Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector

Preliminary Characterization of Mus musculus–Derived Pathogenic Strains of Leptospira borgpetersenii Serogroup Ballum in a Hamster Model

Human and animal leptospirosis caused by Leptospira spp. belonging to serogroup Ballum has increased worldwide in the past decade. We report the isolation and serologic and molecular characterization of four L. borgpetersenii serogroup Ballum isolates obtained from Mus musculus, and preliminary virulence studies. These isolates are useful for diagnosis of leptospirosis and for epidemiologic studies of its virulence and pathogenic mechanisms

New Trends on Antineoplastic Therapy Research: Bullfrog (Rana catesbeiana Shaw) Oil Nanostructured Systems

Bullfrog oil is a natural product extracted from the Rana catesbeiana Shaw adipose tissue and used in folk medicine for the treatment of several diseases. The aim of this study was to evaluate the extraction process of bullfrog oil, to develop a suitable topical nanoemulsion and to evaluate its efficacy against melanoma cells. The oil samples were obtained by hot and organic solvent extraction processes and were characterized by titration techniques and gas chromatography mass spectrometry (GC-MS). The required hydrophile-lipophile balance and the pseudo-ternary phase diagram (PTPD) were assessed to determine the emulsification ability of the bullfrog oil. The anti-tumoral activity of the samples was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for normal fibroblast (3T3) and melanoma (B16F10) cell lines. Both extraction methods produced yielded around 60% and the oil was mainly composed of unsaturated compounds (around 60%). The bullfrog oil nanoemulsion obtained from PTPD presented a droplet size of about 390 nm and polydispersity = 0.05 and a zeta potential of about −25 mV. Both the bullfrog oil itself and its topical nanoemulsion did not show cytotoxicity in 3T3 linage. However, these systems showed growth inhibition in B16F10 cells. Finally, the bullfrog oil presented itself as a candidate for the development of pharmaceutical products free from cytotoxicity and effective for antineoplastic therapy