Biomedical subjects

P M Alves

Publications and source records attributed to P M Alves.

At least 19 recordsLinked to original sources

PubMed2006

Downstream processing of triple layered rotavirus like particles.

Rotavirus like particles (RLPs) constitute a potential vaccine for the prevention of rotavirus disease, responsible for the death of more than half a million children each year. Increasing demands for pre-clinical trials material require the development of reproducible, scaleable and cost-effective purification strategies as alternatives to the traditional laboratory scale CsCl density gradient ultracentrifugation methods commonly used for the purification of these complex particles. Self-assembled virus like particles (VLPs) composed by VP2, VP6 and VP7 rotavirus proteins (VLPs 2/6/7) were produced in 5l scale using the insect cells/baculovirus expression system. A purification process using depth filtration, ultrafiltration and size exclusion chromatography as stepwise unit operations was developed. Removal of non-assembled rotavirus proteins, concurrently formed particles (RLP 2/6), particle aggregates and products of particle degradation due to shear was achieved. Particle stability during storage was studied and assessed using size exclusion chromatography as an analytical tool. Formulations containing either glycerol (10% v/v) or trehalose (0.5 M) were able to maintain 75% of intact triple layered VLPs, at 4 degrees C, up to 4 months. The overall recovery yield was 37% with removal of 95% of host cell proteins and 99% of the host cell DNA, constituting a promising strategy for the downstream processing of other VLPs.

Animals↗

PubMed2006

Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability.

The use of Moloney murine leukaemia virus (MoMLV) derived retroviral vectors in gene therapy requires the production of high titer preparations. However, obtaining high titers of infective MoMLV retroviral vectors is difficult due to the vector inherent instability. In this work the effect of the cell culture medium osmotic pressure upon the virus stability was studied. The osmolality of standard medium was raised from 335 up to 500 mOsm/kg using either ionic (sodium chloride) or non-ionic osmotic agents (sorbitol and fructose). It was observed that, independently of the osmotic agent used, the infectious vector inactivation rate was inversely correlated with the osmolality used in the production media; therefore, the use of high medium osmolalities enhanced vector stability. For production purposes a balance must be struck between cell yield, cell productivity and retroviral stability. From the conditions tested herein sorbitol addition, ensuring osmolalities between 410 and 450 mOsm/kg, yields the best production conditions; NaCl hampered the viral infectious production while fructose originates lower cell yields. Lipid extractions were performed for cholesterol and phospholipid analyses showing that more stable viral vectors had a 10% reduction in the cholesterol content. A similar reduction in cholesterol was observed in the producer cells. A detailed analysis of the major phospholipids composition, type and fatty acid content, by mass spectrometry did not show significant changes, confirming the decrease in the cholesterol to phospholipids ratio in the viral membrane as the major reason for the increased vector stability.

Cell Line↗

PubMed2006

Effect of medium sugar source on the production of retroviral vectors for gene therapy.

The production of gene therapy retroviral vectors presents many difficulties, mainly due to vector instability and low cell productivities hampering the attainment of high titers of infectious viral vectors. The objective of this work is to increase the production titers of retroviral vectors by manipulating the sugar carbon sources used in bioreaction. Four sugars were tested (glucose, galactose, sorbitol, and fructose) on an established Moloney murine leukemia virus (MoMLV) producer cell line. Galactose and sorbitol did not support cell growth or vector production. Glucose supplemented at 25 mM supported the highest cell growth; however, the use of glucose or fructose at 83 and 140 mM have shown to improve the infectious vector titer three to fourfold. The reasons for the titer improvements were further analyzed and, although, the cell-specific productivity in viral transgene RNA and reverse transcriptase were augmented 5- and 6-fold for glucose at 140 mM and 14- and 16-fold for fructose at 140 mM, comparing with glucose at 25 mM, these increases did not seem sufficient to account for the 14- (140 mM glucose) and 32- (140 mM fructose) fold increment obtained for the infectious particles-specific productivity. Further accounting the enhancement in the titers was the improvement in the viral stability, the half-life of the vectors was enhanced by 30-60%. This resulted in a product quality with a superior ratio of infectious to total particles, thus reducing the most problematic contaminant in the production of retroviral vectors, non-infectious retroviral particles.

Carbohydrates↗

PubMed2006

Retrovirus producer cell line metabolism: implications on viral productivity.

The production of retroviral vectors by human cell lines is still hampered by low titers making it relatively difficult to produce very large quantities of this vector of high interest for clinical gene therapy applications. Thus, to improve vector production, we studied the influence of different sugars alone or combinations of sugars on cell growth, vector titers, and metabolism of the producer cell. The use of fructose at 140 mM or a mixed medium (with glucose at 25 mM and fructose at 140 mM) improved the virus titer three- to fourfold, respectively, and the producer cell productivity by fivefold. The increase in the cell productivity was due to a 1.5-fold increase in the vector stability, the remaining increase being due to higher cell specific productivity. The increase in the productivity was associated with lower glucose oxidation and an increase in the lactate and alanine yield. In the mixed medium, an increase in fatty acids derived from the glucose was observed in parallel with a reduction of glutamate and glutamine synthesis via the tricarboxylic acid (TCA) cycle acetyl-CoA and alpha-ketoglutarate, respectively. Although the higher productivities were associated with severe changes in the glycolysis, TCA cycle, and glutaminolysis, the cell energetic status monitored by phosphocreatine and adenosine triphosphate levels was not significantly affected. The synthesis of fatty acids and phospholipids were enhanced in the fructose or mixed media and are possibly key parameters in retroviral vector production.

Adenosine Triphosphate↗

PubMed2006

Relationship between retroviral vector membrane and vector stability.

The present work studies the physico-chemical properties of retroviral vector membrane, in order to provide some explanation for the inactivation kinetics of these vectors and to devise new ways of improving transduction efficiency. For this purpose, vectors with an amphotropic envelope produced by TE Fly A7 cells at two culture temperatures (37 and 32 degrees C) were characterized by different techniques. Electron paramagnetic resonance (EPR) results showed that vectors produced at 32 degrees C are more rigid than those produced at 37 degrees C. Further characterization of vector membrane composition allowed us to conclude that the vector inactivation rate increases with elevated cholesterol to phospholipid ratio. Differential scanning calorimetry (DSC) showed that production temperature also affects the conformation of the membrane proteins. Transduction studies using HCT116 cells and tri-dimensional organ cultures of mouse skin showed that vectors produced at 37 degrees C have higher stability and thus higher transduction efficiency in gene therapy relevant cells as compared with vectors produced at 32 degrees C. Overall, vectors produced at 37 degrees C show an increased stability at temperatures below 4 degrees C. Since vector membrane physico-chemical properties are affected in response to changes in culture temperature, such changes, along with alterations in medium composition, can be used prospectively to improve the stability and the transduction efficiency of retroviral vectors for therapeutic purposes.

Animals↗

PubMed2005

Purification of adenoviral vectors using expanded bed chromatography.

The increasing numbers of pre-clinical and clinical trials where recombinant adenoviral vectors are used for gene therapy and vaccination require the development of cost-effective and reproducible large scale purification strategies of the biologically active particles. Alternatives to the traditional laboratory scale CsCl density gradient ultracentrifugation method, such as fixed bed chromatography strategies, have been developed, but the yields of final recovery remain too low due mainly to the capture and concentration steps taking place before and between the chromatographic stages. In this study, a rapid and efficient scale-able purification protocol allowing to obtain concentrated, pure and bioactive adenoviral vectors was developed. This allows efficient levels of binding to the column media and vector purification without centrifugation or filtration steps. Expanded bed chromatography followed by hollow fiber concentration allows the capture of viral particles directly from cellular extracts with high efficiency and vector purification is achieved in less than one working day with a minimal amount of sample handling, thus presenting an improvement over existing processes. The overall process yield reached 32%, representing an eight-fold improvement over results reported previously, while the purity is comparable to that obtained with the CsCl method.

Adenoviridae↗

PubMed2005

Effect of re-feed strategies and non-ammoniagenic medium on adenovirus production at high cell densities.

Recombinant adenoviruses became one of the vectors of choice for delivery and expression of foreign proteins for gene therapy and vaccination purposes. Nevertheless, the production of adenovirus is currently limited by the so-called "cell density effect", i.e., a drop in cell specific productivity concomitant with increased cell concentration at infection (CCI). This work describes the characterisation and optimisation of the infection process in order to improve recombinant adenovirus type 5 yields at high cell densities. For that purpose, 293 cells adapted to suspension were grown in 2l bioreactors and infected at different cell concentrations, using different re-feed strategies, while evaluating cell metabolism. The consumption of amino acids is enhanced during infection, although no amino acid limitation was detected for cells infected at concentrations in the range of 2 x 10(6)cell/ml, for which the highest volumetric productivity was obtained in batch mode. Conversely, infecting at cell concentrations in the range of 3 x10(6)cell/ml led to complete depletion of glucose, glutamine and threonine before the optimal harvesting time, a significant decrease in volumetric productivity being observed; the effect of amino acids and glucose addition at infection time on cell specific and volumetric productivity of adenovirus was assessed, no improvement on adenovirus production being achieved. The effect of ammonia, present in high concentrations at 3 x10(6)cell/ml, was evaluated and seem to be detrimental; an 1.8-fold increase on adenovirus volumetric productivity was obtained for infections performed at 3 x10(6)cell/ml when non-ammoniagenic medium was used.

Adenoviridae↗

PubMed2005

Modelling and optimization of a recombinant BHK-21 cultivation process using hybrid grey-box systems.

In this work a model-based optimization study of fed-batch BHK-21 cultures expressing the human fusion glycoprotein IgG1-IL2 was performed. It was concluded that due to the complexity of the BHK metabolism it is rather difficult to develop a kinetic model with sufficient accuracy for optimization studies. Many kinetic expressions and a large number of parameters are involved resulting in a complex identification problem. For this reason, an alternative more cost-effective methodology based on hybrid grey-box models was adopted. Several model structures combining the a priori reliable first principles knowledge with black-box models were investigated using data from batch and fed-batch experiments. It has been reported in previous studies that the BHK metabolism exhibits modulation particularities when compared to other mammalian cell lines. It was concluded that these mechanisms were effectively captured by the hybrid model, this being of crucial importance for the successful optimization of the process operation. A method was proposed to monitor the risk of hybrid model unreliability and to constraint the optimization results to acceptable risk levels. From the optimization study it was concluded that the process productivity may be considerably increased if the glutamine and glucose concentrations are maintained at low levels during the growth phase and then glutamine feeding is increased.

Animals↗

PubMed2005

Use of adenoviral vectors as veterinary vaccines.

Vaccines are the most effective and inexpensive prophylactic tool in veterinary medicine. Ideally, vaccines should induce a lifelong protective immunity against the target pathogen while not causing clinical or pathological signs of diseases in the vaccinated animals. However, such ideal vaccines are rare in the veterinary field. Many vaccines are either of limited effectiveness or have harmful side effects. In addition, there are still severe diseases with no effective vaccines. A very important criterion for an ideal vaccine in veterinary medicine is low cost; this is especially important in developing countries and even more so for poultry vaccination, where vaccines must sell for a few cents a dose. Traditional approaches include inactivated vaccines, attenuated live vaccines and subunit vaccines. Recently, genetic engineering has been applied to design new, improved vaccines. Adenovirus vectors are highly efficient for gene transfer in a broad spectrum of cell types and species. Moreover, adenoviruses often induce humoral, mucosal and cellular immune responses to antigens encoded by the inserted foreign genes. Thus, adenoviruses have become a vector of choice for delivery and expression of foreign proteins for vaccination. Consequently, the market requirements for adenovirus vaccines are increasing, creating a need for production methodologies of concentrated vectors with warranted purity and efficacy. This review summarizes recent developments and approaches of adenovirus production and purification as the application of these vectors, including successes and failures in clinical applications to date.

Adenoviridae↗

PubMed2005

Semisolid topical formulations containing nimesulide-loaded nanocapsules, nanospheres or nanoemulsion: development and rheological characterization.

The objective of this work was to develop and characterize semisolid topical formulations containing nimesulide-loaded nanospheres, nanocapsules or nanoemulsion. The nanoprecipitation and spontaneous emulsification methods were used to prepare the colloidal suspensions and the nanoemulsion. The hydrodynamic diameters were 282 nm for the nanoemulsion, 293 nm for the nanocapsules and 191 nm for the nanospheres containing nimesulide. The encapsulation efficiencies were close to 99% in all cases and pH values ranged between 5.1 and 5.3. Each drug-loaded nanocarrier formulation was incorporated in Carbopol 940 gels. The semisolid dosage forms showed yellowish, glossy and homogeneous aspect after the incorporation of the colloidal suspensions and nanoemulsion. The recovery of nimesulide and the pH values for the gels containing nanoemulsion, nanospheres or nanocapsules remained constant during storage (120 days). For all formulations, the rheograms exhibited a non-Newtonian behavior presenting pseudoplastic characteristics and shear thinning. The rheograms were adjusted to Ostwald's model showing regression coefficients higher than 0.9900. None thixotropic phenomenon was experimentally detected under the test conditions for all formulations.

Algorithms↗

PubMed2004

Quantitation of MLV-based retroviral vectors using real-time RT-PCR.

Murine leukaemia virus-based vectors quantitation is a time consuming process that can take up to five days. In order to reduce this time a real-time RT-PCR was developed. This method quantifies vectors without an RNA extraction step, using AMV reverse transcriptase and LightCycler technology. Besides a low quantitation time, this method has the advantages of using a plasmid DNA standard curve with good reproducibility, and of having a high sensitivity (3 x 10(2) particles/microl) as well as an excellent intra- and inter-assay reproducibility. Although the method described quantifies vector particles with RNA whether these particles are infectious or not, it is possible to use it to determine infectious particles concentration after the establishment of a correlation between particles with RNA and infectious particles, for a given set of conditions. This method can also be used to study vector stability by comparison of infectious particles, total particles and particles with RNA.

Genetic Vectors↗

PubMed2001

Effect of ethanol on the metabolism of primary astrocytes studied by (13)C- and (31)P-NMR spectroscopy.

Nuclear magnetic resonance was used as the primary technique to investigate the effect of ethanol (40, 80, and 160 mM) on the levels of high-energy phosphates, glycolytic flux, anaplerotic and oxidative fluxes to the tricarboxylic acid (TCA) cycle, the contribution of the pentose phosphate pathway (PPP), and the uptake and release of amino acids on primary cultures of rat astrocytes. On line (31)P-NMR spectroscopy showed that long-term exposure to ethanol caused a drop in the levels of ATP and phosphocreatine. The ratio between the fluxes through the pyruvate dehydrogenase and pyruvate carboxylase reactions also decreased, whereas the glycolytic flux and the ratio between formation of lactate and glucose consumption increased when cells were exposed to acute doses of ethanol. Flux through the pentose phosphate pathway was not affected. The uptake of cysteine and the release of glutamine were stimulated by ethanol, whereas the release of methionine was inhibited. Moreover, the fractional enrichment in serine was enhanced. The changes in the amino acid metabolism are interpreted as a response to oxidative stress induced by ethanol.

Adenine Nucleotides↗

PubMed2001

Use of automated external defibrillators in a Brazilian airline. A 1-year experience.

After the incorporation of automated external defibrillators by other airlines and the support of the Brazilian Society of Cardiology, Varig Airlines began the onboard defibrillation program with the initial purpose of equipping wide-body aircrafts frequently used in international flights and that airplanes use in the Rio - São Paulo route. With all flight attendants trained, the automated external defibrillation devices were incorporated to 34 airplanes of a total fleet of 80 aircrafts. The devices were installed in the baggage compartments secured with velcro straps and 2 pairs of electrodes, one or which pre-connected to the device to minimize application time. Later, a portable monitor was address to the resuscitation kit in the long flights. The expansion of the knowledge of the basic life support fundamentors and the corrected implantation of the survival chain and of the automated external defibrillators will increase the extense of recovery of cardiorespiratory arrest victims in aircrafts.

Adult↗

PubMed2000

Metabolic shifts do not influence the glycosylation patterns of a recombinant fusion protein expressed in BHK cells.

BHK-21 cells expressing a human IgG-IL2 fusion protein, with potential application in tumor-targeted therapy, were grown under different nutrient conditions in a continuous system for a time period of 80 days. At very low-glucose (< 0.5 mM) or glutamine (< 0. 2 mM) concentrations, a shift toward an energetically more efficient metabolism was observed. Cell-specific productivity was maintained under metabolically shifted growth conditions and at the same time an almost identical intracellular ATP content, obtained by in vivo (31)P NMR experiments, was observed. No significant differences in the oligosaccharide structures were detected from the IgG-IL2 fusion protein preparations obtained by growing cells under the different metabolic states. By using oligosaccharide mapping and MALDI/TOF-MS, only neutral diantennary oligosaccharides with or without core alpha1-6-linked fucose were detected that carried no, one or two beta1-4-linked galactose. Although the O-linked oligosaccharide structures that are present in the IL2 moiety of the protein were studied with less detail, the data obtained from the hydrazinolysis procedure point to the presence of the classical NeuAcalpha2-3Galbeta1-3GalNAc structure. Here, it is shown that under different defined cellular metabolic states, the quality of a recombinant product in terms of O- and N-linked oligosaccharides is stable, even after a prolonged cultivation period. Moreover, unaffected intracellular ATP levels under the different metabolic states were observed.

Animals↗

PubMed2000

NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia.

Changes in high-energy phosphate metabolites (ATP and phosphocreatine) were monitored, in real time, by 31P-nuclear magnetic resonance in primary cell cultures of neurons and astrocytes during periods of hypoxia, ischemia and hypoglycemia, and also during the recovery periods following the re-establishment of standard conditions. Cells were immobilized in basement membrane gel threads and perfused with oxygen-depleted medium (oxygen concentration below 30 microM), to create hypoxic conditions, or with aerobic medium (oxygen concentration approximately 460 microM) containing different concentrations of glucose (hypoglycemia). Ischemic conditions were imposed by stopping perfusion for different periods of time (15 min to 2 h). The experimental set-up enabled the acquisition of 31P-spectra with high signal-to-noise ratio within 10-20 min for both cell types. The effect of hypoxia on glucose metabolism was assessed by 13C-NMR using [1-13C]glucose as substrate. The levels of ATP and PCr in astrocytes were unaffected during hypoxia (up to 2 h), but decreased notably under ischemia. In neurons, hypoxic periods caused a sharp drop of the ATP and PCr levels, and considerable damage to the capacity of neurons to replenish the ATP and PCr pools upon returning to normoxic conditions. However, neurons were remarkably less sensitive to ischemic conditions, the ATP and PCr pools being restored quickly, even after 2 h under challenging conditions. The data show that neurons were more resistant to ischemia than astrocytes, and suggest that the capacity to sustain the pools of ATP and PCr was part of the neuronal protective strategy.

Animals↗

PubMed1999

Proteolytic activity in infected and noninfected insect cells: degradation of HIV-1 Pr55gag particles.

In this work the proteolytic activity in the supernatant and inside insect cells in culture was evaluated for different multiplicities of infection (MOI) and times of infection (TOI). Several methods to detect proteolytic activity in insect cells were tested and that using fluorescein thiocyanite-casein as a substrate was chosen. It was observed that infection caused not only a reduction in the concentration of proteases by decreasing their synthesis but also an inhibition of the intracellular proteolytic activity by increasing the intracellular ATP level (measured by in vivo nuclear magnetic resonance, NMR). The maximum proteolytic activity in the supernatant was observed at 72 hpi except when the cells were infected in the late exponential growth phase or with very low MOI, yielding a nonsynchronous infection. The proteolytic degradation of Pr55gag particles was studied during culture and after harvest. In this particular case it was concluded that the supernatant should be stored at low temperature or quickly purified, since the degradation after 24 h is only 3% at 4 degrees C while at 27 degrees C this value rises to 23%. There is a complex relationship between MOI, TOI, proteolytic activity, and product titer and quality. Thus, the optimal conditions for each case will be a compromise between the final product titer, the desired product quality, and operational issues like process time and capacity, requiring proper integration between bioreaction and downstream processing.

Adenosine Triphosphate↗

PubMed1996

Immobilization of primary astrocytes and neurons for online monitoring of biochemical processes by NMR.

Immobilization of primary astrocytes and cerebellar granule neurons in basement membrane gel threads and on porous microcarriers, respectively, is described. Five different porous microcarriers were examined for their capabilities to support maturation of neurons, as evaluated by determinations of protein content and glucose uptake. These systems proved suitable for perfusion in the NMR tube for long periods of time. The energetic status of the cells was monitored by 31P-NMR, and a very good temporal resolution (less than 30 min) was achieved with superfused astrocytes; thus, this provides a valuable method for real time NMR investigation of biochemical processes in primary astrocytes. Significantly longer acquisition times (approximately 8 h) were required in order to obtain a spectrum of neurons with a reasonably good signal to noise ratio, even so, the results presented here and obtained with this difficult system are very encouraging.

Animals↗

PubMed1995

Lactate metabolism in mouse brain astrocytes studied by [13C]NMR spectroscopy.

Astrocytes possess at least two pathways for pyruvate and thus lactate formation involving precursors derived from mitochondria. The present results suggest that malic enzyme is the preferred route for this process. Although overall lactate release appeared to be independent of extracellular lactate concentration, the incorporation of mitochondrial precursors was decreased by starvation, which is known to deplete astrocyte glycogen stores. Using 1-[13C]glucose in the presence of 10 mM lactate led to label incorporation into extracellular lactate which increased from 3.4 +/- 0.4 to 6.5 +/- 0.5% during 4 and 6 h incubation periods, respectively. Lactate production from glycolysis proceeded virtually unaffected by the extracellular lactate concentration. The total amount of lactate in the medium decreased, however, demonstrating that lactate was used as a substrate.

Animals↗