The use of mice as animal model for testing acute toxicity (LD-50) of toxic shock syndrome toxin / Utilização de camundongos como modelo animal para a verificação da toxicidade aguda da toxina-1 da síndrome do choque tóxico

Acute toxicity test (LD-50) using toxic shock syndrome toxin (TSST-1) was tested in BALB/c, C57BL/6 and Swiss mice. Animals (n = 10) were intraperitoneally injected with TSST-1 (0.01-10.0µg/mouse) followed 4h later by potentiating dose of lipopolysaccharide (75.0µg of LPS - E. coli O111:B4) and cumulative mortality was recorded over 72h. Control animals received either TSST-1 or LPS alone. The data were submitted to qui-Square test and acute toxicity test was calculated by probit analysis (confidence limits expressed as µg toxin/kg). BALB/c mice was the most sensitive (20.0µg/kg, 95 percent confidence limits: 9.0-92.0) followed by C57BL/6 (38.5µg/kg, 95 percent confidence limits: 9.11- 401.6). Data from Swiss mice was not conclusive, indicating only low sensitivity. Selection of the animal model and standardization of the experiment are fundamental for the development of serum neutralization tests used for final quality control of vaccine production.

A toxicidade aguda (DL-50) da toxina da síndrome do choque tóxico (TSST-1) foi testada em linhagens de camundongos BALB/c, C57BL/6 e Suíça. Os animais (n=10) inoculados intraperitoneal com doses crescentes de toxina (0,01 - 10,0µg/animal) receberam 4h após 75µg de LPS (E. coli O111: B4). A toxicidade aguda (DL50) foi observada por um período de 72h e os dados submetidos ao teste de qui- quadrado. Os resultados e os limites de confiança foram expressos em µg de toxina/kg. A linhagem BALB/c apresentou maior sensibilidade (20µg/kg - limite de confiança a 95 por cento entre 9,0- 92,0), seguida da C57BL/6 (38,5µg/kg - limite de confiança a 95 por cento entre 9,11 - 401,6). A amplitude dos limites de confiança deve-se à natureza da toxina, ao mecanismo de ação, a via de inoculação e ao animal utilizado. A seleção do modelo animal e a padronização do experimento são fundamentais para o desenvolvimento de testes de soro neutralização para fins de controle de qualidade do processo de produção de vacinas.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 æM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.

Determination of the neutralizing potency of horse antivenom against bothropic and crotalic venoms by indirect enzyme immunoassay

The use of ELISA to determine antisnake venom potency of horse immune sera should provide benefits of cost and reproducibility compared to in vivo assays. In the present investigation we evaluated the correlation between ELISA antibody levels and in vivo neutralization assays. For the indirect ELISA method, 0.016 µg/well of Bothrops jararaca or Crotalus durissus terrificus venom were used to coat the plates and 100 µl/well of each sample of antibothropic or anticrotalic venom sera were used at 1:10,000 dilution. Sheep anti-horse IgG conjugated to peroxidase was added and the substrate H202/o-phenylenediamine produced the color that was read at 492 nm. A correlation coefficient of r=0.97 was found for anticrotalic venom antibodies and no significant correlation was observed for antibothropic venom sera using 16 serum samples from immunized horses. However, when three antibothropic venom sera showing high in vivo neutralization...