A high efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors

Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50 000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10 000 g for 2 h at 4 degreesC. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10 000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.

Reversal of cardiac and renal fibrosis by pirfenidone and spironolactone in streptozotocin-diabetic rats

1 Fibrosis leads to chronic impairment of cardiac and renal function and thus reversal of existing fibrosis may improve function and survival. This project has determined whether pirfenidone, a new antifibrotic compound, and spironolactone, an aldosterone antagonist, reverse both deposition of the major extracellular matrix proteins, collagen and fibronectin, and functional changes in the streptozotocin(STZ)-diabetic rat. 2 Streptozotocin (65 mg kg(-1) i.v.)-treated rats given pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone; approximately-200 mg kg(-1) day(-1) as 0.2-2g l(-1) drinking water) or spironolactone (50 mg kg(-1) day(-1) s.c.) for 4 weeks starting 4 weeks after STZ showed no attenuation of the increased blood glucose concentrations and increased food and water intakes which characterize diabetes in this model. 3 STZ-treatment increased perivascular and interstitial collagen deposition in the left ventricle and kidney, and surrounding the aorta. Cardiac, renal and plasma fibronectin concentrations increased in STZ-diabetic rats. Passive diastolic stiffness increased in isolated hearts from STZ-diabetic rats. Both pirfenidone and spironolactone treatment attenuated these increases without normalizing the decreased + dP/dt(max) of STZ-diabetic hearts. 4 Left ventricular papillary muscles from STZ-treated rats showed decreased maximal positive inotropic responses to noradrenaline, EMD 57033 (calcium sensitizer) and calcium chloride; this was not reversed by pirfenidone or spironolactone treatment. STZ-treatment transiently decreased GFR and urine flow rates in isolated perfused kidneys; pirfenidone but not spironolactone prevented the return to control values. 5 Thus, short-term pirfenidone and spironolactone treatment reversed cardiac and renal fibrosis and attenuated the increased diastolic stiffness without normalizing cardiac contractility or renal function in STZ-diabetic rats.

A flexible method for rapid force computation in elliptical magnets

The design of open-access elliptical cross-section magnet systems has recently come under consideration. Obtaining values for the forces generated within these unusual magnets is important to progress the designs towards feasible instruments. This paper presents a novel and flexible method for the rapid computation of forces within elliptical magnets. The method is demonstrated by the analysis of a clinical magnetic resonance imaging magnet of elliptical cross-section and open design. The analysis reveals the non-symmetric nature of the generated Maxwell forces, which are an important consideration, particularly in the design of superconducting systems.

An inverse design method for elliptical MRI magnets

An inverse, current density mapping (CDM) method has been developed for the design of elliptical cross-section MRI magnets. The method provides a rapid prototyping system for unusual magnet designs, as it generates a 3D current density in response to a set of target field and geometric constraints. The emphasis of this work is on the investigation of new elliptical coil structures for clinical MRI magnets. The effect of the elliptical aspect ratio on magnet performance is investigated. Viable designs are generated for symmetric, asymmetric and open architecture elliptical magnets using the new method. Clinically relevant attributes such as reduced stray field and large homogeneous regions relative to total magnet length are included in the design process and investigated in detail. The preliminary magnet designs have several novel features.

On a possible mechanism for peripheral nerve stimulation during magnetic resonance imaging scans

When patients undergo a magnetic resonance imaging scan, they are subject to both strong static and temporal magnetic fields. The temporal fields are designed to vary at each point in the region being imaged. This is achieved by the use of gradient coils. However, when the gradient coils are switched very rapidly, the strongly time-varying magnetic fields produced can be responsible for stimulating nerves in the peripheral regions of the body. This paper gives a somewhat novel explanation for this phenomenon. The physical mechanism suggested is supported by an illustrative theoretical calculation.

Energy cost of physical activity in cystic fibrosis

Objective: The purpose of this study was to compare the energy cost of standardized physical activity (ECA) between patients with cystic fibrosis (CF) and healthy control subjects. Design: Cross-sectional study using patients with CF and volunteers from the community. Setting: University laboratory. Subjects: Fifteen patients (age 24.6 +/- 4.6 y) recruited with consent from their treating physician and 16 healthy control subjects (age 25.3 +/- 3.2) recruited via local advertisement. Interventions. Patients and controls walked on a computerised treadmill at 1.5 km/h for 60 min followed by a 60 min recovery period and, on a second occasion, cycled at 0.5 kp (kilopond), 30 rpm followed by a 60 min recovery. The ECA was measured via indirect calorimetry. Resting energy expenditure (REE), nutritional status, pulmonary function and genotype were determined. Results: The REE in patients was significantly greater than the REE measured in controls (P = 0.03) and was not related to the severity of lung disease or genotype. There was a significant difference between groups when comparing the ECA for walking kg root FFM (P = 0.001) and cycling kg root FFM (P = 0.04). The ECA for each activity was adjusted (ECA(adj)) for the contribution of REE (ECA kJ kg root FFM 120 min(-1) - REE kJ kg root FFM 120 min(-1)). ECA(adj) revealed a significant difference between groups for the walking protocol (P = 0.001) but no difference for the cycling protocol (P = 0.45). This finding may be related to the fact that the work rate during walking was more highly regulated than during cycling. Conclusions ECA in CF is increased and is likely to be explained by an additional energy-requiring component related to the exercise itself and not an increased REE. Sponsorship. The Prince Charles Hospital Foundation; MLR was in receipt of a QUTPRA Scholarship.

Comparison of measured sleeping metabolic rate and predicted basal metabolic rate during the first year of life: evidence of a bias changing with increasing metabolic rate

Objective: To compare measurements of sleeping metabolic rate (SMR) in infancy with predicted basal metabolic rate (BMR) estimated by the equations of Schofield. Methods: Some 104 serial measurements of SMR by indirect calorimetry were performed in 43 healthy infants at 1.5, 3, 6, 9 and 12 months of age. Predicted BMR was calculated using the weight only (BMR-wo) and weight and height (BMR-wh) equations of Schofield for 0-3-y-olds. Measured SMR values were compared with both predictive values by means of the Bland-Altman statistical test. Results: The mean measured SMR was 1.48 MJ/day. The mean predicted BMR values were 1.66 and 1.47 MJ/day for the weight only and weight and height equations, respectively. The Bland-Altman analysis showed that BMR-wo equation on average overestimated SMR by 0.18 MJ/day (11%) and the BMR-wh equation underestimated SMR by 0.01 MJ/day (1%). However the 95% limits of agreement were wide: - 0.64 to - 0.28MJ/day (28%) for the former equation and - 0.39 to +0.41 MJ/day (27%) for the latter equation. Moreover there was a significant correlation between the mean of the measured and predicted metabolic rate and the difference between them. Conclusions: The wide variation seen in the difference between measured and predicted metabolic rate and the bias probably with age indicates there is a need to measure actual metabolic rate for individual clinical care in this age group.