent relaxation in vessels from cholesterol-fed rabbits. CONCLUSIONS: Adenovirus-mediated gene transfer of eNOS to carotid arteries of cholesterol-fed animals improves endothelium-dependent relaxation when an optimal viral titer is administered.

Animals↗
PubMed2000

Familial splenomegaly: macrophage hypercatabolism of lipoproteins associated with apolipoprotein E mutation [apolipoprotein E (delta149 Leu)].

Splenomegaly with sea-blue histiocytes is not associated with dyslipidemia, except in severe cases of hypertriglyceridemia, Tangier disease, or lecithin cholesterol acyltransferase deficiency. We describe two kindreds in which the sea-blue histiocyte syndrome was associated with an apoE variant in the absence of severe dyslipidemia. Both patients presented with mild hypertriglyceridemia and splenomegaly. After splenectomy both patients developed severe hypertriglyceridemia. Pathological evaluation of the spleen revealed the presence of sea-blue histiocytes. A mutation of apoE was demonstrated, with a 3-bp deletion resulting in the loss of a leucine at position 149 in the receptor-binding region of the apoE molecule [apoE (delta149 Leu)]. Although both probands were unrelated, they were of French Canadian ancestry, suggesting the possibility of a founder effect. In summary, we describe two unrelated probands with primary sea-blue histiocytosis who had normal or mildly elevated serum triglyceride concentrations that markedly increased after splenectomy. In addition, we provide evidence linking the syndrome to an inherited dominant mutation in the apoE gene, a 3-bp deletion on the background of an apoE 3 allele that causes a derangement in lipid metabolism and leads to splenomegaly in the absence of severe hypertriglyceridemia.

Adult↗