Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor.

Lentiviral vectors (LVs) are promising tools for gene therapy. However, scaling up the production methods of LVs in order to produce high-quality vectors for clinical purposes has proven to be difficult. In this article, we present a scalable and efficient method to produce LVs with transient transfection of adherent 293T cells in a fixed-bed bioreactor. The disposable iCELLis bioreactors are scalable with a large three-dimensional (3D) growth area range between 0.53 and 500 m2, an integrated perfusion system, and a controllable environment for production. In this study, iCELLis Nano (2.67-4 m2) was used for optimizing production parameters for scale-up. Transfections were first done using traditional calcium phosphate method, but in later runs polyethylenimine was found to be more reliable and easier to use. For scalable LV production, perfusion rate control by measuring cell metabolite concentrations in the bioreactor leads to higher productivity and reduced costs. Optimization of cell seeding density for targeted cell concentration during transfection, use of low compaction fixed-bed and lowering the culture pH have a positive effect on LV productivity. These results show for the first time that iCELLis bioreactor is scalable from bench level to clinical scale LV production.

Purification of metal finishing waste waters with zeolites and activated carbons.

Sixteen zeolites and 5 activated carbons were tested for the removal of nickel, zinc, cadmium, copper, chromium, and cobalt from waste simulants mimicking effluents produced in metal plating plants. The best performances were obtained from 4 zeolites: A, X, L, and ferrierite types and from 2 carbon types made from lignite and peat. The distribution coefficients for these sorbents were in the range of 10,000-440,000 ml/g. Column experiments showed that the most effective zeolites for Zn, Ni, Cu, and Cd were A and X type zeolites. The activated carbons, Hydrodarco 3000 and Norit Row Supra, exhibited good sorption properties for metals in aqueous solutions containing complexing agents.

The effect of physical training on hormonal status and exertional hormonal response in patients with chronic congestive heart failure.

BACKGROUND: Physical training improves exercise capacity in patients with chronic heart failure. It decreases plasma noradrenaline at rest, which may be prognostically favourable. The effect on atrial natriuretic peptide, another prognostic factor, and on catabolic and anabolic hormones remains unknown. Furthermore, to our knowledge, the contribution of exertional hormonal responses to the improved exercise capacity has not been evaluated. METHODS: 27 patients with stable chronic heart failure (New York Heart Association class II-III) were randomized to training (n=12) and control (n=15) groups. The training group exercised on a bicycle ergometer for 30 min three times a week for 3 months. The load corresponded to 50-60% of their peak oxygen consumption. For the next 3 months they exercised at home according to personal instructions. The control group did not change its physical activities. The levels of hormones regulating the cardiovascular system and metabolism were determined at rest and after graded maximal exercise and during exercise with constant submaximal workload. RESULTS: Submaximal exercise capacity increased significantly and peak oxygen consumption tended to improve by 12% in the training group. The plasma noradrenaline at rest tended to decrease by 19%. The plasma level of N-terminal pro atrial natriuretic peptide did not change. Serum cortisol, a catabolic hormone, was normal at baseline and remained unchanged. The serum levels of anabolic hormones, growth hormone and insulin, as well as dehydroepiandrosteronesulfate and free testosterone were within a normal range at baseline. They were not altered by training. The dehydroepiandrosteronesulfate/cortisol, and the free testosterone/cortisol ratios, reflecting anabolic/catabolic balance, did not change, either. Training resulted in a higher peak noradrenaline response during graded maximal exercise. The rise in serum cortisol during exercise tended to attenuate. CONCLUSION: Physical training, which improves exercise capacity, does not have an unfavourable effect on anabolic/catabolic balance or neurohumoral activation in patients with congestive heart failure. It decreases plasma noradrenaline at rest. Minor changes in hormonal responses during exercise emerged after physical training which unlikely contribute to the improved exercise capacity.

Effect of ethanol drinking, hangover, and exercise on adrenergic activity and heart rate variability in patients with a history of alcohol-induced atrial fibrillation.

To elucidate the mechanism of alcohol-induced atrial fibrillation (AF) we studied the heart rate variability and parameters of the adrenergic system during alcohol intake, hangover, and exercise in 6 men (mean age 43 years) prone to alcohol-induced AF, together with 6 age-matched controls. The ambulatory (15 hour) electrocardiogram was recorded and blood samples were taken for lymphocytic beta adrenoceptor, plasma catecholamine, and cyclic adenosine monophosphate (cAMP) measurements before and after alcohol intake (blood alcohol 1.5 per thousand), during hangover, and after a standardized bicycle exercise test. The beta-adrenoceptor density in lymphocytes was unchanged in the control group after alcohol intake or during hangover. Each of the AF patients had an increase in beta-adrenoceptor density after ethanol drinking (mean increase 29%, p <0.05). The hangover or exercise beta-receptor values did not differ from those in corresponding controls. Plasma adrenaline concentration tended to decrease and noradrenaline to increase after drinking and during hangover in both groups. Plasma cAMP levels were lower in patients after drinking than in controls (p <0.05). The exercise values of the adrenergic parameters were very similar in AF patients whether or not preceded by alcohol. Analysis of ambulatory electrocardiography showed a very low rate of ectopic beats in both AF patients and controls. Analysis of heart rate variability revealed a tendency toward an increase in sympathetic/parasympathetic component ratio (low-frequency/high-frequency ratio) in AF patients, but not in controls, after ethanol drinking. In conclusion, no signs of arrhythmogenic cardiac disease were detected in patients with AF to explain the tendency toward AF. Increases in beta-adrenoceptor density and low-frequency/high-frequency ratio during ethanol intoxication in patients with AF suggest an exaggerated sympathetic reaction.

Haemodynamic, neuroendocrine and metabolic correlates of circulating cytokine concentrations in congestive heart failure.

OBJECTIVES: Increased activity of pro-inflammatory cytokines in the circulation has been observed in many, though not all, patients with congestive heart failure. To identify the predictors of cytokine activation in congestive heart failure, we assessed the relationship of peripheral and hepatic venous cytokines to central haemodynamics, neuroendocrine status and intermediary metabolism in patients with moderate or severe congestive heart failure. PATIENTS AND METHODS: Concentrations of tumour necrosis factor-alpha, soluble tumour necrosis factor-receptor II and interleukin 6 were measured from peripheral and hepatic venous plasma in 58 adult cardiac patients, of whom 44 had congestive heart failure, undergoing heart catheterization, echocardiography and assessment of selected neuroendocrine and metabolic characteristics. RESULTS: Peripheral venous soluble tumour necrosis factor-receptor II was directly related to NYHA class (rs = 0.46, P < 0.001) and inversely to 6-min walking distance (rs = -0.46, P < 0.001). Peripheral venous tumour necrosis factor-alpha was related to 6-min walking distance (rs = -0.37, P < 0.01), but like soluble tumour necrosis factor-receptor II, was unrelated to other haemodynamic and neuroendocrine measurements. Peripheral venous interleukin 6 correlated with NYHA class (rs = 0.66, P < 0.001) and 6-min walking distance (rs = -0.52, P < 0.001). In addition, interleukin 6 was related to right atrial pressure (rs = 0.55, P < 0.001), pulmonary artery wedge pressure (rs = 0.50, P < 0.001) and left ventricular ejection fraction (rs = -0.39, P < 0.01); in multivariate analysis, only right atrial pressure was an independent predictor of interleukin 6 concentration (P < 0.001). Comparisons between patients with and without congestive heart failure showed significantly higher hepatic venous tumour necrosis factor-alpha, soluble tumour necrosis factor-receptor II and interleukin 6 in the heart failure group; the differences in peripheral venous cytokines were less consistent. CONCLUSIONS: In cardiac patients, increased plasma tumour necrosis factor-alpha and soluble tumour necrosis factor-receptor II are associated with symptoms of heart failure and poor exercise capacity, while the most important predictor of increased interleukin 6 is elevated systemic venous pressure. Different but still unknown mechanisms may be responsible for the increased release of cytokines in congestive heart failure.

Autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA: clinical, biochemical, and molecular genetic features of the 10q-linked disease.

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disease characterized by accumulation of multiple large deletions of mtDNA in patients' tissues. We previously showed that the disease is genetically heterogeneous by assigning two nuclear loci predisposing to mtDNA deletions: one on chromosome 10q 23.3-24.3 in a Finnish family and one on 3p 14.1-21.2 in three Italian families. To reveal any locus-specific disease features, we report here the clinical, biochemical, and molecular genetic characteristics of the 10q-linked disease in the single family reported to date. All seven patients and four asymptomatic subjects had ragged-red fibers and multiple deletions of mtDNA in their muscle. Ptosis and external ophthalmoplegia were the major clinical findings, and depression or avoidant personality traits were frequently, but not consistently, present in the subjects carrying mutant mtDNA. In six of the subjects with mutant mtDNA, the activities of the respiratory chain complexes I or IV, or both, were below or within the low normal range. Two autopsy studies revealed the characteristic distribution of mutant mtDNA in these patients: highest proportion of mutant mtDNA is found in different parts of the brain, followed by the skeletal and ocular muscle, and the heart.

Skeletal muscle lactate accumulation and creatine phosphate depletion during heavy exercise in congestive heart failure. Cause of limited exercise capacity?

OBJECTIVE: To study the mechanisms of limited exercise capacity and skeletal muscle energy production in male patients with congestive heart failure. DESIGN: Muscle biopsy study. PATIENTS: Skeletal muscle metabolic response to maximal bicycle exercise was studied in 10 patients with chronic congestive heart failure (ejection fraction 0.22 +/- 0.05; peak oxygen consumption, VO2 15.1 +/- 4.9 ml.min-1.kg-1) and in nine healthy subjects (peak VO2 33.5 +/- 6.7 ml.min-1.kg-1). Activities of skeletal muscle enzymes were measured from the vastus lateralis muscle of 48 patients (ejection fraction 0.24 +/- 0.06, peak VO2 17.4 +/- 5.4 ml.min-1.kg-1) and 36 healthy subjects (peak VO2 38.3 +/- 8.4 ml.min-1.kg-1). RESULTS: Although blood lactate levels were lower in patients than in healthy subjects (2.2 +/- 0.3 vs 5.2 +/- 0.6 mmol.l-1; P < 0.001) at peak exercise (96 +/- 11 W for patients and 273 +/- 14 W for controls), skeletal muscle lactate was similarly elevated (25.6 +/- 3.2 vs 22.7 +/- 2.7 mmol.kg-1) and creatine phosphate was equally depressed (P < 0.02) to low levels (7.0 +/- 1.9 vs 6.7 +/- 0.9 mmol.kg-1). The muscle ATP decreased by 21% (P < 0.05) and 8% (P < 0.01) in the patients and controls, respectively. Activities of rate limiting enzymes of the citric acid cycle (alpha-ketoglutarate dehydrogenase) and oxidation of free fatty acids (carnitine palmitoyltransferase II) were 48% and 21% lower than in controls, but the mean phosphofructokinase activity was unchanged in congestive heart failure. CONCLUSIONS: It seems that the main limiting factor of exercise performance during heavy exercise is the same in congestive heart failure and healthy subjects, a high rate of skeletal muscle lactate accumulation and high-energy phosphate depletion. In congestive heart failure, the low activity of aerobic enzymes is likely to impair energy production and lead to lactate acidosis at low workloads.

Effect of physical training on exercise capacity and gas exchange in patients with chronic heart failure.

Decreased exercise capacity is the main factor restricting the daily life of patients with chronic congestive heart failure (CHF). We performed a controlled, randomized study to evaluate the effect of dynamic exercise training of moderate intensity on exercise capacity and gas exchange in patients with CHF. Twenty-seven patients with stable CHF, New York Heart Association (NYHA) functional class II and III, were randomized to training (n = 12) and control (n = 15) groups. During a 3-month period, the training group underwent a supervised physical training program using a bicycle ergometer for 30 min 3 times a week at a load corresponding to 50 to 60% of their peak oxygen consumption. Thereafter, they were advised to continue training at home for the next 3 months. The control group did not change their previous physical activity. A graded maximal exercise test with respiratory gas analysis and an endurance test with constant submaximal workload were performed at baseline and after 3 and 6 months. The exercise endurance increased from 14.7 +/- 2.0 to 27.8 +/- 2.7 min (p < 0.01) and the peak oxygen consumption tended to improve from 19.3 +/- 1.6 to 21.7 +/- 2.3 mL/kg/min (p = 0.09) during the supervised training period. At submaximal workloads, minute ventilation was reduced by 16% per se (p < 0.01) and by 7% in proportion to carbon dioxide production (p < 0.05). Oxygen consumption at the anaerobic threshold increased from 10.5 +/- 0.8 to 12.7 +/- 1.0 mL/kg/min (p < 0.05). The positive training effects were associated with an improvement in the NYHA functional class. The effects of supervised training were preserved during the home-based training period. The results indicate that physical training of moderate intensity significantly improves the exercise capacity and reduces the exaggerated ventilatory response to exercise, particularly at submaximal working levels in patients with CHF. This is associated with alleviation of symptoms.

Blood ketone bodies in congestive heart failure.

OBJECTIVES: The present study was designed to assess whether blood ketone bodies are elevated in congestive heart failure (CHF) and whether ketonemia is related to the hemodynamic and neurohumoral abnormalities of CHF. BACKGROUND: In CHF, consumption of the body's fat stores may become abnormally high, contributing to the development of cardiac cachexia. Increased mobilization of free fatty acids could, in theory, augment ketogenesis, but whether patients with CHF are prone to ketosis remains unknown. METHODS: Forty-five patients with chronic CHF (mean age [+/- SD] 57 +/- 13 years) and 14 control subjects free of CHF (mean age 53 +/- 13 years) underwent invasive and noninvasive cardiac studies and determination of blood ketone bodies (acetoacetate plus beta-hydroxybutyrate), circulating free fatty acids, glucose, lactate, insulin, glucagon, growth hormone, cortisol, norepinephrine, N-terminal proatrial natriuretic peptide, tumor necrosis factor-alpha and interleukin-6 after an overnight fast. RESULTS: Patients with CHF had elevated blood ketone bodies (median 267 mumol/liter, range 44 to 952) compared with control subjects (median 150 mumol/liter, range 31 to 299, p < 0.05). In the total study group, blood ketone bodies were related to pulmonary artery wedge pressure (r5 = 0.45, p < 0.001), left ventricular ejection fraction (r3 = -0.37, p < 0.01), right atrial pressure (r3 = 0.36, p < 0.01) and circulating concentrations of free fatty acids (r5 = 0.52, p < 0.001), glucose (r5 = -0.39, p < 0.001), norepinephrine (r3 = 0.45, p < 0.001), growth hormone (r5 = 0.30, p < 0.05) and interleukin-6 (r3 = 0.27, p < 0.05). In multivariate analysis, left ventricular ejection fraction, serum free fatty acids and serum glucose were independent predictors of ketonemia. CONCLUSIONS: Blood ketone bodies are elevated in CHF in proportion to the severity of cardiac dysfunction and neurohormonal activation. This may be at least partly attributable to increased free fatty acid mobilization in response to augmented neurohormonal stimulation. Additional studies are needed to identify the detailed mechanisms and clinical implications of CHF ketosis.

Effect of beta-blocking agents with and without intrinsic sympathomimetic activity on work efficiency in healthy men.

In order to evaluate the effect of beta-blocking agents with and without intrinsic sympathomimetic activity (ISA) on work efficiency in healthy subjects, we studied the haemodynamic and gas exchange parameters, as well as blood lactate concentrations, during a graded maximal bicycle exercise test performed after perorally given propranolol (PRO) and pindolol (PIN) in seven healthy men. The medications (PRO: 80 mg x 2/day, PIN: 10 mg x 2/day, for seven days) were given in a placebo (PLA) controlled, double-blind, randomized, cross-over fashion. Both the drugs reduced heart rate and blood pressure during exercise equally compared with the placebo. The oxygen uptake at submaximal work loads, as well as at the maximum, was constantly and equally reduced by PRO and PIN compared with PLA. The anaerobic threshold was reached at a slightly lower oxygen uptake for both the drugs compared with the placebo (P < 0.05). No significant difference was, however, observed in the work levels at which the ventilatory anaerobic threshold was reached. Moreover, the gross efficiency, i.e. the amount of work performed at a certain energy consumption level (aerobic + anaerobic), was increased by both PRO (26.7 +/- 0.5%, P < 0.02 vs PLA: 24.7 +/- 0.5%) and PIN (26.5 +/- 0.5%, P < 0.05 vs PLA) at a submaximal work load of 240 W. The results indicate that beta-blocking agents propranolol and pindolol slightly and equally reduce maximal work performance, but increase the efficiency of submaximal work in a way that a certain amount of external work can be done with smaller consumption of oxygen. These findings may contribute to the benefit of beta-blocking agents in patients with coronary heart disease.

Reversal of autonomic derangements by physical training in chronic heart failure assessed by heart rate variability.

Decreased heart rate variability has been associated with chronic congestive heart failure (CHF). We evaluated the effect of physical training on heart rate variability in 20 patients with CHF (NYHA class II-III) randomized to training (n = 8) and control (n = 12) groups. The training group underwent 3 months of physical training, by exercising on a bicycle ergometer for 30 min three times a week at a load corresponding to 50-60% of their peak oxygen consumption. Heart rate variability was assessed from 20-h ambulatory ECG recordings in the frequency domain, determined by high (0.15-0.40 Hz), low (0.04-0.15 Hz) and very low frequency (0.008-0.04 Hz) components. The high frequency component increased by 22-55% in the training group during the day (P = 0.0001) but not at night. The increase was seen during both sedentary and active periods. The low frequency/high frequency ratio attenuated in the training group during the day (P = 0.05) whereas an increase was seen in the control group throughout the day (P = 0.0003). Training lengthened the exercise duration by 71% at a submaximal workload (P = 0.01) and tended to increase the peak oxygen consumption by 15% (P = 0.09). These remained unchanged in the control group. In conclusion, physical training, which improves exercise capacity, ameliorates the autonomic derangement in CHF by increasing the parasympathetically mediated component of heart rate variability. It may thus influence favourably the prognosis of the disease.