Prediction of disease severity in neuromyelitis optica by the levels of interleukin (IL)-6 produced during remission phase.

T helper type 17 (Th17) cytokines have been implicated in the pathogenesis of neuromyelitis optica (NMO). As humanized anti-interleukin (IL)-6R (tocilizumab) immunoglobulin (Ig)G has been used as disease-modifying therapy for NMO, the objective of our study was to investigate the role of endogenous IL-6 on NMO-derived CD4(+) T cell behaviour. High production of IL-6, IL-17 and IL-21 by CD4(+) T-cells was detected in NMO patients. Further, IL-21 and IL-6 levels were related directly to the level of neurological disabilities. The addition of anti-IL-6R IgG not only reduced directly the production of these cytokines, but also almost abolished the ability of activated autologous monocytes in enhancing IL-6, IL-17 and IL-21 release by CD4(+) T cells. In contrast, the production of IL-10 was amplified in those cell cultures. Further, anti-IL-6R monoclonal antibodies (mAb) also potentiated the ability of glucocorticoid in reducing Th17 cytokines. Finally, the in-vivo and in-vitro IL-6 levels were significantly higher among those patients who experienced clinical relapse during 2-year follow-up. In summary, our results suggest a deleterious role of IL-6 in NMO by favouring, at least in part, the expansion of corticoid-resistant Th17 cells.

Effect of ammonia production on intracellular pH: Consequent effect on adenovirus vector production.

Recombinant adenoviral vectors (AdV) have proven to be highly efficient for the delivery and expression of foreign genes in a broad spectrum of cell types and species both for vaccination and gene therapy in a number of specific applications. In this study, the effect of ammonia production on intracellular pH (pH(i)) and consequently inhibition of AdV production at high cell densities is assessed. Different specific ammonia production rates were obtained for 293 cells adapted to grow in glutamate supplemented medium (non-ammoniagenic medium) as compared with 293 cells growing in glutamine supplemented medium (ammoniagenic medium); pH(i) was observed to be lower during cell growth and AdV production at both high and low CCI in the ammoniagenic medium, where the specific ammonia production rate is higher. In addition, after infection at CCI of 3x10(6)cell/ml, the cell viability decreased significantly in the ammoniagenic medium, attributed to the activation of an acidic pathway of apoptosis. Furthermore, AdV DNA was observed to be degraded at the observed pH(i) in the ammoniagenic medium, decreasing significantly the amount of AdV DNA available for encapsulation. To elucidate the pH(i) effect upon AdV production, 293 cells were infected at a CCI of 1 x 10(6)cell/ml in the non-ammoniagenic medium with a manipulated pH(i) as observed at the time of infection at CCI of 3 x 10(6)cell/ml in the ammoniagenic (pH(i) 7.0) and non-ammoniagenic (pH(i) 7.3) media; AdV volumetric productivities were observed to be lower when the cells were exposed to the lower pH(i). Thus, the importance of controlling all the factors contributing to pH(i) on AdV production, such as ammonia production, has been established.

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.

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.

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.