Effective protection of mice against Shigella flexneri with a new self-adjuvant multicomponent vaccine.

PURPOSE: The aim of this study was to develop an immunogenic protective product against Shigella flexneri by employing a simple and safe heat treatment-based strategy. METHODOLOGY: The physicochemical characteristics of naturally produced (OMV) and heat-induced (HT) outer-membrane vesicles from S. flexneri were examined, including a comparison of the protein content of the products. Toxicological and biodistribution studies, and a preliminary experiment to examine the protective effectiveness of HT in a murine model of S. flexneri infection, were also included. RESULTS: This method simultaneously achieves complete bacterial inactivation and the production of the HT vaccine product, leading to a safe working process. The obtained HT complex presented a similar morphology (electron microscopy) and chemical composition to the classical OMV, although it was enriched in some immunogens, such as lipoproteins, OmpA or OmpC, among others. The HT formulation was not toxic and biodistribution studies performed in mice demonstrated that the vaccine product remained in the small intestine after nasal administration. Finally, a single dose of HT administered nasally was able to protect mice against S. flexneri 2a. CONCLUSION: The convenient and safe manufacturing process, and the preliminary biological evaluation, support the use of the self-adjuvanted HT complex as a new vaccine candidate to face shigellosis. Further development is required, such as additional immune analyses, to evaluate whether this new subunit vaccine can be useful in achieving full protection against Shigella.

Validation of a UHPLC-FLD analytical method for the simultaneous quantification of aflatoxin B1 and ochratoxin a in rat plasma, liver and kidney.

A rapid and simple method for the simultaneous quantification of AFB1 and OTA in rat plasma, liver and kidney by UHPLC-FLD has been successfully validated according to the following criteria: selectivity, stability, linearity, precision, accuracy, recovery, robustness and limits of quantification and detection. The extraction method, calibration curves and chromatographic conditions are common for the three matrices. Plasma and homogenized tissue samples (100 µL) were extracted with acetonitrile:formic acid mixture (99:1) (300 µL). Chromatographic separation was performed with a mixture of water and acetonitrile:methanol (50:50), both acidified with 0.5% of formic acid using a gradient profile. The method avoids the use of immunoaffinity columns and allows reduction of sample and solvent volumes as well as toxic wastes. The detection is based on a photochemical reaction which enhances the AFB1 response without affecting the OTA signal before reaching the fluorescent detector. The mycotoxin recovery for each matrix was very efficient, between 93% and 96% for AFB1 and between 94% and 96% for OTA. For both mycotoxins the LOQs were 2µg/L in plasma and 8µg/kg in liver and kidney. The method has successfully been applied to rat samples after a single oral administration of a mixture of AFB1 and OTA and it could be a useful tool in toxicokinetic and toxicological studies.

Genetic toxicology and preliminary in vivo studies of nitric oxide donor tocopherol analogs as potential new class of antiatherogenic agents.

Nitric oxide donor tocopherol analogs were found to be incorporated in low-density lipoprotein to release nitric oxide into the hydrophobic core of the lipoprotein, thus inhibiting lipid oxidation processes associated with atheroma plaque formation. Previously, we studied their cytotoxicity against human and murine macrophages as first selection for in vivo studies. Herein, we examined both the in vitro mutagenic and DNA-damage effects of selected compounds to further evaluate drug potential. While the compounds of interest were nongenotoxics in both experimental tests (Ames and alkaline comet), one of the potential blood metabolites exhibited genotoxicity (alkaline comet test), and the furazan derivative was mutagenic (Ames test). Two selected (nitrooxy and furoxan) compounds were studied in long- and short-term in vivo treatment, and in these conditions, animal toxicity was not evidenced, suggesting the possibility of these compounds as potential antiatherogenic drugs.

Cytotoxic palladium complexes of bioreductive quinoxaline N1,N4-dioxide prodrugs.

Four new palladium(II) complexes with the formula Pd(L)(2), where L are quinoxaline-2-carbonitrile N(1),N(4)-dioxide derivatives, were synthesized as a contribution to the chemistry and pharmacology of metal compounds with this class of pharmacologically interesting bioreductive prodrugs. Compounds were characterized by elemental, conductometric and thermogravimetric analyses, fast atom bombardment mass spectrometry (FAB-MS) and electronic, Fourier transform infrared (FTIR) and (1)H-nuclear magnetic resonance spectroscopies. The complexes were subjected to cytotoxic evaluation on V79 cells in hypoxic and aerobic conditions. In addition, a preliminary study on interaction with plasmid DNA in normoxia was performed. Complexes showed different in vitro biological behavior depending on the nature of the substituent on the quinoxaline ring. Pd(L1)(2) and Pd(L2)(2), where L1 is 3-aminoquinoxaline-2-carbonitrile N(1),N(4)-dioxide and L2 is 3-amino-6(7)-methylquinoxaline-2-carbonitrile N(1),N(4)-dioxide, showed non selective cytotoxicity, being cytotoxic either in hypoxic or in aerobic conditions. On the other hand, Pd(L3)(2), where L3 is 3-amino-6(7)-chloroquinoxaline-2-carbonitrile N(1),N(4)-dioxide, resulted in vitro more potent cytotoxin in hypoxia (P=5.0 microM) than the corresponding free ligand (P=9.0 microM) and tirapazamine (P=30.0 microM), the first bioreductive cytotoxic drug introduced into clinical trials. In addition, it showed a very good selective cytotoxicity in hypoxic conditions, being non-cytotoxic in normoxia. Its hypoxic cytotoxicity relationship value, HCR, was of the same order than those of other hypoxia selective cytotoxins (i.e., Mitomycine C, Misonidazole and the N-oxide RB90740). Interaction of the complexes with plasmid DNA in normoxia showed dose dependent ability to relax the negative supercoiled forms via different mechanisms. Pd(L2)(2) introduced a scission event in supercoiled DNA yielding the circular relaxed form. Meanwhile, both Pd(L1)(2) and Pd(L3)(2) produced the loss of negative supercoils rendering a family of topoisomers with reduced electrophoretic mobility. Pd(L3)(2) showed a more marked effect than Pd(L1)(2). Indeed, for the highest doses assayed, Pd(L3)(2) was even able to introduce positive supercoils on the plasmid DNA.