Evaluation of beta-galactosidase activity in tissue in the presence of blood

The reporter gene for beta -galactosidase is frequently used to determine the efficiency of gene transfer in arteries. However, blood is often present in arterial explants and may compromise the results by the presence of hemoglobin. The light absorption of hemoglobin is similar to the absorption of several colorimetric products of the commonly used beta -galactosidase substrates, including o-nitrophenyl-beta -D-galactopyranoside (ONPG) and chlorophenol red galactopyranoside (CPRG), This may result in false-positive measurements of beta -galactosidase enzyme activity. The aim of this investigation was to determine the most appropriate method for quantification of beta -galactosidase activity in the presence of blood. Colorimetric substrates (ONPG, CPRG) or the chemiluminescent Galacton-Plus substrate were used, and light absorption was measured at different concentrations of erythrocyte extract. Among the beta -galactosidase substrates tested, CPRG was the most appropriate, allowing detection of enzyme activity at concentrations as low as 0.05 mU, independent of blood contamination. Addition of reducer stabilized enzyme activity for at least 5 h. Endogenous beta -galactosidase activity was evaluated and used to correct results. CPRG substrate, in combination with the reducer agent mercaptoethanol, was found to be the optimal reagent for quantifying beta -galactosidase activity in the presence of blood after nonviral in vivo reporter gene transfection, even with a relatively low transfer efficiency. Copyright (C) 2000 S. Karger AG, Basel

Immune-Complex Allergic Vasculitis in Association with the Immune-Complex Allergic Vasculitis in Association with the Development of Transverse Myelitis: A Case Report

A severe vasculitis, probably therapy related, in a sixty-four-year-old man being treated for possible subacute bacterial endocarditis, was associated with the development of transverse myelitis. It is hypothesized that the vasculitis affected the small vessels to the spinal cord in the same way that systemic vasculitis can also cause a transverse myelitis

Use of Nuclear Magnetic Resonance Imaging Angiography to Follow-Up Arterial Remodeling in an Animal Model

Appropriately sized arteries in small animals may be possible models for studying the remodeling process as occurs after arterial balloon injury in humans. Magnetic resonance imaging (MRI) is able to noninvasively image tissue in vivo. To date, small animal angiog raphy models have mostly used research-dedicated instruments and resolution, which are not universally available.Experiments were carried out on a rat aorta model of remodeling in vivo (n=40). Arteries were injured by oversized balloon dilation; control arteries were uninjured. Angiography imaging was performed immediately before sacrifice with an unmodified clinical MRI unit, a 1.5 Tesla MR tomograph with a 20-cm-diameter coil. Longitudinal MRI pictures of the aorta and morphometry of tissue sections to measure luminal and arterial wall areas were analyzed with use of computer-assisted techniques.Comparison of dimensions demonstrated correlation between MRI and histology measurements of the lumen. MRI and morphometry showed a gradual increase in mean luminal area over 6 weeks following injury. The lumen increase correlated with total arterial area and thickness.In this rat aorta model, remodeling documented at histology was followed-up in vivo. The use of such clinical MRI scanners has potential to reduce animal numbers needed to follow-up the remodeling process after therapeutic intervention

Small poly-L-lysines improve cationic lipid-mediated gene transfer in vascular cells in vitro and in vivo

The potential of two small poly-L-lysines ( sPLLs), low molecular weight sPLL ( LMW-L) containing 7 - 30 lysine residues and L18 with 18 lysine repeats, to enhance the efficiency of liposome-mediated gene transfer ( GT) with cationic lipid DOCSPER {[}1,3- dioleoyloxy- 2-( N-5-carbamoyl-spermine)-propane] in vascular smooth muscle cells ( SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA ( 50 100 nm). At high ionic strength, large complexes ( 11 mu m) were observed without any significant differences in particle size between lipoplexes ( DOCSPER/ DNA) and lipopolyplexes ( DOCSPER/ sPLL/ DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/ sPLL/ DNA formulations enhanced GT in vitro up to 5- fold, in a porcine model using local periadventitial application up to 1.5- fold. Both sPLLs significantly increased liposome- mediated GT. Poly-L-lysine L18 was superior to LMW-L since it enabled maximal GT at a 10-fold lower concentration. Thus, sPLLs may serve as enhancers for GT applications in SMCs in vitro and in vivo using local delivery. Copyright (c) 2007 S. Karger AG, Basel

Embolisation of Vertebral Artery Damaged Following Cervical Disc Removal

Introduction: The incidence of vertebral artery (VA) injury during cervical spine surgery is rare. Even though tamponade is effective in many cases, early consultation of an endovascular team is recommended if bleeding cannot be controlled. We report a case of emergent endovascular embolisation of left VA due to iatrogenic injury during anterior cervical disc removal and fusion. Case: A 47-year-old woman was admitted to our emergency department with serious arterial bleeding from the neck only hours after undergoing anterior cervical disc removal and fusion surgery. She was intubated and mechanically ventilated, however hemorrhage could not be successfully controlled by packing with surgical hemostatic agents. Cranial computed tomography, computed tomography of the cervical spine and CT angiography confirmed the suspected diagnosis of injury to the VA. Emergent endovascular embolisation successfully stopped the bleeding. Occlusion of the vessel was achieved by vascular plugging. The patient was discharged from our hospital 14 days after the intervention, receiving a revision surgery of the cervical spine on the day of embolisation. At the date of discharge she presented without any focal neurological deficit. Conclusion: Pre-operative radiographic imaging of the cervical spine should be used routinely to identify anatomic abnormalities of the vertebral arteries. Endovascular embolisation appears to be effective in treating acute iatrogenic dissection of the vertebral arteries

238 A randomized, double-blind placebo-controlled study of NV1FGF gene therapy in critical limb ischemia patients (TAMARIS Study) Rationale, design and baseline patient characteristics

BackgroundPatients with critical limb ischemia (CLI) unsuitable for revascularization have a high rate of amputation and mortality (30% and 25% at 1 year respectively Local gene therapy using plasmid DNA encoding acidic fibroblast growth factor (NV1FGF, riferminogene pacaplasmid) showed promising results in a phase II trial on amputation free survival. This report provides the rationale, design and baseline characteristics of CLI patients enrolled to the pivotal phase III trial (TAMARIS). It also describes baseline characteristics by diabetes status and region of origin.Table: Comparison of 6 modes of ABI calculation to predict the 5-years mortality (abstract 237).ABI mode of calculationHigh/HighMean/HighLow/HighHigh/MeanMean/MeanLow/MeanAUC0.632*0.6200.6150.6100.6180.598Sensitivity with 0.9055.7%56.7%60.4%53.6%60.8%58.8%Specificity with 0.9065.1%62.3%60.2%63.6%58.5%58.6%Optimal cutpoint0.940.970.921.000.970.92Sensitivity for optimal cutpoint63.9%72.2%64.6%72.2%74.2%62.9%Specificity for optimal cutpoint60.3%50.6%58.7%48.3%50.3%57.5%*p<0.05 vs. High/Mean and Low/MeanMethodsAn international, double-blind, placebo-controlled, randomized study included 525 CLI patients worldwide who were unsuitable for revascularization and had non-healing skin lesions, to evaluate whether repeated intramuscular administration of NV1FGF results in reduction of major amputations or deaths at 1 year.ResultsMean age of the population was 70 ± 10 years including 70% males and 53% diabetic patients. Fifty four percent of the population had previous lower extremity revascularization and 22% had previous minor amputation of the index leg. Ninety six percent of patients had an ankle pressure < 70mmHg and/or a toe pressure < 50mmHg or a TcPO2 < 30mmHg. In 94% the index leg had distal occlusive disease affecting arteries below the knee. Statins were prescribed in 54% of patients, and antiplatelet drugs in 80%. Variation in region of origin resulted in only minor demographic imbalance. Patients with diabetes had more risk factors including history of coronary artery disease, but were similar to non-diabetic patients regarding limb haemodynamics and vascular lesions.ConclusionThe clinical and vascular anatomy presentation of patients with CLI with ischemic skin lesions who were unsuitable for revascularization was homogeneous with little imbalance according to region of origin or diabetic status. The findings from this large CLI cohort are important for the understanding of the epidemiology of the disease

G-CSF/SCF reduces inducible arrhythmias in the infarcted heart potentially via increased connexin43 expression and arteriogenesis

Granulocyte colony-stimulating factor (G-CSF), alone or in combination with stem cell factor (SCF), can improve hemodynamic cardiac function after myocardial infarction. Apart from impairing the pump function, myocardial infarction causes an enhanced vulnerability to ventricular arrhythmias. Therefore, we investigated the electrophysiological effects of G-CSF/SCF and the underlying cellular events in a murine infarction model

Effectiveness of revascularisation of the ulcerated foot in patients with diabetes and peripheral artery disease: A systematic review

In patients with diabetes, foot ulceration and peripheral artery disease (PAD), it is often difficult to determine whether, when and how to revascularise the affected lower extremity. The presence of PAD is a major risk factor for non-healing and yet clinical outcomes of revascularisation are not necessarily related to technical success. The International Working Group of the Diabetic Foot updated systematic review on the effectiveness of revascularisation of the ulcerated foot in patients with diabetes and PAD is comprised of 64 studies describing >13000 patients. Amongst 60 case series and 4 non-randomised controlled studies, we summarised clinically relevant outcomes and found them to be broadly similar between patients treated with open vs endovascular therapy. Following endovascular revascularisation, the 1 year and 2 year limb salvage rates were 80% (IQR 78-82%) and 78% (IQR 75-83%), whereas open therapy was associated with rates of 85% (IQR 80-90%) at 1 year and 87% (IQR 85-88%) at 2years, however these results were based on a varying combination of studies and cannot therefore be interpreted as cumulative. Overall, wound healing was achieved in a median of 60% of patients (IQR 50-69%) at 1 year in those treated by endovascular or surgical therapy, and the major amputation rate of endovascular vs open therapy was 2% vs 5% at 30days, 10% vs 9% at 1 year and 13% vs 9% at 2years. For both strategies, overall mortality was found to be high, with 2% (1-6%) perioperative (or 30day) mortality, rising sharply to 13% (9-23%) at 1 year, 29% (19-48%) at 2years and 47% (39-71%) at 5years. Both the angiosome concept (revascularisation directly to the area of tissue loss via its main feeding artery) or indirect revascularisation through collaterals, appear to be equally effective strategies for restoring perfusion. Overall, the available data do not allow us to recommend one method of revascularisation over the other and more studies are required to determine the best revascularisation approach in diabetic foot ulceration.Peer reviewe

ESVM Guideline on peripheral arterial disease

International audienc