A specific and sensitive liquid chromatography (LC)-tandem mass spectrometric method for quantitative determination of methylprednisolone (MP) in rat plasma and liver was developed and validated using triamcinolone acetonide as an internal standard. Liquid-liquid extraction using tertbutyl methyl ether was used to extract the drug and the internal standard from plasma and liver. The separation of MP was performed on a C 18 column with a mobile phase of acetonitrile:0.5% formic acid aqueous solution (85:15, v/v) over 4 min. The assay was based on the selected reaction monitoring transitions at m/z 375→161 for MP in plasma, 375→357 for MP in liver, and 435→415 for internal standard in both plasma and liver. The lower limit of quantification was 20 ng/mL based on 100 μL of plasma or liver homogenate. Intra-and inter-day assay variations were ≤15%, and the accuracy values were between 85.8 and 118%. The extraction recoveries ranged from 76.8 to 79.2% for plasma and 76.8 to 80.8% for liver across the calibration curve range. The method was successfully applied to measurement of low concentrations of regenerated MP in plasma and liver after intravenous administration of a single dose (5 mg/kg) of a liver-targeted dextran prodrug of MP to rats.

We previously described that DNA vaccination with the gene encoding amastigote surface protein 2 (ASP-2) protects approximately 65% of highly susceptible A/Sn mice against the lethal Trypanosoma cruzi infection. Here, we explored the possibility that bacterial recombinant proteins of ASP-2 could be used to improve the efficacy of vaccinations. Initially, we compared the protective efficacy of vaccination regimens using either a plasmid DNA, a recombinant protein, or both sequentially (DNA priming and protein boosting). Survival after the challenge was not statistically different among the three mouse groups and ranged from 53.5 to 75%. The fact that immunization with a recombinant protein alone induced protective immunity revealed the possibility that this strategy could be pursued for vaccination. We investigated this possibility by using six different recombinant proteins representing distinct portions of ASP-2. The vaccination of mice with glutathione S-transferase fusion proteins representing amino acids 261 to 500 or 261 to 380 of ASP-2 in the presence of the adjuvants alum and CpG oligodeoxynucleotide 1826 provided remarkable immunity, consistently protecting 100% of the A/Sn mice. Immunity was completely reversed by the in vivo depletion of CD8 ؉ T cells, but not CD4 ؉ T cells, and was associated with the presence of CD8 ؉ T cells specific for an epitope located between amino acids 320 and 327 of ASP-2. We concluded that a relatively simple formulation consisting of a recombinant protein with a selected portion of ASP-2, alum, and CpG oligodeoxynucleotide 1826 might be used to cross-prime strong CD8 ؉ -T-cell-dependent protective immunity against T. cruzi infection.

Background: Visceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis.

Trypanosoma cruzi expresses several proteins containing antigenic amino acid repeats. Here we characterized TcRpL7a and TcRBP28, which carry similar repeat motifs and share homology to the eukaryotic L7a ribosomal protein and to a Trypanosoma brucei RNA binding protein, respectively. Analyses of the full length and truncated recombinant TcRpL7a showed that the humoral response of patients with Chagas disease is directed towards its repetitive domain. Sequence analyses of distinct copies of TcRpL7a genes present in the genome of six T. cruzi strains indicate that the number of repeats is higher in proteins from T. cruzi II than T. cruzi I strains. A serum panel of 59 T. cruzi infected patients showed that 73% reacted with TcRpL7a, 71% reacted with TcRBP28 and 80% reacted with 1:1 mixture of both antigens. Synthetic peptides harboring the TcRpL7a repeat motif reacted with 46% of the serum samples. Antibodies raised against both antigens identified equivalent amounts of the native proteins in all three stages of the parasite life cycle. Analyses of subcellular fractions indicated that TcRBP28 is present in the cytoplasm whereas TcRpL7a co-fractionates with polysomes. Confirming their predicted cellular localization, GFP fusions showed that, whereas GFP::TcRBP28 localizes in the cytoplasm, GFP::TcRpL7a accumulates in the nucleus, where ribosome biogenesis occurs.

The effects of the alkylaminoalkanethiosulfuric acids (AAATs), new schistosomicidal drugs, on Schistosoma mansoni ATP diphosphohydrolase isoforms, members of the NTPDase family, were analyzed. Pre-incubation of worm adult tegument with AAATs derivatives, with small apolar alkyl groups and an apolar alkane portion of 6 or 8 carbon atoms linked to the amino group, inhibited ATPase activity with a Ki 100-1000 μM. Little inhibition (20%) was observed when ADP was the substrate. The 2-[(tert-butyl)amino]-1-ethanethiosulfuric acid (100 μM) which has a less lipophilic structure, inhibited 28% ATPase and 12% ADPase activities, suggesting that the lipophilicity, although important, is not the only requisite for enzyme activity inhibition. The N-(sec-butyl)-2-bromo-1-octanaminium bromide, which contains a bromide atom instead of thiosulphate, inhibited b 10% of the enzyme activity, suggesting the involvement of cysteine residue(s) from S. mansoni ATP diphosphohydrolase isoforms in a mixed disulfide formation. Treatment of parasite tegument with 5 mM iodoacetamide or 1 mM dithiothreitol protected ATPase and ADPase activities against inhibition by AAATs, corroborating the participation of disulfide interchange in the AAATs mechanism. Since S. mansoni ATP diphosphohydrolase isoforms and potato apyrase share structural similarities, the latter enzyme was also tested. ADPase activity from potato apyrase was inhibited by 55%, showing a higher sensitivity to 1 mM AAATs than that shown by ADPase activity from the tegument, while the ATPase activities from both samples showed similar inhibition levels. Furthermore, sulfhydryl reagents protected potato apyrase activity. Therefore, it is possible that both soluble S. mansoni ATP diphosphohydrolase and membrane-associated isoforms are targets for the AAATs. Published by Elsevier B.V.

We recently described that vaccination of mice with a gluthatione S transferase fusion protein representing amino acids 261-500 of the Amastigote Surface Protein-2 efficiently cross-primed protective CD8 + T cells against a lethal challenge with the human protozoan parasite Trypanosoma cruzi. In this study, we initially established that this protective immunity was long lived. Subsequently, we studied the importance of TLR9 agonist CpG ODN 1826, TLR4 and CD4 + T cells for the generation of these protective CD8 + T cells. We found that: (i) the TLR9 agonist CpG ODN 1826 improved the efficiency of protective immunity; (ii) TLR4 is not relevant for priming of specific CD8 + T cells; (iii) CD4 + T cells are critical for priming of memory/protective CD8 + T cells.

The kinetics of effector CD8 ؉ -T-cell responses to specific Trypanosoma cruzi epitopes was investigated after challenge. Our results suggest that the delayed kinetics differs from that observed in other microbial infections and facilitates the establishment of the disease in naïve mice. In contrast, in vaccinated mice, the swift CD8 ؉ -T-cell response helps host survival after challenge.

This paper discusses the development of a series of sulfur-containing compounds that show an interesting in vivo activity against infection by Schistosoma mansoni. These substances include the aminoalkanethiols, aminoalkanethiosulfuric acids and aminoalkyl disulfides, among others. Although the aminoethanethiols and their disulfide derivatives have presented a relatively high toxicity for the host animal, the aminoalkanethiosulfuric acids have a low toxicity and a high specificity for the adult female S. mansoni worms. In vitro studies with schistosomula, lung-phase schistosomula and adult worms have demonstrated effects on the tegument and the metabolism on these different stages of S. mansoni worms. The encapsulation of these drugs in a nanoemulsion has resulted in an increase in the in vitro activity.