Immunohistochemical study of the vaginal inflammatory response in experimental trichomoniasis.

In the present paper, the acute and subchronical inflammatory processes of the vaginal epithelial were studied in mice experimentally infected with two Trichomonas vaginalis strains of different pathogenicity, by means of histological and immunological methods. There was an increase in the stratified epithelium layers as well as edema produced by the increase of vascularization in the propia submucosa and infiltration of leukocytes. The proliferation of the vaginal epithelium favors the settlement and persistence of the parasitic infection. All of the findings corresponded with signs of a systemic disease being observed in the animals, including significant weight loss and also intestinal invasion. The entire inflammatory process has been corroborated by studies of adhesion molecules such as E-Selectin, VCAM-1 and PECAM-1. A correlation between the time of appearance and the perseverance of the inflammatory process with E-Selectin and VCAM-1 expression was observed, but not with PECAM-1. The strain with a higher pathogenicity was able to invade deep vaginal tissues and thus, parasites could not be detected by vaginal washings. This may be an important cause of diagnosis and treatment failure. Also, by the different localization of trichomonads, it appeared that the battle between host and parasite took place in different areas dependent upon the characteristics of the strain.

Effect of piroxicam, metamizol, and S-adenosylmethionine in a murine model of experimental trichomoniasis.

Biological effects of piroxicam, metamizol, and S-adenosylmethionine (S-AMET) have been tested in NMRI mice infected intraperitoneally with Trichomonas vaginalis. An intraperitoneal treatment during ten preinfection days with piroxicam (10 mg/Kg/day), or metamizol (275 mg/Kg/day), but not with S-AMET (117 mg/Kg/day) induced a significant decrease of abdominal lesions and mortality, assessed by means of a pathogenicity index. The trichomonicidal activity of piroxicam, metamizol, and S-AMET was tested in vitro at the concentration of 300 microM, but found ineffective. These assays have shown the usefulness of the experimental trichomoniasis model for the study of the immunomodulating activity of synthetic drugs.

A new pharmacological screening assay with Trypanosoma cruzi epimastigotes expressing beta-galactosidase.

We have developed a new pharmacological screening assay for epimastigotes of Trypanosoma cruzi (clone CL-B5) that express the Escherichia coli LacZ gene. The assay is based on determining the activity of the cytoplasmic beta-galactosidase released into the culture on membrane lysis in the presence of the substrate chlorophenol red beta-D-galactopyranoside (CPRG). The experimental conditions were adjusted to find those in which the relationship between epimastigote number and CPRG absorbance was linear over the widest possible range. Absorbance was significantly correlated with the number epimastigote from 5x10(3) to 1.2x10(6) parasites/ml (r=0.98, P<0.01). The optimal final concentration of CPRG was 200 microM and the optimal incubation period was 6 h when parasites were incubated for 3 days. Once the assay was standardized, the trypanocidal activities of nifurtimox and benznidazole were determined both by CPRG assay and microscopic counting, demonstrating the methods utility for drug-screening. The efficacy obtained was comparable to that obtained with the manual method.

Trichomonas vaginalis: determination of acid phosphatase activity as a pharmacological screening procedure.

A simple method to screen trichomonacides, based on the quantification of acid phosphatase (AP) activity, has been designed. Using p-nitrophenyl phosphate as chromogenic substrate, we first determined the optimal conditions for enzyme reaction. After seeding, a linear correlation between number of trichomonads and optical densities at 405 nm was obtained at 24 hr but not at 48 hr. Then, the inhibitory effect of metronidazole was assessed both by microscope counts and by AP determination. Similar values for 50% inhibitory concentrations (2.6 microM), with 95% confidence limits of 1.91-3.33 for microscopic and 2.21-3.05 for colorimetric method, were obtained. We concluded that the colorimetric method described in this investigation is suitable for pharmacological studies and for the screening of new, potential antitrichomonal agents.

Modulation by Polypodium leucotomos extract of cytokine patterns in experimental trichomoniasis model.

The immunomodulating effects of Anapsos, an aqueous hydrosoluble extract obtained from the rhizomes of the fern Polypodium leucotomos, on both pathogenicity and cytokine levels in serum (IFN-gamma/IL-4) were assayed in a Trichomonas vaginalis experimental model (BALB/c mice infected with 10(7) trichomonads and examined at day 15 after infection). Doses of 20 mg/kg/day administered for 10 days before the infection with the parasite induced a decrease of the experimental pathogenicity approximately 10-20% compared to controls. Gross histopathologic changes at abdominal organs and mortality rate, as a consequence of pathogenicity of the protozoa and the immune response of the host, were evaluated. IFN-gamma and IL-4 cytokines were determined on days -5, 0, 5, 10, and 15 postinfection by indirect ELISA. Treatment with PAL before infection modulates and downregulates the IFN-gamma concentration, while anticipates and upregulates the IL-4 level. The assays performed have showed the utility of the murine model of experimental trichomoniasis for the evaluation of immunomodulatory activity of synthetic or natural products.

Effect of Anapsos in a murine model of experimental trichomoniasis.

Immunomodulator effect of Anapsos (Polypodium leukotomas extract) in NMRI (US Naval Medical Research Institute) outbred mice infected by the intraperitoneal route with 10(7) Trichomonas vaginalis has been tested. Gross histopathologic changes in abdominal organs and mortality rate, as a consequence of the pathogenicity of the protozoa and the immune response of the host, were evaluated. Among the different treatment regimes assayed, Anapsos at doses of 20 mg/Kg/day administered for 10 days before infection decreases the parasite pathogenicity index (PI) in the treated animals when compared to those of the untreated control group. The immunosuppressor treatments with azathioprine (100 mg/Kg/day x 1), cyclophosphamide (100 mg/Kg/day x 1), and FK-506 (10 mg/Kg/day x 10) significantly decreased the PI, while an immunostimulant treatment with glycophosphopeptical (13 mg/Kg/day x 10) increased it. These assays have shown the usefulness of the murine model of experimental trichomoniasis for the study of immunomodulator activity of natural or synthetic drugs.

New thiadiazine derivatives with activity against Trypanosoma cruzi amastigotes.

The cytotoxicity of 18 new 1,2,6-thiadiazin-3,5-dione 1,1-dioxides was evaluated. This group of products was previously assayed against epimastigotes of Trypanosoma cruzi and some of them showed a high antiprotozoal activity. Thereafter 13 compounds with a high anti-epimastigote activity and low cytotoxicity were selected to be assayed against amastigotes. Some of the products showed the same or even lower cytotoxicity than nifurtimox and benznidazole, but most of them were very toxic for macrophages at 100 microg/ml. Only one of the compounds had an anti-amastigote activity similar to that of reference drugs at 10 microg/ml, but unfortunately this disappeared at lower concentrations.

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.

Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains.

Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD) analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis) was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.

In vitro screening of american plant extracts on Trypanosoma cruzi and trichomonas vaginalis.

From the beginning of this decade and with the revival of the phytotherapy, biological research about immunomodulatory, anti-inflammatory and antiprotozoal effects of Central and South American plants have been in progress. Our objective was to determine the antiprotozoal activity of 79 extracts from different plant families, including Asteraceae, Araceae, Moraceae, Solanaceae, Rhamnaceae, Zingiberaceae, Leguminosae and Sapotaceae. Once matching with herbarium specimens authenticated the plants, selected parts were separated, dried carefully and reduced to powder. Most of the screened extracts were aqueous. Two protozoa with different metabolic pathways, Trypanosoma cruzi and Trichomonas vaginalis were used as experimental models. Trypanocidal activity of plants was assayed on epimastigote cultures in liver infusion tryptose (LIT). Anti-Trichomonas activity was determined over cultures of the parasite in Diamond medium. In both cases, microscopic counting of parasites, after their incubation in the presence of different concentrations of the crude extracts, were made in order to determine the cytocidal and cytostatic activities respect to control cultures. Of the nine extracts that showed antiprotozoal activity, those from Mikania cordifolia and Philodendron bipinnatifidum were then fractionated, and again, were assayed the organic and aqueous phases obtained.

Evaluation of drug activity against intracellular forms of Trypanosoma cruzi employing enzyme immunoassay.

OBJECTIVE: To describe application of a new method for the evaluation of anti-Trypanosoma cruzi activity against intracellular forms. METHOD: Vero fibroblasts in 96-well tissue culture plates were infected with trypomastigote forms of T. cruzi. Amastigotes growth was estimated after 24 and 96 h both by microscopic counts of Giemsa-stained monolayers and enzyme-linked immunoIsorbent assay (ELISA). ELISA was performed directly on the fixed cultures using a rabbit anti-T. cruzi immunoIglobulin as the first antibody and a peroxidase-labelled antirabbit immunoglobulin as the second antibody. Three chemical series of structural analogous of gentian violet, thiadiazines and derivatives of 5-nitrothiophene-2-carbaldehyde as well as three reference compounds (nifurtimox, benznidazole and gentian violet) were then assayed. The anti-T. cruzi activity of all of them had been determined previously by microscopic counting of Giemsa-stained infected cultures. RESULTS: None of the assayed compounds showed better activity than the reference ones, but the application of the enzyme immunoassay to quantify the inhibition of growth amastigotes is of great interest, as it yielded results comparable with microscopic counts. CONCLUSION: ELISA can be applied to pharmacological screening, with some advantages over the microscopic examination, including possible automation, rapidity and objectivity in assessment.

Evaluation of a murine model of experimental trichomoniasis.

By using a reference strain of Trichomonas vaginalis and the intraperitoneal route for infecting animals, the influence of the strain of mice, the time observation and the inoculation doses were followed in order to standardize the optimal conditions for the evolution of experimental trichomoniasis. Our results suggest that the inoculation of BALB/c mice with 10(7) trichomonads and the semiquantitative assessment at day 15 postinfection of the gross-pathologic changes in the abdominal cavity--peritoneum, spleen, pancreas, stomach and liver--as well as the presence of ascitic fluid and mortality, maybe a suitable laboratory model of trichomoniasis.

The relationship of experimental pathogenicity in vivo with in vitro cytoadherence and cytotoxicity of 6 different isolates of Trichomonas vaginalis.

The experimental pathogenic effects in vivo and in vitro of 6 different isolates of Trichomonas vaginalis were studied following their inoculation into NMRI mice and on to adherent cultures of HeLa cells. Contact between the parasite and the adherent monolayer of cells was necessary to induce the monolayer to detach. The strains which were more virulent to mice also showed a greater weighted index of adherence; the weighted index of cytotoxicity in vitro did not, on the other hand, correlate with experimental pathology in vivo.

Activity assays of thiadiazine derivatives on Trichomonas vaginalis and amastigote forms of Trypanosoma cruzi.

Eight thiadiazine 1,1-dioxide derivatives were evaluated for antitrichomonal and antitrypanosomal activities. In vivo tests were performed on a murine model for trichomoniasis standardized in our laboratory. The capacity of compounds to clear visceral lesions in experimentally infected animals as well as their effects on the mortality time of mice were used as criteria for activity. One of the thiadiazines (compound 7b) showed an efficacy similar to that obtained with the reference drug metronidazole, although higher doses were required. Its toxicity on cell proliferation in tissue culture was moderate. In vitro assays on amastigote forms of Trypanosoma cruzi were carried out using cultures of Vero cells infected with metacyclic trypomastigotes. For one compound (1) trypanocidal activity resembled that of nifurtimox as assessed by microscopic counts of infected and uninfected cells. Unfortunately, this compound showed a high degree of cytotoxicity on Vero cell cultures.

Research for new antichagasic drugs.

A series of ten 1-[(5-nitrothenylidene)amino]azoles has been synthesized by the reaction of 5-nitrothiophene-2-carbaldehyde with 1-aminopyrazole, 1-aminoimidazole, 1- and 4- amino-1,2,4-triazoles, 1-aminoindole, 1- and 2-aminoindazoles, 1-aminobenzimidazole and 1- and 2-aminobenzotriazoles. Physical data, spectroscopic characteristics and biological properties of all the derivatives have been examined. The antiprotozoal activity has been tested against Trypanosoma cruzi, comparative to Nifurtimox (Lampit).

New derivatives of 5-nitroimidazole: synthesis and antiparasitic activity.

The synthesis and the antiparasitic evaluation of twelve new 5-nitroimidaole derivatives has been carried out. The most effective compounds were the less hydrophilic pyridinium and imidazolium salts (IV), (V) and (X), and above all the tetrahydropyridine derivatives (XII) and (XIII).

Antiparasitic activity of nine pyrazole derivatives against Trichomonas vaginalis, Entamoeba invadens and Plasmodium berghei.

Nine nitropyrazole derivatives were prepared and tested against Trichomonas vaginalis in vitro and in vivo, Entamoeba invadens in vitro and Plasmodium berghei in vivo. Three of the compounds, 4-4-nitropyrazole, 1-methyl-4-nitropyrazole and 4,4'-dinitro-1,1'-methylenedipyrazole, have an activity similar to that of metronidazole (used as the reference compound) against T. vaginalis and E. invadens after 48 hours of incubation. All the compounds tested were inactive against P. berghei.

3,5-Diamino-1,2,6-thiadiazine 1,1-dioxide derivatives: synthesis and antiparasitic activity.

The synthesis of new 3,5-diamino-1,2,6-thiadiazine 1,1-dioxide derivatives is described and their structures discussed on the basis of 1H and 13C-N.M.R. data. The antiparasitic activity of these and related compounds was evaluated. The bacterial mutagenicity of the parent compound (I) was studied.