Effects of extrusion, lipid concentration and purity on physico-chemical and biological properties of cationic liposomes for gene vaccine applications

We developed cationic liposomes containing DNA through a conventional process involving steps of (i) preformation of liposomes, (ii) extrusion, (iii) drying and rehydration and (iv) DNA complexation. Owing to its high prophylactic potentiality against tuberculosis, which had already been demonstrated in preclinical assays, we introduced modifications into the conventional process towards getting a simpler and more economical process for further scale-up. Elimination of the extrusion step, increasing the lipid concentration (from 16 to 64 mM) of the preformed liposomes and using good manufacturing practice bulk lipids (96-98% purity) instead of analytical grade purity lipids (99.9-100%) were the modifications studied. The differences in the physico-chemical properties, such as average diameter, zeta potential, melting point and morphology of the liposomes prepared through the modified process, were not as significant for the biological properties, such as DNA loading on the cationic liposomes, and effective immune response in mice after immunisation as the control liposomes prepared through the conventional process. Beneficially, the modified process increased productivity by 22% and reduced the cost of raw material by 75%.

Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains.

Control of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, processes. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of"emerging" LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demonstrates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storage.

Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains.

Control of lipid droplet (LD) nucleation and copy number are critical, yet poorly understood, processes. We use model peptides that shift from the endoplasmic reticulum (ER) to LDs in response to fatty acids to characterize the initial steps of LD formation occurring in lipid-starved cells. Initially, arriving lipids are rapidly packed in LDs that are resistant to starvation (pre-LDs). Pre-LDs are restricted ER microdomains with a stable core of neutral lipids. Subsequently, a first round of"emerging" LDs is nucleated, providing additional lipid storage capacity. Finally, in proportion to lipid concentration, new rounds of LDs progressively assemble. Confocal microscopy and electron tomography suggest that emerging LDs are nucleated in a limited number of ER microdomains after a synchronized stepwise process of protein gathering, lipid packaging, and recognition by Plin3 and Plin2. A comparative analysis demonstrates that the acyl-CoA synthetase 3 is recruited early to the assembly sites, where it is required for efficient LD nucleation and lipid storage.

Structural study of BSA/poly(ethylene glycol) lipid conjugate complexes

In this work we report the structural characteristics of bovine serum albumin/poly(ethylene glycol) lipid conjugate (BSA/PEG(2000)-PE) complexes under physiological conditions (37 degrees C and pH 7.4) for particular fractions of BSA to PEG-lipid concentration, CBSA/C-PEG2000-PE. Ultraviolet fluorescence spectroscopy (UV) results shown that PEG(2000)-PE is associated to BSA, leading to;protein unfolding for fixed C-BSA = 0.01 wt % and variable C-PEG2000-PE = 0.0015-0.6 wt %. Tryptophan groups on the BSA surface are in contact with the PEG-lipid at C-PEG2000-PE = 0.0015 wt %, while they are exposed to water at C-PEG2000-PE (>)0.0015 wt %. Dynamic and static light scattering (DLS and SLS) and small-angle neutron scattering (SANS) point out the existence of individual BSAIPEG-lipid complexes in the system for fixed C-BSA = 1 wt % and variable C-PEG2000-PE = 0.15-2 wt %. DLS shows that there is only one BSA molecule per protein/PEG-lipid complex, while SLS shows that the PEG-lipid associates to the BSA without promoting aggregation between adjacent protein/ polymer-lipid conjugate complexes. SANS was used to show that BSA/PEG(2000)-PE complexes adopt an oblate ellipsoidal shape. Partially unfolded BSA is contained in the core of the oblate ellipsoid, which is surrounded by an external shell containing the PEG(2000)-PE.

Ionization and Structural Changes of the DMPG Vesicle along Its Anomalous Gel-Fluid Phase Transition: A Study with Different Lipid Concentrations

Dispersions of saturated anionic phospholipid dimyristoyl phosphatidylglycerol (DMPG) have been extensively studied regarding their peculiar thermostructural behavior. At low ionic strength, the gel-fluid transition is spread along nearly 17 degrees C, displaying several thermal events in the calorimetric profile that is quite different from the single sharp peak around 23 degrees C found for higher ionic strength DMPG dispersions. To investigate the role of charge in the bilayer transition, we carefully examine the temperature dependence of the electrical conductivity of DMPG dispersions at different concentrations, correlating the data with the corresponding differential scanning calorimetry (DSC) traces. Electrical conductivity together with electrophoretic mobility measurements allowed the calculation of the dependence of the degree of ionization of DMPG vesicles on lipid concentration and temperature. It was shown that there is a decrease in vesicle charge as the lipid concentration increases, which is probably correlated with the increase in the concentration of bulk Na(+). Apart from the known increase in the electrical conductivity along the DMPG temperature transition region, a sharp rise was observed at the bilayer pretransition for all lipid concentrations studied, possibly indicating that the beginning of the chain melting process is associated with an increase in bilayer ionization. It is confirmed here that the gel-fluid transition of DMPG at low ionic strength is accompanied by a huge increase in the dispersion viscosity. However, it is shown that this measured macroviscosity is distinct from the local viscosity felt by either charged ions or DMPG charged aggregates in measurements of electrical conductivity or electrophoretic mobility, Data presented here give support to the idea that DMPG vesicles, at low ionic strength, get more ionized along the temperature transition region and could be perforated and/or deformed vesicle structures.

Growth, lipogenesis and body composition of piracanjuba (Brycon orbignyanus) fingerlings fed different dietary protein and lipid concentrations

Piracanjuba (Brycon orbignyanus) is a Brazilian migratory fast-growing omnivore, very appreciated as a sport fish, which is threatened to extinction in Southern Brazil due to stock over exploitation and dam building. Therefore, efforts have been made to raise this fish in captivity for reintroduction and aquaculture purposes. In the present study, the effects of different dietary protein and lipid concentrations on piracanjuba fingerlings growth performance, feed utilization, body composition, hepatosomatic index (HSI) and activity of the lipogenic enzymes fatty acid synthetase (FAS), glucose-6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) were investigated using a 2 x 3 factorial experiment. Six casein-gelatin based diets were prepared combining two protein (30% and 32%) and three lipid concentrations (5.5%, 8.8% and 12.1%). Eleven fish, average weight 11.30 +/- 0.1 g, were held in each of 18 100-1 aquaria, supplied with recirculating freshwater. Each diet was randomly assigned to triplicate groups of fish and fed to apparent satiation, twice a day for 100 d. Piracanjuba fingerlings' daily weight gain (0.36-0.40 g), specific growth rate (1.43-1.51%), feed utilization and HSI were not influenced by dietary protein or lipid concentration. However, body composition was directly affected by dietary treatment. An increase in body fat and dry matter was observed as dietary lipid increased, for both dietary protein concentrations tested. The activity of FAS was depressed by increasing dietary fat levels but the G6PD activity did not differ among dietary treatments, although ME activity showed some regulation by dietary protein. These results indicate that an increase from 5.5% to 12.1% in the dietary lipid, at a dietary protein concentration of 30% or 32%, promotes body fat accumulation in piracanjuba fingerlings with no improvement in growth, suggesting that the lipid requirement for this species should be 5% or less, when raised for commercial purposes. However, the additional energy reserve from body fat accumulation could be desirable for piracanjuba fingerlings produced for stock enhancement. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

In vitro evaluation of permeation, toxicity and effect of praziquantel-loaded solid lipid nanoparticles against Schistosoma mansoni as a strategy to improve efficacy of the schistosomiasis treatment

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Growth, lipogenesis and body composition of piracanjuba (Brycon orbignyanus) fingerlings fed different dietary protein and lipid concentrations

Piracanjuba (Brycon orbignyanus) is a Brazilian migratory fast-growing omnivore, very appreciated as a sport fish, which is threatened to extinction in Southern Brazil due to stock over exploitation and dam building. Therefore, efforts have been made to raise this fish in captivity for reintroduction and aquaculture purposes. In the present study, the effects of different dietary protein and lipid concentrations on piracanjuba fingerlings growth performance, feed utilization, body composition, hepatosomatic index (HSI) and activity of the lipogenic enzymes fatty acid synthetase (FAS), glucose-6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) were investigated using a 2 x 3 factorial experiment. Six casein-gelatin based diets were prepared combining two protein (30% and 32%) and three lipid concentrations (5.5%, 8.8% and 12.1%). Eleven fish, average weight 11.30 +/- 0.1 g, were held in each of 18 100-1 aquaria, supplied with recirculating freshwater. Each diet was randomly assigned to triplicate groups of fish and fed to apparent satiation, twice a day for 100 d. Piracanjuba fingerlings' daily weight gain (0.36-0.40 g), specific growth rate (1.43-1.51%), feed utilization and HSI were not influenced by dietary protein or lipid concentration. However, body composition was directly affected by dietary treatment. An increase in body fat and dry matter was observed as dietary lipid increased, for both dietary protein concentrations tested. The activity of FAS was depressed by increasing dietary fat levels but the G6PD activity did not differ among dietary treatments, although ME activity showed some regulation by dietary protein. These results indicate that an increase from 5.5% to 12.1% in the dietary lipid, at a dietary protein concentration of 30% or 32%, promotes body fat accumulation in piracanjuba fingerlings with no improvement in growth, suggesting that the lipid requirement for this species should be 5% or less, when raised for commercial purposes. However, the additional energy reserve from body fat accumulation could be desirable for piracanjuba fingerlings produced for stock enhancement. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

Dietary salt restriction increases plasma lipoprotein and inflammatory marker concentrations in hypertensive patients

Background: Dietary salt restriction has been reported to adversely modify the plasma lipoprotein profile in hypertensive and in normotensive subjects. We investigated the effects of the low sodium intake (LSI) on the plasma lipoprotein profile and on inflammation and thrombosis biomarkers during the fasting and postprandial periods. Methods: Non-obese, non-treated hypertensive adults (n=41) were fed strictly controlled diets. An initial week on a control diet (CID, Na=160 mmol/day) was followed by 3 weeks on LSI (Na=60mmol/day). At admission and on the last day of each period, the 24-h ambulatory blood pressure was monitored and blood was drawn after an overnight fasting period and after a fat-rich test meal. Results: The dietary adherence was confirmed by 24-h urinary sodium excretion. Fasting triglyceride (TG), chylomicron-cholesterol, hsC-reactive protein (CRP), tumor necrosis factor-a (TNF-alpha). interleukin-6 (IL-6) concentrations, renin activity, aldosterone, insulin, and homeostasis model assessment insulin resistance (HOMA-IR) Values were higher, but non-esterified fatty acids (NEFA) were lower on LSI than on CD. For LSI, areas under the curve (AUC) of TG, chylomicron-cholesterol, apoB and the cholesterol/apoB ratio were increased, whereas AUC-NEFA was lowered. LSI did not modify body weight, hematocrit, fasting plasma cholesterol, glucose, adiponectin, leptin, fibrinogen and factor VII (FVII), and AUC of lipoprotein lipase and of lipoprotein remnants. Conclusion: LSI induced alterations in the plasma lipoproteins and in inflammatory markers that are common features of the metabolic syndrome. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Preparation and cytotoxicity of cisplatin-containing liposomes

We encapsulated cisplatin into stealth pH-sensitive liposomes and studied their stability, cytotoxicity and accumulation in a human small-cell lung carcinoma cell line (GLC4) and its resistant subline (GLC4/CDDP). Since reduced cellular drug accumulation has been shown to be the main mechanism responsible for resistance in the GLC4/CDDP subline, we evaluated the ability of this new delivery system to improve cellular uptake. The liposomes were composed of dioleoylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethyleneglycol 2000 (DSPE-PEG2000) and were characterized by determining the encapsulation percentage as a function of lipid concentration. Among the different formulations, DOPE/CHEMS/DSPE-PEG liposomes (lipid concentration equal to 40 mM) encapsulated cisplatin more efficiently than other concentrations of liposomes (about 20.0%, mean diameter of 174 nm). These liposomes presented good stability in mouse plasma which was obtained using a 0.24-M EDTA solution (70% cisplatin was retained inside the liposomes after 30 min of incubation). Concerning cytotoxic effects, they are more effective (1.34-fold) than free cisplatin for growth inhibition of the human lung cancer cell line A549. The study of cytotoxicity to GLC4 and GLC4/CDDP cell lines showed similar IC50 values (approximately 1.4 µM), i.e., cisplatin-resistant cells were sensitive to this cisplatin formulation. Platinum accumulation in both sensitive and resistant cell lines followed the same pattern, i.e., approximately the same intracellular platinum concentration (4.0 x 10-17 mol/cell) yielded the same cytotoxic effect. These results indicate that long-circulating pH-sensitive liposomes, also termed as stealth pH-sensitive liposomes, may present a promising delivery system for cisplatin-based cancer treatment. This liposome system proved to be able to circumvent the cisplatin resistance, whereas it was not observed when using non-long-circulating liposomes composed of phosphatidylcholine, phosphatidylserine, and cholesterol.

Interventions infirmières relatives à l'allaitement maternel de nouveau-nés prématurés

Problématique. Basée sur les constats effectués lors d’un essai clinique randomisé qui visait à évaluer les effets du lait maternel de fin d’expression sur la croissance et le développement de nouveau-nés prématurés et qui s’est soldé par un recrutement infructueux, une intervention de soutien à la lactation chez les mères de nouveau-nés prématurés a été développée. La mère d’un nouveau-né prématuré est en effet trois fois plus à risque qu’une autre de présenter une production lactée insuffisante. Il est donc crucial de soutenir ces mères dans l’établissement et le maintien d’une production lactée adéquate. Le but de cette étude pilote est d’estimer les effets d’une intervention de soutien à la lactation sur l’expression de lait maternel et la production lactée de mères ayant donné naissance prématurément ainsi que d’évaluer les aspects d’acceptabilité et de faisabilité de l’intervention, de l’étude et de ses procédures. Hypothèse de recherche. Les mères de nouveau-nés prématurés qui reçoivent une intervention de soutien à la lactation expriment leur lait significativement plus longtemps et plus fréquemment et produisent significativement un plus grand volume de lait à plus grande concentration lipidique sur une base quotidienne que celles qui reçoivent les soins usuels. Méthode. Devis : Projet-pilote de type essai clinique randomisé. Échantillon: Quarante mères de nouveau-nés prématurés de <30 semaines de gestation admis à une unité de soins intensifs néonatals. Procédures: Les mères du groupe témoin reçoivent les soins usuels alors que celles du groupe expérimental reçoivent une intervention de soutien à la lactation. Cette dernière comporte quatre volets: une séance d’enseignement portant sur l’établissement et le maintien d’une production lactée suffisante, un suivi téléphonique, une ligne d’aide téléphonique et le prêt d’un tire-lait électrique double pompage. Dans les deux groupes, les mères sont amenées à tenir un journal de bord de leurs séances d’expression et du volume de lait maternel exprimé. Résultats. L’étude et ses procédures de même que l’intervention de soutien sont acceptables et faisables. Les résultats observés en lien avec l’hypothèse de recherche sont orientés dans la même direction que cette dernière à l’exception de la concentration lipidique du lait maternel. Recommandations. Une étude à plus grande échelle doit être réalisée afin d’évaluer les effets de l’intervention de soutien à la lactation sur la production lactée de mères de nouveau-nés prématurés. Quant à la clinique, des actions concertées doivent être menées afin de créer un contexte propice et des conditions favorables à l’expression de lait maternel chez les mères de nouveau-nés prématurés.

Fish flesh agar medium - a suitable experimental medium for the detection of spoilage bacteria

The spoilage characteristics of bacterial strains were studied by growing them at 28 _+ 2 °C in agar and broth media prepared with sterile fish and prawn flesh homogenates. The percentage of spoilers found among the bacterial isolates tested, as shown by odour production and halo zone formation, was independent of the source of flesh used. Indole and fluorescent pigment production were also observed in the broth. Pseudomonas, Vibrio and Acinetobacter exhibited faster growth in flesh media than in the usual artificial media. Decrease of protein and lipid concentration in the clear zone of agar media suggests the utilization of the available substrate by spoilage bacteria.

Differential effects of temperature on fruit development and bean quality of contrasting genotypes of cacao (Theobroma cacao)

The effects of temperature and light integral on fruit growth and development of five cacao genotypes (Amelonado, AMAZ 15/15, SCA 6, SPEC 54/1 and UF 676) were studied in semi-controlled environment glasshouses in which the thermal regimes of cacao-growing regions of Brazil, Ghana and Malaysia were simulated. Fruit losses because of physiological will (cherelle will) were greater at higher temperatures and also differed significantly between genotypes, reflecting genetic differences in competition for assimilates between vegetative and reproductive components. Short-term measurements of fruit growth indicated faster growth rates at higher temperatures. In addition, a significant negative linear relationship between temperature and development time was observed. There was an effect of genotype on this relationship, such that time to fruit maturation at a given temperature was greatest for the clone UF 676 and least for AMAZ 15/15. Analysis of base temperatures, derived from these relationships indicated genetic variability in sensitivity of cacao fruit growth to temperature (base temperatures ranged from 7.5 degrees C for Amelonado and AMAZ 15/15 to 12.9 for SPEC 54/1). Final fruit size was a positive function of beam number for all genotypes and a positive function of light integral for Amelonado in the Malaysia simulated environment (where the temperature was almost constant). In simulated environments where temperature was the main variable (Brazil and Ghana) increases in temperature resulted in a significant decrease in final pod size for one genotype (Amelonado) in Brazil and for two genotypes (SPEC 54/1 and UF 676) in Ghana. It was hypothesised that pod growth duration (mediated by temperature), assimilation and beam number are all determinants of final pod size but that under specific conditions one of these factors may override the others. There was variability between genotypes in the response of beam size and beam lipid content to temperature. Negative relationships between temperature and bean size were found for Amelonado and UF 676. Lipid concentration was a curvilinear function of temperature for Amelonado and UF 676, with optimal temperatures of 23 degrees C and 24 degrees C, respectively. The variability observed here of different cacao genotypes to temperature highlights the need and opportunities for appropriate matching of planting material with local environments.

Consumption of mate tea (Ilex paraguariensis) decreases the oxidation of unsaturated fatty acids in mouse liver

Mate (Ilex paraguariensis) is rich in polyphenolic compounds, which are thought to contribute to the health benefits of tea. Mate tea was administered orally to mice at a dose of 0.5, 1.0 or 2.0 g/kg for 60 d, and changes both in serum lipid concentration and fatty acid composition of liver and kidney were examined. The effects of mate tea on serum and tissue lipid peroxidation were assessed by the evaluation of thiobarbituric acid-reactive substances (TBARS). In tea-consuming mice, both MUFA (18: 1n-9) and PUFA (18: 2n-6 and 20: 4n-6) were increased (P<0.05) in the liver lipid (approximately 90 and 60%, respectively), whereas only MUFA (approximately 20%) were increased in the kidney lipid. The most altered PUFA class was n-6 PUFA, which increased by approximately 60-75 % (P<0.05). This difference in the fatty acid profile in the liver is reflected in the increased PUFA:SFA ratio. Consistent with these results, mice fed with mate tea had much lower TBARS in the liver. No differences (P>0.05) were found in the levels of serum cholesterol, HDL-cholesterol and TAG under the conditions of the present study. These results suggest that treatment with mate tea was able to protect unsaturated fatty acids from oxidation and may have selective protective effects within the body, especially on the liver.