Anti-parasitic action and elimination of intracellular Toxoplasma gondii in the presence of novel thiosemicarbazone and its 4-thiazolidinone derivatives

Toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. In the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of Toxoplasma. The compounds were tested in infected Vero cell culture using concentration screens (0.1 to 20 mM). The final concentration of 1 mM was chosen for biological assay. The following results were obtained: 1) These new derivatives decreased T. gondii infection with an in vitro parasite IC50 percent of 0.2-0.7 mM, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) The main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) Ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) Vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) The death and elimination of T. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. Our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mM concentration for 24 h of treatment.

[Neutralization of helicobacter pylori cytotoxicity on vero cells by omeprazole]

Omeprazole is a gastric parietal cells proton pump inhibitor that is also active against H. pylori in vitro. This study was designed to examine the neutralization of H. pylori cytotoxicity on Vero cells by omeprazole micronized in strains isolated from gastritis, ulcer, cancer and Barrett's ulcer, to determine whether omeprazole can inhibit vacuolation of the Vero cells induced by cytotoxin of H. pylori or by urease. The effect of omeprazole on motility of H. pylori was assessed using concentrations lower than MIC. The antimicrobial activity of omeprazole micronized was studied by determining the MICs for 15 H. pylori strains. Water extract of the bacteria [concentrated culture supernatant] and different concentrations of omeprazole were added to Vero cells in culture. Also extracted urease from H. pylori strains with urea [10 mM] and omeprazole were added to Vero cells in culture. The inhibitory effect of omeprazole on motility of H. pylori was tested in semi-solid medium. MIC of omeprazole micronized was 20 micro g/ml. Omeprazole could inhibit induced vacuolation by the water extract of H. pylori strains in Vero cells. It could also inhibit vacuolation induced by urease. Inhibition of vacuolation strains was assessed microscopically and by the neutral red method. It was also found that omeprazole inhibits the motility of H. pylori strains at concentrations lower than MIC. The results of this study suggest that omeprazole micronized could neutralize the vacuolation effect of H. pylori cytotoxin on Vero cells probably by targeting v-type ATPase. The bacterial motility was also inhibited by low concentrations of omeprazole. The results of this study considers omeprazole micronized as an effective drug which targets important virulence factors of H. pylori including vacuolating cytotoxin, urease, and motility

Biological characterization of Trypanosoma cruzi strains

Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans

Determination of antigenic domain in GST fused major surface protein (Nc-p43) of Neospora caninum

The antigenic domain of the major surface protein (Nc-p43) of Neospora caninum was examined by polymerase chain reaction of its gene fragments and recombinant expression as GST fusion proteins. The fragments of Nc-p43 were as follow: a total open reading frame (OFR), T; OFR without signal sequence and C-terminal hydrophobic sequence, S; N-terminal 2/3 parts of S, A; C-terminal 2/3 parts, P; N-terminal 1/3 part, X; middle 1/3 part, Y; and C-terminal 1/3 part, Z, respectively. The DNA fragments were cloned into pGEX-4T vector. Recombinant plasmids transformed into Escherichia coli of BL21 pLysS (DE3) strain were induced to express GST or GST fused fragments of Nc-p43 such as 69 kDa protein for T, 66 kDa for S, 52 kDa for A, 53 kDa for P, and 40 kDa proteins for X, Y, and Z, respectively in SDS-PAGE. The Nc-p43 fragments of T, S, and P reacted with a bovine serum of neosporosis while those of A, X, Y, and Z together with GST did not in the western blot. These findings suggest that the antigenic domain of Nc-p43 of N. caninum may be localized in the C-terminal 2/3 parts. Together with A19 clone in SAG1 of Toxoplasma gondii (Nam et al., 1996), the P fragment of Nc-p43 could be used as efficient antigens to diagnose and differentiate those infections with both species.