Biomedical subjects

Timothy O'Brien

Publications and source records attributed to Timothy O'Brien.

At least 19 recordsLinked to original sources

PubMed2006

The characteristics of gait in Charcot-Marie-Tooth disease types I and II.

Certain typical gait characteristics such as foot-drop and foot supination are well described in Charcot-Marie-Tooth disease. These are directly related to the primary disease and due to the weakness of ankle dorsiflexors and everters characteristic of this hereditary neuropathy. We analysed 16 subjects aged 8-52 years old (11 with type I, 5 with type II Charcot-Marie-Tooth disease) using three-dimensional gait analysis and identified kinematic features previously unreported. These patients showed a combination of tight tendo achillei, foot-drop, failure of plantar flexion and increased foot supination, but also presented with excessive internal rotation of the knee and/or tibia, knee hyperextension in stance, excessive external rotation at the hips and decreased hip adduction in stance (typical of a broad based gait). These proximal features could have been an adaptation to or consequence of the disrupted ankle and foot biomechanics, however a direct relation to the neuropathy is also possible since sub-normal muscle power was observed at the proximal levels in most subjects on both manual testing and kinetic analysis. Gait analysis is a useful tool in defining the characteristic gait of patients with Charcot-Marie-Tooth disease.

Adolescent↗

PubMed2006

Optimization of a fibrin scaffold for sustained release of an adenoviral gene vector.

One of the major limitations of adenovirus as an efficient gene delivery vehicle is its transient nature of transgene expression. At present, no biodegradable release system exists that permits the sustained release of gene delivery. This study aimed to optimize a biodegradable fibrin scaffold for sustained adenoviral mediated gene delivery. Scaffold formulations were fabricated incorporating an adenoviral vector encoding beta-galactosidase, and each scaffold was incubated in elution fluid. The level of transfection of cells was measured to assess virus diffusion from each scaffold at different time periods. Results showed that viral particles were present in the elution fluid and had the ability to transfect cells at different time points. Depending on the scaffold formulation, variable rates of diffusion were seen. Analysis of scaffold microarchitecture using stereo pairs showed that variable pore depth is seen in different fibrin scaffold concentration, which may be a determining factor in the diffusion rate. It was concluded that sustained release of the adenovirus could be attained up to 192 h at the optimal concentration of 60 mg/mL fibrinogen and 4 IU thrombin.

Adenoviridae↗

PubMed2006

Adenoviral-mediated gene transfer of nitric oxide synthase isoforms and vascular cell proliferation.

OBJECTIVE: Many vascular diseases are associated with reduced nitric oxide (NO) bioavailability. Nitric oxide synthase (NOS) gene therapy to the vasculature is a possible treatment for vascular disease as a means of increasing NO bioavailability, and this may be achieved using any of the NOS isoforms. The aim of our study was to compare the effects of adenoviral-mediated overexpression of the most commonly used NOS isoforms eNOS and iNOS on vascular cell proliferation. METHODS: Human coronary artery smooth muscle cells (HCSMCs) and human umbilical vein endothelial cells (HUVECs) were transduced with adenoviral vectors encoding eNOS or iNOS at a multiplicity of infection of 100. Control cells were exposed to AdNull (empty vector) or diluent alone. Transgene expression was sought by Western blotting. The Greiss assay was used to measure nitrite levels. Cell proliferation was assessed by cell counting on days 0, 3 and 6. Apoptosis was sought using FACS analysis. Angiogenesis was measured using a commercially available in vitro kit. RESULTS: Expression of both isoforms was detected in transduced cells by Western blot at all three time points. NOS transduction resulted in increased nitrite levels with higher levels seen in iNOS- compared to eNOS-transduced cells. Cell proliferation was diminished in AdeNOS- and AdiNOS-transduced cells compared with non-transduced cells on days 3 and 6 in both HCSMCs and HUVECs. Apoptosis was not detected in either cell line with either of the isoforms at any timepoint studied. Both eNOS and iNOS gene transfer caused a reduction in angiogenesis. CONCLUSIONS: NOS gene transfer to both endothelial and vascular smooth muscle cells is antiproliferative and antiangiogenic. The biological effect is identical with both isoforms and there is no evidence to support a differential effect on endothelial and vascular smooth muscle cell biology.

Adenoviridae↗

PubMed2006

Endothelial progenitor cells: diagnostic and therapeutic considerations.

Endothelial progenitor cells (EPCs) may be defined as adherent cells derived from peripheral blood- or bone marrow-derived mononuclear cells demonstrating acLDL uptake and isolectin-binding capacity. The number of circulating EPCs inversely correlates with the number of cardiovascular risk factors and is reduced in cardiovascular disease. This measurement may therefore serves as a surrogate marker for cardiovascular disease risk. EPC numbers can be modified by various means. However, the effectiveness of risk-factor modification on EPC number and function is currently unknown. Furthermore, EPCs may be used as a potential therapy for a variety of vascular disease states including ischaemia, restenosis and pulmonary hypertension. This review provides an update on multiple factors that affect EPC number as well as highlighting the potential use of EPCs as a novel marker of vascular dysfunction. Furthermore, potential gene- and/or EPC-based approaches to a number of vascular disease states are explored.

Animals↗

PubMed2006

Gene-eluting stents: comparison of adenoviral and adeno- associated viral gene delivery to the blood vessel wall in vivo.

Gene-eluting stents are being evaluated in animals as an alternative approach to inhibiting in-stent restenosis. Adeno-associated virus type 2 (AAV2) and adenovirus are commonly used for gene transfer applications. We tested the hypothesis that these vectors can achieve prolonged and localized gene delivery to the vessel wall, using stents as delivery platforms. AdbetaGal (5 x 10(9) plaque-forming units) and AAV2betaGal (5.3 x 10(9) DNase-resistant particles) were used to coat BiodivYsio stents with matrix HI coating (Abbott Vascular Devices, Galway, Ireland). After balloon injury, external iliac arteries of New Zealand White rabbits were stented. The reverse transcription-polymerase chain reaction was used to assess viral spread. Expression of LacZ was demonstrated with both vectors at five time points (3, 7, 14, 21, and 28 days). In the adenovirus group the median percentage of cells expressing the transgene on day 3 was 2.73%, which increased to a median expression of 7.31% at 28 days (p > 0.05). Expression was localized to medial cells on day 3, but was observed predominantly in neointimal cells on day 28. In the AAV group, day 3 expression was 5.78%, which decreased to 2.12% on day 28 (p = 0.05). No systemic dissemination of virus was seen in any group. Adenovirus- and AAV2-coated stents can be used to deliver genes to the blood vessel wall for up to 28 days.

Adenoviridae↗

PubMed2005

Design and analysis of drug combination experiments.

In this paper we present and discuss a novel, simple and easy to implement parametric modeling approach to assess synergy. An extended three parameter log-logistic model is used to analyse the data and calculate confidence intervals of the interaction indices. In addition the model corrects for the bias due to plate-location effects. The analysis is performed with PROC NLMIXED and SAS-code is provided. The approach is illustrated using data coming from an oncology study in which the inhibition effect of a combination of two compounds is studied using 96-well plates and a fixed-ratio design.

Algorithms↗

PubMed2005

Analysis of superoxide anion production in tissue.

Endothelial production of oxygen free radicals, especially superoxide anion (O(2)-), is an important mechanism of vascular dysfunction in hypertension. Overproduction of oxygen free radicals, mainly O(2)- occurs in human hypertension and in a wide variety of animal models. Thus, analysis of O(2)- generation represents a useful tool for identifying oxidative stress in hypertension. Among the methods used for O(2)- detection, the chemiluminescent probe lucigenin has been widely shown to be a useful method for detecting and quantifying the O(2)- formation. On the other hand, staining by the oxidative fluorescent probe dihydroethidine, which is freely permeable to cell membranes, is suitable to monitor in situ production of O(2)- and to provide a reliable marker of its intracellular presence. Dihydroethidine is oxidized in the presence of O(2)- to a fluorescent marker product, which is rapidly intercalated into DNA. Thus, nuclei are the primary fluorescent structures labeled. By simply incubating experimental samples in the presence of dihydroethidine followed by analysis of fluorescence, this method allows rapid and specific detection of intracellular oxidative stress due to superoxide anion generation.

Animals↗

PubMed2005

Gene transfer in endothelial dysfunction and hypertension.

Gene transfer represents a method for treatment of several cardiovascular disorders, including endothelial dysfunction and hypertension. For effective and safe gene therapy in vascular disease, a suitable therapeutic gene needs to be identified and delivered to the vasculature by appropriate delivery devices. In this chapter, we review the different vectors used, both viral and nonviral, suitable genes identified, and associated delivery devices. Several genes have been identified with a view to improve endothelial dysfunction, and we have elaborated the advantages and disadvantages of these approaches. Strategies to treat hypertension, both systemic and pulmonary, have also been described. The optimal vector has not yet been discovered although a wide variety of choices is available, each with properties that may render it suitable for specific applications. The individual characteristics of these vectors are described in relation to the proposed therapeutic paradigm. Although there are several unanswered questions in this arena, the future application of gene transfer technology to diseases of the vasculature holds significant promise.

Endothelium, Vascular↗

PubMed2004

The serine protease stratum corneum chymotryptic enzyme (kallikrein 7) is highly overexpressed in squamous cervical cancer cells.

OBJECTIVE: To determine whether the Stratum Corneum Chymotryptic Enzyme (SCCE), a novel serine protease known to contribute to the cell shedding process by catalyzing the degradation of intercellular cohesive structures at the skin surface, is overexpressed in human cervical tumors. METHODS: SCCE expression was evaluated in 18 cervical cancer cell lines (i.e., 10 primary and 8 established cell lines) as well as in 8 normal cervical keratinocyte cultures by RT-PCR. In addition, SCCE expression was evaluated by immunohistochemistry on paraffin-embedded tumor tissue. RESULTS: Normal cervical keratinocytes did not express SCCE. In contrast, 50% of the primary and 50% of the established cervical cancer cell lines expressed SCCE by RT-PCR. Eighty percent (i.e., four of five) of primary squamous cervical tumors and 20% (i.e., one of five) of primary adenocarcinomas expressed SCCE. Five out of five (100%) of the patients harboring SCCE-positive tumors were found to have metastatic involvement of the pelvic tumor draining lymph nodes. Immunohistochemistry staining of paraffin-embedded cervical cancer specimens confirmed SCCE expression in tumor cells and its absence on normal cervical epithelial cells. CONCLUSION: Squamous cervical cancer expressed high levels of SCCE, suggesting that this protease may play an important role in invasion and metastasis. Because SCCE appears only in abundance in tumor tissue and contains a secretion signal sequence, suggesting that SCCE is secreted, it may prove to be a useful diagnostic/prognostic tool for the detection of metastatic or recurrent disease or as a novel molecular target for cervical cancer therapy.

Adult↗

PubMed2004

Textile dye allergic contact dermatitis following paraphenylenediamine sensitization from a temporary tattoo.

Textile dye allergy is frequently caused by azo dyes, which can cross-react with structurally similar compounds, including paraphenylenediamine. A case of allergic contact dermatitis to azo textile dyes, presenting principally as a sock dermatitis, is presented. The patient also gave a history of an episode of scalp dermatitis consistent with contact allergy to paraphenylenediamine in hair dye. It is proposed that paraphenylenediamine sensitization from a temporary skin tattoo may have been the primary sensitizing event for these reactions.

Adult↗

PubMed2003

The novel serine protease tumor-associated differentially expressed gene-15 (matriptase/MT-SP1) is highly overexpressed in cervical carcinoma.

BACKGROUND: Tumor-associated differentially expressed gene-15 (TADG-15/matriptase/MT-SP1) is a novel transmembrane serine protease involved in numerous biologic processes, including activation of growth and angiogenic factors and degradation of extracellular matrix components. To assess the value of TADG-15 as a possible marker for tumor detection and/or as a target for therapeutic intervention, the authors investigated the frequency of expression of TADG-15 in human cervical tumors. METHODS: TADG-15 expression was evaluated in 19 cervical carcinoma cell lines (i.e., 11 primary tumor cell lines and 8 established cell lines) and in 8 normal cervical keratinocyte control cultures using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, to validate gene expression data at the protein level, TADG-15 expression was evaluated by immunohistochemistry on paraffin embedded tissue from which all 11 primary tumor cell lines were established. RESULTS: TADG-15 was expressed at high levels in 8 of 11 (73%) primary cervical carcinoma cell lines and in 6 of 8 (75%) established cervical carcinoma cell lines by RT-PCR. Expression of TADG-15 was found in 6 of 6 (100%) primary squamous cell cervical carcinomas, whereas 2 of 5 (40%) primary adenocarcinomas expressed TADG-15. In contrast, none of the normal cervical keratinocyte control cultures (n = 4) or flash-frozen normal cervical biopsy specimens (n = 4) expressed TADG-15. Immunohistochemistry staining of paraffin embedded cervical carcinoma specimens confirmed TADG-15 expression in tumor cells and its absence on normal cervical epithelial cells. CONCLUSIONS: Cervical carcinoma cells expressed high levels of TADG-15, suggesting that this protease may play an important role in invasion and metastasis. Because TADG-15 appears only in abundance in squamous tumor tissue and contains a proteolytic cleavage site, suggesting that the TADG-15 protease domain is released, it may prove to be a useful diagnostic tool for the early detection of recurrent/persistent cervical carcinoma after standard treatment or as a novel molecular target for therapy in patients with cervical carcinoma.

Adenocarcinoma↗

PubMed2003

In vivo expression and function of recombinant GTPCH I in the rabbit carotid artery.

Tetrahydrobiopterin (BH4) is an essential co-factor for endothelial nitric oxide synthase enzymatic activity. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme in BH4 synthesis. This study set out to test the hypothesis that in vivo gene transfer of GTPCH I to endothelial cells could increase bioavailability of BH4, enhance biosynthesis of nitric oxide and thereby enhance endothelium-dependent relaxations mediated by nitric oxide. In vivo gene transfer was carried out by adenovirus (Ad)-mediated delivery into rabbit carotid arteries. Each artery was transduced by 20-min intraluminal incubation of 10(9) plaque-forming units of Ad-encoding GTPCH I (AdGTPCH) or beta-galactosidase as a control. The rabbits were euthanized 72 h later, and vasomotor function of isolated arteries was assessed by isometric force recording. GTPCH I enzymatic activity, BH4, and oxidized biopterin levels were detected with the use of HPLC, and cGMP was measured with the use of radioimmunoassay. Expression of recombinant proteins was detected predominantly in endothelial cells. Both GTPCH I activity and BH4 levels were increased in arteries transduced with AdGTPCH. However, contraction to phenylephrine (10(-5) to 10(-9) M), endothelium-dependent relaxation to acetylcholine (10(-5) to 10(-9) M) and cGMP levels were not significantly affected by increased expression of GTPCH I. Our results suggest that expression of GTPCH I in vascular endothelium in vivo increases intracellular concentration of BH4. However, under physiological conditions, it appears that this increase does not affect nitric oxide production in endothelial cells of the carotid artery.

Acetylcholine↗

PubMed2003

In vivo gene transfer of inducible nitric oxide synthase to carotid arteries from hypercholesterolemic rabbits.

BACKGROUND AND PURPOSE: Hypercholesterolemia is associated with endothelial dysfunction. Inducible nitric oxide synthase (iNOS) is upregulated in atherosclerotic vessels. However, its role in the regulation of vascular function is not completely understood. We examined the effect of adenovirus-mediated gene transfer of iNOS to the hypercholesterolemic rabbit carotid artery in vivo. METHODS: Rabbits were fed a high-cholesterol or chow diet for 10 weeks. Two doses (1x10(8) and 1x10(9) plaque-forming units [pfu]/mL) of adenoviral vectors encoding iNOS (AdiNOS) or beta-galactosidase (Ad(beta)gal) were luminally delivered to the carotid arteries from chow- and cholesterol-fed animals. Vascular reactivity and superoxide levels were assessed in Ad(beta)gal- and AdiNOS-transduced vessels from chow- and cholesterol-fed animals after 3 days. RESULTS: Endothelium-dependent vasorelaxation was impaired in the carotid artery from cholesterol-fed animals. In AdiNOS-transduced arteries, transgene expression was demonstrated by positive immunostaining in the endothelium. Transduction with low-dose (1x10(8) pfu/mL) AdiNOS did not affect vascular reactivity in arteries from chow- and cholesterol-fed animals. In contrast, high-dose (1x10(9) pfu/mL) AdiNOS significantly reduced endothelium-dependent relaxation in vessels from cholesterol- but not chow-fed rabbits. After both low- and high-dose iNOS gene transfer, levels of O2*(-) were significantly (P<0.05) elevated in carotid arteries from cholesterol-fed animals. Incubation with an O2*(-) scavenger did not reverse vascular dysfunction in these arteries. CONCLUSIONS: Adenoviral-mediated overexpression of iNOS results in increased production of O2*(-) in carotid arteries from cholesterol- but not chow-fed animals. High-dose AdiNOS gene transfer is associated with reduced endothelium-dependent and -independent relaxation in vessels from cholesterol-fed animals.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed2003

Rituximab treatment for relapsed autoimmune hemolytic anemia in Evans syndrome.

A case is reported of idiopathic relapsed autoimmune hemolytic anemia successfully treated with rituximab. A 43-year-old white male patient with past medical history of Evans syndrome was found to have recurrent autoimmune hemolytic anemia. Previous treatments included steroids, splenectomy, intravenous immunoglobulin, plasmapheresis, staphylococcal Protein A immunoadsorption (Prosorba column), and chemotherapeutic agents (cytoxan and vincristine). Rituximab was given weekly at 375 mg/m2 for 4 doses. The drug was well tolerated and the patient remains in remission 9 months after completion of therapy.

Adult↗

PubMed2003

Mastopathy and diabetes.

Sclerosing lymphocytic lobulitis or "diabetic mastopathy" is strongly associated with type 1 diabetes but may occur occasionally in its absence. It is characterized by keloid-type fibrosis, lymphocytic lobulitis, and perivasculitis and epithelioid cells. Infiltrating cells are predominantly B lymphocytes. It may be associated with retinopathy and neuropathy, but more research is needed to verify this association. The etiopathogenesis is unknown and the disorder does not seem to predispose to breast carcinoma or lymphoma.

Adult↗

PubMed2003

Multigene adenoviral therapy for the attenuation of ischemia-reperfusion injury after preservation for cardiac transplantation.

OBJECTIVE: The protective effect of adenovirus-mediated ex vivo multigene transfer with superoxide dismutase, a free radical scavenger, and nitric oxide, a vasodilator with anti-inflammatory properties, was examined in the rat heart during experimental ischemia-reperfusion mimicking preservation for cardiac transplantation. METHODS: Donor rat hearts (n = 6 per group) were perfused with solution containing adenoviral vector carrying genes for beta-galactosidase (group A), endothelial nitric oxide synthase (group B), manganese superoxide dismutase (group C), or both endothelial nitric oxide synthase and manganese superoxide dismutase (group D). Hearts were then implanted heterotopically into the abdomens of recipient rats. Four days later, transplanted hearts were collected, connected to a Langendorff perfusion apparatus, and subjected to 6 hours of ischemia followed by 1 hour of reperfusion. Cardiac function was evaluated with an intraventricular balloon at the beginning of Langendorff perfusion and after ischemia-reperfusion. RESULTS: Effective gene transfection was confirmed with X-gal staining in group A hearts. Positive immunoreactivity for endothelial nitric oxide synthase, manganese superoxide dismutase, or both was present predominantly in cardiomyocytes in group B, C, and D hearts. Percentage recovery of preischemic left ventricular developed pressure was 62.1% +/- 7.36% in group A; recoveries were increased to 79.6% +/- 6.4%, 86.8% +/- 9.1%, and 79.4% +/- 6.2% in groups B, C, and D, respectively. CONCLUSION: These results indicate that adenoviral gene transfer of manganese superoxide dismutase and endothelial nitric oxide synthase can attenuate myocardial ischemia-reperfusion injury, with the former providing the most significant protection. Combined overexpression of manganese superoxide dismutase and endothelial nitric oxide synthase did not enhance myocardial recovery any further.

Adenoviridae↗

PubMed2003

Near misses in bladder cancer - an airline safety approach to urology.

INTRODUCTION: Traditionally, surgical audit has identified and highlighted the incidence of adverse events complicating a patient's care. The airline industry has taken this concept a step further back by identifying and studying near misses, i.e. events that have the potential to do harm. We have applied this approach to patients with known or suspected bladder cancer. PATIENTS AND METHODS: A prospective study was performed by two urology firms on all patients with known or suspected bladder cancer over a 3-week period. Patients presented to either a central (hub) hospital, or to an associated (spoke) hospital. Four stages in bladder cancer care were considered: (i) diagnostic or check flexible cystoscopy; (ii) admission to hospital prior to TURBT; (iii) peri-operative period; and (iv) first out-patient consultation. A separate proforma, comprising various aspects of management was used for each of these stages of care. If any one criterion was not met, the episode was recorded as a near miss. Near misses were classified as due to capacity limitations in the system, clerical error, equipment failure, clinical error and patient failure. RESULTS: A total of 115 completed episodes were recorded. A near miss was recorded in 65 (56.5%) of all episodes. Capacity limitations accounted for 54%, clinical error for 23%, clerical error for 16%, patient failure for 5% and equipment failure for 2% of all recorded near misses. Of particular note is that near misses relating to diagnosis were more common at the spoke hospital, delayed referral from GPs accounted for more than 25% of clinical error, diagnosis of 5 new bladder tumours was delayed and availability of upper tract imaging was a problem at all phases of patient management. CONCLUSIONS: Near misses are very common in the management of patients with bladder cancer, and their identification should provide a useful framework for identifying potential areas for improvement in patient care.

Ambulatory Care↗

PubMed2002

Inhibitory effect of recombinant iNOS gene expression on vasomotor function of canine basilar artery.

The present study was designed to determine the effect of recombinant inducible nitric oxide (NO) synthase (iNOS) gene expression on vasomotor function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 min at 37 degrees C) to an adenoviral vector [10(7), 10(8), or 10(9) plaque-forming units (pfu)/ml] encoding either the iNOS gene or the beta-galactosidase reporter gene. Twenty-four hours after transduction, Western blot analysis demonstrated expression of iNOS protein only in iNOS (10(9) pfu/ml)-transduced arteries. Immunohistochemical analysis localized iNOS expression predominantly in adventitia. Vascular reactivity of isolated basilar arteries was studied by isometric force recording. Concentration-response curves to UTP (10(-9)-10(-3) M) and diethylaminodiazen-1-ium-1,2-dioate (10(-10)-10(-5) M) were significantly shifted to the right in iNOS gene (10(9) pfu/ml)-transduced rings compared with control and beta-galactosidase-transduced rings (P < 0.05, n = 5-6). Endothelium-dependent relaxation to bradykinin was significantly attenuated in iNOS-transduced rings (P < 0.001, n = 8). The basal level of cGMP and superoxide anion (O(2)(-).) production were elevated in iNOS-transduced rings (P < 0.05, n = 7 for cGMP; P < 0.01, n = 6-9 for O(2)(-). production). Our results suggest that expression of recombinant iNOS in cerebral arteries reduces vasomotor reactivity to both vasoconstrictor and vasodilator agonists. Attenuation of contractions is most likely due to functional antagonism between UTP and cGMP. Reduction of endothelium-dependent relaxation to bradykinin appears to be mediated in part by reduced reactivity of smooth muscle cells to NO.

Animals↗