Mifepristone-inducible transgene expression in neural progenitor cells in vitro and in vivo.

Numerous gene and cell therapy strategies are being developed for the treatment of neurodegenerative disorders. Many of these strategies use constitutive expression of therapeutic transgenic proteins, and although functional in animal models of disease, this method is less likely to provide adequate flexibility for delivering therapy to humans. Ligand-inducible gene expression systems may be more appropriate for these conditions, especially within the central nervous system (CNS). Mifepristone's ability to cross the blood-brain barrier makes it an especially attractive ligand for this purpose. We describe the production of a mifepristone-inducible vector system for regulated expression of transgenes within the CNS. Our inducible system used a lentivirus-based vector platform for the ex vivo production of mifepristone-inducible murine neural progenitor cells that express our transgenes of interest. These cells were processed through a series of selection steps to ensure that the cells exhibited appropriate transgene expression in a dose-dependent and temporally controlled manner with minimal background activity. Inducible cells were then transplanted into the brains of rodents, where they exhibited appropriate mifepristone-inducible expression. These studies detail a strategy for regulated expression in the CNS for use in the development of safe and efficient gene therapy for neurological disorders.

Optimizing the outcome of vascular intervention for Takayasu arteritis.

BACKGROUND: Takayasu arteritis (TA) predisposes to the development of arterial stenoses and aneurysms, and is associated with considerable morbidity and mortality amongst young patients. The aims of this study were to analyse indications and outcomes of surgical intervention, and to assess the potential benefits of immunosuppression and the use of perioperative imaging. METHODS: This was a retrospective review of patients with TA referred between 2001 and 2012. RESULTS: A series of 97 patients with TA, seen at a single tertiary centre, is reported. Immunosuppression was required in 87 patients (90 per cent). Thirty-seven (38 per cent) underwent 64 procedures: 27 patients underwent 33 open surgical procedures and 20 patients had 31 endovascular procedures. After a median follow-up of 6 years, the overall success rate was 79 per cent for open surgery (mean graft patency 9.4 years) and 52 per cent for endovascular procedures (P = 0.035). Procedural failure was significantly reduced in patients receiving preoperative immunosuppression, and particularly endovascular procedures (P = 0.001). In addition to clinical examination and measurement of acute-phase reactants, combination non-invasive imaging including Doppler ultrasonography, [18F]fluorodeoxyglucose combined positron emission and computed tomography (CT), magnetic resonance angiography and CT angiography was used to identify arterial lesions, establish the diagnosis and monitor treatment outcomes. CONCLUSION: Outcomes of vascular intervention in TA may be improved by detailed preoperative assessment including measurement of disease activity, and by ensuring optimal immunomodulatory therapy before and after the procedure.

Herpesvirus vector gene transfer and expression of beta-glucuronidase in the central nervous system of MPS VII mice.

Genetic disorders affecting the central nervous system (CNS) can potentially be treated by gene transfer using vectors which infect and express genes in post-mitotic neurons. Herpesviruses establish latent infections in neurons during which only one viral gene (LAT) is expressed, thus the LAT promoter may express foreign genes in latently infected CNS cells. Expression of a beta-glucuronidase gene driven by the LAT promoter was tested in mice lacking this enzyme, which are a model for a human genetic disease affecting the CNS (mucopolysaccharidosis VII, Sly disease). Cells expressing the missing enzymatic activity were present in the trigeminal ganglia and brainstems of latently infected animals, up to four months post-inoculation, demonstrating the potential of this approach for the long-term expression of foreign genes in the CNS.

Decreased lysosomal storage in the adult MPS VII mouse brain in the vicinity of grafts of retroviral vector-corrected fibroblasts secreting high levels of beta-glucuronidase.

A deficiency of beta-glucuronidase (GUSB) causes the multisystem progressive degenerative syndrome, mucopolysaccharidosis (MPS) type VII (Sly disease), which includes mental retardation. Animal homologues of MPS VII (ref. 3, 4) are models for testing somatic gene transfer approaches to treat the central nervous system in this and other lysosomal storage disorders. Previous attempts to correct murine MPS VII by gene therapy have successfully treated lesions in some organs but not in the brain. Other experimental modalities have forestalled some disease progression in the brain, but only if done at birth, before the onset of severe lesions, when the animals are phenotypically normal. We tested whether therapeutic amounts of GUSB could be delivered to the diseased adult brain by transplanting cells engineered to super-secrete the normal enzyme for export to surrounding neural tissues. Lysosomal distention was cleared from neurons and glial cells in the vicinity of the grafts, showing that the secreted enzyme could reach the diseased cells and reverse lesions in the severely diseased brain. The ability to correct established lesions will be important for the treatment of many lysosomal storage diseases affecting the brain, because most patients are not diagnosed until lesions are advanced enough to affect phenotype or developmental milestones in early childhood, and some forms of the diseases do not become apparent until later in life.

Identification of a variant of gross leukemia virus that induces disease in mice inoculated as adults.

Gross murine leukemia virus normally induces leukemia (thymic lymphoma) in mice inoculated as neonates, but not as adults. We have isolated an apparent variant of this virus which induces thymomas when inoculated i.p. into susceptible adult mice. Using H-2 congenic BALB and C57BL mice, susceptibility to virus-induced thymomagenesis was found to be linked to the H-2 complex. In addition, a radioresistant immune mechanism leading to inhibition of tumor growth was observed in mice with a C57BL but not a BALB background.

Assessment of surgical competence at carotid endarterectomy under local anaesthesia in a simulated operating theatre.

BACKGROUND: Methods of surgical training that do not put patients at risk are desirable. A high-fidelity simulation of carotid endarterectomy under local anaesthesia was tested as a tool for assessment of vascular surgical competence, as an adjunct to training. METHODS: Sixty procedures were performed by 30 vascular surgeons (ten junior trainees, ten senior trainees and ten consultants) in a simulated operating theatre. Each performed in a non-crisis scenario followed by a crisis scenario. Performance was assessed live by means of rating scales for technical and non-technical skills. RESULTS: There was a significant difference in technical skills with ascending grade for both generic and procedure-specific technical skill scores in both scenarios (P < 0.001 for all comparisons). Similarly, there was also a significant difference in non-technical skill with ascending grade for both scenarios (P < 0.001). There was a highly significant correlation between technical and non-technical performance in both scenarios (non-crisis: r(s) = 0.80, P < 0.001; crisis: r(s) = 0.85, P < 0.001). Inter-rater reliability was high (alpha > or = 0.80 for all scales). CONCLUSION: High-fidelity simulation offers competency-based assessment for all grades and may provide a useful training environment for junior trainees and more experienced surgeons.

Hybrid treatment of complex aortic arch disease with supra-aortic debranching and endovascular stent graft repair.

BACKGROUND: Aortic arch disease has conventionally been the domain of open surgical repair. Hybrid open and endovascular repair has evolved as an alternative, less invasive, treatment option with promising results. A systematic literature review and analysis of the reported outcomes was undertaken. METHODS: An Internet-based literature search using MEDLINE was performed to identify all studies reporting on hybrid aortic arch repair with supra-aortic branch revascularisation and subsequent stent graft deployment. Debranching should involve at least one carotid artery, so that patients merely requiring a carotid-subclavian bypass were not included. Only reports of five patients or more were included in the analysis. Outcome measures were technical success, perioperative, 30-day and late morbidity and mortality. RESULTS: Eighteen studies fulfilled our search criteria, and data from 195 patients were entered for the analysis. No comparative studies of hybrid aortic arch repair with other conventional or innovative treatment modalities were identified. Complete arch repair was performed in 122 patients (63%). The overall technical success rate was 86% (167/195). The most common reason for technical failure was endoleak (9%, 17/195). Overall perioperative morbidity and mortality rates were 21% (41/195) and 9% (18/195), respectively. The most common perioperative complication was stroke (7%, 14/195). Four aneurysm-related deaths were reported during follow-up (2%). No long-term data on hybrid aortic arch repair were identified. CONCLUSIONS: Hybrid repair of complex aortic arch disease is an alternative treatment option with acceptable short-term results. Stroke remains a frequent complication and mortality rates are significant. Further research with large comparative studies and longer follow-up is required.

Hybrid repair of the aortic arch in patients with extensive aortic disease.

OBJECTIVE: To evaluate the outcome of hybrid treatment of the aortic arch with supra-aortic debranching and endovascular stent-graft repair in a selected group of patients with complex disease. DESIGN: Case series study with retrospective analysis of prospectively collected non-randomised data. METHODS: Patients with hybrid repair of complex arch disease at a single centre over a 6-year period were enrolled in the study. Only patients with extensive arch pathologies requiring debranching of at least the left carotid artery were considered. Patients were divided into those who underwent complete and partial supra-aortic revascularisation. The χ2 test was used to evaluate differences in outcomes. Logistic regression analyses were applied to identify predictors of poor outcome. RESULTS: A total of 33 patients were included in the study. Complete and partial arch repair was performed in nine and 24 patients, respectively. The aortic disease extended to the thoracic and abdominal aorta in 39% and 52% of the patients, respectively. One-third of the patients (30%) were treated on an urgent/emergency basis. Elective 30-day mortality and morbidity rates were 13% and 35%, respectively. Early mortality was significantly higher in the complete arch repair group (p=0.046). Pre-existing renal impairment was identified as a poor prognostic factor. All extra-anatomic bypasses remained patent and no aortic disease-related deaths occurred during a mean follow-up period of 23 months (range, 1.5-58 months). Complete arch repair was associated with an increased incidence of late endoleak (p=0.018). CONCLUSIONS: Hybrid treatment of the aortic arch provides a feasible alternative treatment in patients who are high risk for conventional open surgical repair. Careful selection of patients is required to achieve satisfactory results.

Long-term AAV vector gene and protein expression in mouse brain from a small pan-cellular promoter is similar to neural cell promoters.

The neurogenetic, lysosomal enzyme (LSE) deficiency diseases are characterized by storage lesions throughout the brain; therefore, gene transfer needs to provide widespread distribution of the normal enzyme. Adeno-associated virus (AAV) vectors can be effective in the brain despite limited transduction because LSEs are exported to neighboring cells (cross-correction) to reverse the metabolic deficit. The extent of correction is determined by a combination of the total amount of LSE produced by a vector and the spatial distribution of the vector within the brain. Neuron-specific promoters have been used in the brain because AAV predominantly transduces neurons. However, these promoters are large, using up a substantial amount of the limited cloning capacity of AAV vector genomes. A small promoter that is active in all cells, from the LSE beta-glucuronidase (GUSB), has been used for long-term expression in AAV vectors in the brain but the natural promoter is expressed at very low levels. The amount of LSE exported from a cell is proportional to the level of transcription, thus more active promoters would export more LSE for cross-correction, but direct comparisons have not been reported. In this study, we show that in long-term experiments (>6 months) the GUSB minimal promoter (hGBp) expresses the hGUSB enzyme in brain at similar levels as the neuron-specific enolase promoter or the promoter from the latency-associated transcript of herpes simplex virus. The hGBp minimal promoter thus may be useful for long-term expression in the central nervous system of large cDNAs, bicitronic transcription units, self-complimentary or other designs with size constraints in the AAV vector system.

Preliminary pioneer 10 encounter results from the ames research center plasma analyzer experiment.

Preliminary results from the Ames Research Center plasma analyzer experiment for the Pioneer 10 Jupiter encounter indicate that Jupiter has a detached bow shock and magnetopause similar to the case at Earth but much larger in spatial extent. In contrast to Earth, Jupiter's outer magnetosphere appears to be highly inflated by thermal plasma and therefore highly responsive in size to changes in solar wind dynamic pressure.

Preliminary results on the plasma environment of saturn from the pioneer 11 plasma analyzer experiment.

The Ames Research Center Pioneer 11 plasma analyzer experiment provided measurements of the solar wind interaction with Saturn and the character of the plasma environment within Saturn's magnetosphere. It is shown that Saturn has a detached bow shock wave and magnetopause quite similar to those at Earth and Jupiter. The scale size of the interaction region for Saturn is roughly one-third that at Jupiter, but Saturn's magnetosphere is equally responsive to changes in the solar wind dynamic pressure. Saturn's outer magnetosphere is inflated, as evidenced by the observation of large fluxes of corotating plasma. It is postulated that Saturn's magnetosphere may undergo a large expansion when the solar wind pressure is greatly diminished by the presence of Jupiter's extended magnetospheric tail when the two planets are approximately aligned along the same solar radial vector.

Pioneer 11 encounter: preliminary results from the ames research center plasma analyzer experiment.

Pioneer 11 observations of the interaction of Jupiter's magnetosphere with the distant solar wind have confirmed the earlier Pioneer 10 observations of the great size and extreme variability of the outer magnetosphere. The nature of the plasma transitions across Jupiter's bow shock and magnetopause as observed on Pioneer 10 have also been confirmed on Pioneer 11. However, the northward direction of the Pioneer 11 outbound trajectory and the distance of the final magnetopause crossing (80 Jupiter radii) now suggest that Jupiter's magnetosphere is extremely broad with a half-thickness (normal to the ecliptic plane in the noon meridian) which is comparable to or greater than the sunward distance to the nose.

Electron observations and ion flows from the pioneer venus orbiter plasma analyzer experiment.

Additional plasma measurements in the vicinity of Venus are presented which show that (i) there are three distinct plasma electron populations-solar wind electrons, ionosheath electrons, and nightside ionosphere electrons; (ii) the plasma ion flow pattern in the ionosheath is consistent with deflected flow around a blunt obstacle; (iii) the plasma ion flow velocities near the downstream wake may, at times, be consistent with the deflection of plasma into the tail, closing the solar wind cavity downstream from Venus at a relatively close distance (within 5 Venus radii) to the planet; (iv) there is a separation between the inner boundary of the downstream ionosheath and the upper boundary of the nightside ionosphere; and (v) during the first 4.5 months in orbit the measured solar wind plasma speed continued to vary, showing a number of high-speed, but generally nonrecurrent, streams.

Prevention of paraplegia during thoracoabdominal aortic aneurysm repair.

Paraplegia affects up to 22% of patients undergoing thoarcoabdominal aneurysm surgery, producing long-term morbidity and a significant burden to healthcare. This article discusses the mechanisms that may lead to paraplegia during open and endovascular repair from an anatomical and physiological perspective. There are many adjuncts that must be considered to reduce the risk of spinal cord injury, such as revascularisation of intercostal arteries, maintenance of high mean blood pressure, spinal cord drainage and cooling. These adjuncts are discussed, highlighting the evidence available for each method and the practical ways in which they may be used.

Gaining consent for carotid surgery: a simulation-based study of vascular surgeons.

AIM: Despite no formal training in consenting patients, surgeons are assumed to be competent if they are able to perform an operation. We tested this assumption for carotid endarterectomy (CEA). METHODS: Thirty-two surgeons [Group 1: junior surgical trainees--performed 0 CEA's (n=11); 2: senior vascular trainees--1-50 CEA's (n=11); 3: consultant vascular surgeons - > 50 CEA's (n=10)] consented two patients (trained actors) for a local anaesthetic CEA. The performance was assessed at post hoc video review by two independent assessors using a validated rating scale and checklist of risk factors. RESULTS: There was no difference in performance between the junior and senior trainees (1: median 91 range 64-121; 2: median 100.5 range 66-125; p=0.118 1 vs. 2 Mann-Whitney). There was a significant improvement between senior trainees and consultant surgeons (3: median 120 range 89-1 142; p=0.001 2 vs. 3). Few junior (1/11) and senior (2/11) trainees, and most (8/11) consultants, were competent. Inter-rater reliability was high (alpha=0.832). Consultant surgeons were significantly more likely to discuss cranial nerve injuries (p<0.0001 Chi-square test) as well as personal or hospital specific stroke risk (p<0.0001) than their junior counterparts. They were less likely to discuss infection (p<0.0001). CONCLUSION: Senior trainees, despite being able to perform a CEA, were not competent in consent. The majority of consultant surgeons had developed competence in consenting even though they had no formal training.

Treatment of complex aneurysmal disease with fenestrated and branched stent grafts.

OBJECTIVES: To describe our experience of treating juxtarenal (JRAAA's <4mm neck) and thoracoabdominal aortic aneurysms (TAAA's) using fenestrated and branched stent graft technology. DESIGN: Prospective single centre experience. METHODS: Since 2005, 29 fenestrated/branched procedures have been performed. 15 patients are studied with JRAAAs (n=7; median neck length 0mm (IQR 0-3.8)) or TAAAs (type I (n=2), III (n=2), IV (n=4)). ASA grade III in 12/15. Maximum diameter of aneurysm 64 mm (56-74 mm). Aneurysms were excluded using covered stents or branches from the main body to patent visceral vessels (40 target vessels total). Pre-operative and follow-up CT scans (1, 3, and 12 months) were analysed by a single Vascular Interventional Radiologist. RESULTS: Technical success for cannulation and stenting of target vessels was 98%. In-hospital mortality was 0%. One patient underwent conversion to open repair. Five had major complications including one paraplegia (type III TAAA) with subsequent recovery. Median length of stay was 9 days (IQR 7-18.75). At a median follow-up of 12 months (9-14), CT confirmed 36/37 (97%) target vessels remain patent. Sac size increased >5 mm in one patient only. There were no type I endoleaks, three type II endoleaks (one embolised, two under surveillance) and three type III endoleaks (two successfully treated percutaneously, one aneurysm ruptured 18 months after endografting and died). CONCLUSION: In selected patients, fenestrated and branched stents appear to be a safe and effective alternative to surgery for juxtarenal and thoracoabdominal aneurysms. The complication and mortality rates are low. The long-term durability of this procedure, however, needs to be proven.

The visceral hybrid repair of thoraco-abdominal aortic aneurysms--a collaborative approach.

OBJECTIVE: To report the collaborative data of 3 major European Vascular Units using the 'visceral hybrid' procedure for thoraco-abdominal aortic aneurysms and dissections. METHODS: A consecutive series of 107 urgent and elective high-risk patients were included in a prospectively collected database. RESULTS: All stents involved the entire thoracic and abdominal aorta with left subclavian coverage in 19 and revascularisation in 12. The distal landing zone was in the infra-renal aorta in 75% and in the iliac artery in 25%. The 30-day mortality rate was 16/107 (14.95%). 13/107 (12.1%) of the patients suffered spinal cord ischaemia which was complete and permanent in 9/12 (8.4%). 4 patients (3.7%) required long term dialysis and a segment of gut infarction requiring resection occurred in 3 (2.8%). Most patients had visceral bypass grafting and aortic stent-grafting performed in one stage. In 18 patients the stenting was performed later. Three of these patients ruptured before the stenting procedure was undertaken. CONCLUSION: These early results of visceral hybrid repair for high-risk patients with complex thoraco-abdominal aortic aneurysms are encouraging, in a group of patients in whom fenestrated/branched stent-grafting is not an option and open surgery hazardous.

Prostacyclin reversal of lethal endotoxemia in dogs.

Severe endotoxemia, a condition where microembolization and intravascular coagulation are thought to play important roles, was treated experimentally with prostacyclin (PGI(2)). In a study of 24 dogs, 8 control animals injected with 1.75 mg.kg(-1) of endotoxin died within 24 h. Six animals given intravenous aspirin 100 mg/kg, 30 min after endotoxin died. 9 of 10 dogs infused with 100 ng PGI(2).kg(-1).min(-1) for 3 h, given 30 min after the injection of endotoxin survived 24 h (P < 0.025). Injection of endotoxin resulted in a: (a) maximal 62% fall in mean arterial pressure (P < 0.001); (b) transient doubling of mean pulmonary arterial pressure (P < 0.001); (c) initial 70% drop in cardiac index (P < 0.001); (d) decline in blood platelets from 213,700 to 13,700/mm(3) (P < 0.001), and leukocytes from 7,719 to < 750/mm(3) (P < 0.001); (e) depressed urine output (P < 0.001); (f) 34% decrease in blood fibrinogen (P < 0.01) and an increase in fibrin degradation products > 50 mug/ml (P < 0.001); (g) fivefold increase in circulating cathepsin D titer (P < 0.005) and (h) increase in blood norepinephrine (P < 0.005), dopamine (P < 0.005), and epinephrine (P < 0.001). Aspirin treatment led to an increase in mean arterial pressure (P < 0.001) and mean pulmonary arterial pressure (P < 0.005), but cardiac index, urine flow, platelets, leukocytes, fibrin degradation products, and cathepsin D levels remained similar to untreated controls. After infusion of PGI(2) there was a: (a) prompt increase of cardiac index to base-line levels; (b) late increase in mean arterial pressure (P < 0.005) after the discontinuation of PGI(2) treatment (c) restoration of urine output; (d) increase in circulating platelets to levels still below base line but above untreated control animals (P < 0.05); (e) no effect on circulating leukocyte levels; (f) fall in fibrin degradation products to 11.2 mug/ml (P < 0.05); (g) decline in cathepsin D levels to values 60% lower than the untreated controls (P < 0.025); and (h) reduction in plasma norepinephrine levels to base line at 4 h (P < 0.005). Although the mode of PGI(2) action is not clear, it is effective in the treatment of experimental endotoxemia.

Competence assessment of senior vascular trainees using a carotid endarterectomy bench model.

BACKGROUND: Competency-based assessment is being introduced to surgical training. The value of bench-top technical skills assessment using a synthetic carotid endarterectomy (CEA) model was evaluated in vascular trainees and consultants. METHODS: Forty-one surgeons (13 junior trainees, 15 senior trainees and 13 experienced consultants with experience of more than 50 CEAs) performed a three-throw knot-tying exercise on a jig and a CEA on the bench model. A composite score for knot-tying was calculated, incorporating electromagnetic motion analysis. CEA technical skill was assessed using validated rating scales by blinded video analysis. RESULTS: Senior trainees performed better than junior trainees in knot-tying (P = 0.025) as well as generic (P < 0.001) and procedural (P < 0.001) skills on CEA model assessment. There was no difference between senior trainees and consultants on any of these measures. The CEA model interobserver reliability was high for all rating scales (generic alpha = 0.974, procedural alpha = 0.952, end-product alpha = 0.976). CONCLUSION: Senior trainees achieved the same score as consultants, suggesting a similar level of basic technical skill and knowledge required to perform CEA, and were significantly better than junior trainees. Performance on the bench model could provide an early assessment for suitability to proceed to operative training in a competency-based training and assessment programme.

Genes transferred by retroviral vectors into normal and mutant myoblasts in primary cultures are expressed in myotubes.

Retroviral vectors were used to transfer genes efficiently into rat and dog myoblasts in primary cultures under conditions which permitted the transduced myoblasts to differentiate into myotubes expressing the transferred genes. The transduced myotubes expressed normal markers of differentiation and were morphologically indistinguishable from uninfected myotubes. Retroviral vector-mediated gene transfer was also used to correct a genetic enzyme deficiency in mutant canine muscle cells.