Vaccines against toxoplasma gondii : challenges and opportunities

Development of vaccines against Toxoplasma gondii infection in humans is of high priority, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects. Rodent models have been used in research on vaccines against T. gondii over the past decades. However, regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector. Only a few live, attenuated T. gondii strains used for immunization have been able to confer protective immunity, which is measured by a lack of tissue cysts after challenge. Furthermore, challenge with low virulence strains, especially strains with genotype II, will probably be insufficient to provide protection against the more virulent T. gondii strains, such as those with genotypes I or II, or those genotypes from South America not belonging to genotype I, II or III. Future studies should use animal models besides rodents, and challenges should be performed with at least one genotype II T. gondii and one of the more virulent genotypes. Endpoints like maternal-foetal transmission and prevention of eye disease are important in addition to the traditional endpoint of survival or reduction in numbers of brain cysts after challenge

<it>Rhodococcus erythropolis</it> ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration

<p>Abstract</p> <p>Background</p> <p>The suitability of the strain <it>Rhodococcus erythropolis</it> ATCC 25544 grown in a two-liter fermentor as a source of cholesterol oxidase has been investigated. The strain produces both cell-linked and extracellular cholesterol oxidase in a high amount, that can be extracted, purified and concentrated by using the detergent Triton X-114.</p> <p>Results</p> <p>A spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing. Both were similar as far as biomass yield is concerned. Cell-linked cholesterol oxidase was extracted with Triton X-114, and this detergent was also used for purification and concentration, following temperature-induced detergent phase separation. Triton X-114 was utilized to purify and to concentrate the cell-linked and the extracellular enzyme. Cholesterol oxidase was found mainly in the resulting detergent-rich phase. When Triton X-114 concentration was set to 6% w/v the extracellular, but not the cell-extracted enzyme, underwent a 3.4-fold activation after the phase separation process. This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium. Fermentation yielded 360 U/ml (672 U/ml after activation), 36% of which was extracellular (65% after activation). The Triton X-114 phase separation step yielded 11.6-fold purification and 20.3-fold concentration.</p> <p>Conclusions</p> <p>The results of this work may make attractive and cost-effective the implementation of this bacterial strain and this detergent in a purification-based industrial production scheme of commercial cholesterol oxidase.</p

Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and methyljasmonate

In this work, the effect of different inducing factors on trans-resveratrol extracellular production in Monastrell grapevine suspension cultured cells is evaluated. A detailed analysis provides the optimal concentrations of cyclodextrins, methyljasmonate and UV irradiation dosage, optimal cell density, elicitation time and sucrose content in the culture media. The results indicate that trans-resveratrol production decreases as the initial cell density increases for a constant elicitor concentration in Monastrell suspension cultured cells treated with cyclodextrins individually or in combination with methyljasmonate, the decrease observed in cell cultures elicited with cyclodextrins alone being far more drastic than those observed in the combined treatment. trans-Resveratrol extracellular production observed by the joint use of cyclodextrins and methyljasmonate (1447.8 ± 60.4 μmol trans-resveratrol g-1 dry weight) is lower when these chemical compounds are combined with UV light short exposure (669.9 ± 45.2 μmol trans-resveratrol g-1 dry weight). Likewise, trans-resveratrol production is dependent on levels of sucrose in the elicitation medium being the maximal levels observed with 20 g l-1 sucrose and the joint action of cyclodextrins and 100 µM methyljasmonate. The sucrose concentration did not seem limiting the process although significantly affect the specific productivity since the lowest sucrose concentration is (10 g l-1), the highest productivity is reached (100.7 ± 5.8 μmol trans-resveratrol g-1 dry weight g-1 sucrose) using cyclodextrins and 25 µM methyljasmonate.Fil: Belchi-Navarro, Sarai. Universidad de Murcia. Facultad de Biología; EspañaFil: Almagro, Lorena. Universidad de Murcia. Facultad de Biología; EspañaFil: Lijavetzky, Diego Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Bru, Roque. Universidad de Alicante. Facultad de Ciencias. Departamento de Agroquímica y Bioquímica; EspañaFil: Pedreño, María A.. Universidad de Murcia. Facultad de Biología; Españ

Dissecting the transcriptional response to elicitors in Vitis vinifera cells

The high effectiveness of cyclic oligosaccharides like cyclodextrins in the production of trans-resveratrol in Vitis vinifera cell cultures is enhanced in the presence of methyl jasmonate. In order to dissect the basis of the interactions among the elicitation responses triggered by these two compounds, a transcriptional analysis of grapevine cell cultures treated with cyclodextrins and methyl jasmonate separately or in combination was carried out. The results showed that the activation of genes encoding enzymes from phenylpropanoid and stilbene biosynthesis induced by cyclodextrins alone was partially enhanced in the presence of methyl jasmonate, which correlated with their effects on trans-resveratrol production. In addition, protein translation and cell cycle regulation were more highly repressed in cells treated with cyclodextrins than in those treated with methyl jasmonate, and this response was enhanced in the combined treatment. Ethylene signalling was activated by all treatments, while jasmonate signalling and salicylic acid conjugation were activated only in the presence of methyl jasmonate and cyclodextrins, respectively. Moreover, the combined treatment resulted in a crosstalk between the signalling cascades activated by cyclodextrins and methyl jasmonate, which, in turn, provoked the activation of additional regulatory pathways involving the up-regulation of MYB15, NAC and WRKY transcription factors, protein kinases and calcium signal transducers. All these results suggest that both elicitors cause an activation of the secondary metabolism in detriment of basic cell processes like the primary metabolism or cell division. Crosstalk between cyclodextrins and methyl jasmonate-induced signalling provokes an intensification of these responses resulting in a greater trans-resveratrol production