Constituents from stem barks of Luehea ochrophylla Mart and evaluation of their antiparasitic, antimicrobial, and antioxidant activities.

Luehea species are found in almost all Central and South American countries. The present work describes the phytochemical study, isolation, and structural characterisation of friedelin, ß-friedelinol, lupeol, pseudotaraxasterol, ß-sitosterol, betulinic acid, taraxasterol, (-)-epicatechin, ß-sitosterol-3-O-ß-d-glucopyranoside, and (+)-epicatechin-(4ß→8)-epicatechin from stem barks of Luehea ochrophylla Mart. The structural identification of the isolated compounds was mainly performed by NMR analyses and comparison with the data from literature. These compounds were isolated for the first time in the genus Luehea, except ß-sitosterol glucopyranoside, (-)-epicatechin, and lupeol. Hexane extract (HE) and dichloromethane (DF) and ethyl acetate (AF) fractions exhibited antiparasitic activity against amastigote (intracellular) and trypomastigote culture forms of Trypanosoma cruzi. The ethanol extract (EE), DF, and ethanol fraction (EF) exhibited considerable antifungal activity against Candida albicans. Moreover, extracts and fractions exhibited significant percentage of capture free radicals of 2,2-diphenyl-picrylhydrazyl (DPPH) when compared to the standard of ascorbic acid.

Functional analysis of iron superoxide dismutase-A in wild-type and antimony-resistant Leishmania braziliensis and Leishmania infantum lines.

In this work, we characterized the gene encoding iron superoxide dismutase-A (FeSOD-A) in wild-type (WTS) and antimony-resistant (SbR) L. (Viannia) braziliensis and L. (Leishmania) infantum lines, which were selected in vitro. FeSOD-A transcript and protein expression were similar in all tested lines; however, specific enzyme activity analysis revealed higher superoxide dismutase activity in SbIII-resistant LbSbR and LiSbR lines than in the corresponding WTS lines. These parasites were also more tolerant to oxidative stress generated by the herbicide paraquat. Functional analysis showed that in comparison to non-transfected lines, wild-type LbWTS and LiWTS clones overexpressing the FeSOD-A enzyme are 1.6- and 1.7-fold more resistant to SbIII, respectively. Our results suggest that FeSOD-A is involved in the antimony resistance phenotype in L. (V.) braziliensis and L. (L.) infantum.

Influence of the long-term Trypanosoma cruzi infection in vertebrate host on the genetic and biological diversity of the parasite.

The influence of the long-term Trypanosoma cruzi infection in vertebrate host on the biological and genetic properties of the parasite was evaluated. Four T. cruzi isolates obtained from different chronic chagasic dogs infected with Berenice-78 T. cruzi strain during 2 and 7 years were comparatively analyzed. The long-term T. cruzi infection has led to alterations in parasitemia, virulence and pathogenicity of Be-78 strain for mice. These biological parameters varied from low to high in realation to the parental strain. Randomly amplified polymorphic DNA and isoenzyme profiles detected two distinct genetic groups of parasites. The first group included the parental strain and two T. cruzi isolates, and the second group the two other isolates. Interestingly, the isolates of the second group showed a reversibility of the genetic profile to the parental strain after 25 passages in mice. No correlation between the genetic groups and biological properties of the isolates was observed. Our findings confirmed the population heterogeneity of the Be-78 strain, and showed how differently it responds to the long-term infection in the same vertebrate hosts.

A study of experimental reinfection by Trypanosoma cruzi in dogs.

The role of reinfection in the evolution of Chagas' disease was evaluated in dogs alternately infected with the 147 and SC-1 strains of Trypanosoma cruzi. A parasitologic, serologic, clinical, and electrocardiographic follow-up was carried out on the infected and noninfected dogs. The dogs were reinfected five times over a period of 38 months. No deaths were observed during the experiment. They presented a brief oligosymptomatic acute phase. The level of parasitemia decreased progressively with the number of reinfections. Bloodstream parasites were not detectable after the fifth reinfection. All parasite samples isolated during the follow-up were zymodeme B, corresponding to strain 147, irrespective of the strain with which the dogs were first infected and of the triatomine species used for isolation. Conversely, amplification by the polymerase chain reaction of a segment of the T. cruzi mini-exon gene showed the simultaneous presence of both strains in three of the eight reinfected animals. Antibody titers were greater among the dogs successively infected than those infected only once. Neither amastigotes nor T. cruzi DNA were detected in the tissues of the infected dogs. Alterations related to Chagas' disease were identified only in the heart and consisted of chronic focal and discrete myocarditis, compatible with the indeterminate form of Chagas' disease. All infected dogs developed this form of the disease, which was independent of the number of infections.

Kinetics of cytokine gene expression in experimental chagasic cardiomyopathy: tissue parasitism and endogenous IFN-gamma as important determinants of chemokine mRNA expression during infection with Trypanosoma cruzi.

We investigated the kinetics of parasite replication, leukocyte migration, and cytokine/chemokine mRNA expression in the heart tissue from animals infected with the Colombiana strain of Trypanosoma cruzi. Cardiac tissue parasitism was noticeable at 15 days, peaked around 30 days and was dramatically reduced at 120 days postinfection (p.i.). Kinetic studies showed that the inflammatory infiltrate was dominated by the presence of alphabetaT CD3(+ )CD4(+ )CD8(-), alphabetaT CD3(+ )CD4(-)CD8(+ )lymphocytes and macrophages. The mRNA expression of the monokines IL-1beta and IL-12(p40) was elevated at 15 days p.i. and controlled at later time points. In contrast, TNF-alpha mRNA was expressed throughout the infection. Interestingly, we found that at 15 and 30 days p.i. cytokine expression was dominated by the presence of IFN-gamma mRNA, whereas at 60 days or later time points the balance of type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs. The chemokine mRNAs encoding JE, MIP-1alpha, MIP-1beta, KC, and MIP-2 were all mainly expressed at 15 and/or 30 days p.i. and diminished thereafter. In contrast, the expression of RANTES, MIG and IP-10 mRNAs was augmented at 15 days p.i. and persisted at high levels up to 120 days p.i. Taken together, our results indicate that regulation of IFN-gamma and chemokine expression, associated with decreased tissue parasitism, may be largely responsible for the control of inflammation and immunopathology observed in the cardiac tissue of animals infected with T. cruzi.

In-vivo treatment with benznidazole enhances phagocytosis, parasite destruction and cytokine release by macrophages during infection with a drug-susceptible but not with a derived drug-resistant Trypansoma cruzi population.

To stuck the effect of chemotherapy on parasite-macrophage interaction we used the wild-type Y strain (drug-susceptible) of Trypanosoma cruzi and a drug-resistant parasite population derived from the same strain. Trypomastigotes isolated from untreated infected mice, as well as, 3 h after treatment with BZ were incubated with inflammatory macrophages and used to study phagocytosis, parasite destruction, cytokine release and reactive nitrogen intermediates (RN!) synthesis. Phagocytosis and destruction of the drug-susceptible parasites were significant/v enhanced by drug treatment. These enhancements were accompanied by an increase in cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)alpha] and RNI release by murine inflammatory macrophages primed with IFN-gamma. In contrast, BZ treatment of mice infected with drug-resistant T. cruzi population showed no effect whatsoever. The synthesis of IFN-gamma and RNI by splenocytes of mice infected with either susceptible and drug-resistant parasite populations, before and after treatment with BZ were also studied. On/v the splenocytes from mice infected with the drug-susceptible parasites treated with BZ produced high levels of IFN-gamma and RNI. Our findings indicate that BZ acts on the drug-susceptible T. cruzi parasites by enhancing the phagocytosis and the production of cytokines and RN!, thus, favouring the destruction of the intracellular parasites by the cellular compartment of the immune system.

Interleukin-12 enhances in vivo parasiticidal effect of benznidazole during acute experimental infection with a naturally drug-resistant strain of Trypanosoma cruzi.

The roles of gamma interferon (IFN-gamma) and interleukin-12 (IL-12) in mediating and/or enhancing the in vivo trypanosomicidal activity of the nitroheterocyclic derivative benznidazole (Bz) were evaluated during early stages of experimental Chagas' disease. Our results show that treatment of Trypanosoma cruzi-infected mice with anti-cytokine monoclonal antibodies (MAbs) had no apparent effect when the optimal dose of Bz (100 mg/kg of body weight) was used. In contrast, treatment with anti-IL-12 or anti-IFN-gamma MAbs enhanced the parasitemia and accelerated the mortality of mice treated with a suboptimal dose of Bz (25 mg/kg). Simultaneous treatment with a suboptimal dose of Bz and recombinant IL-12 (rIL-12) enhanced the efficacy of drug treatment in terms of parasitemia and mouse survival. Interestingly, we found that drug-resistant T. cruzi strains were found to be poor inducers of IL-12 both in vitro and in vivo compared to strains of T. cruzi which are susceptible or partially resistant to Bz treatment. These results suggest that early activation of the cellular compartment of the immune system by IL-12 may favor in vivo Bz activity against T. cruzi. In order to test this hypothesis mice infected with the drug-resistant Colombiana strain of T. cruzi were treated with 100 mg of Bz per kg plus different concentrations of rIL-12. By using the results of PCR and serological and parasitological methods as the criteria of a cure, our results indicate that a higher percentage of mice treated with Bz combined with rIL-12 than mice treated with Bz alone are cured.

Molecular characterization of susceptible and naturally resistant strains of Trypanosoma cruzi to benznidazole and nifurtimox.

Twenty-seven Trypanosoma cruzi strains, susceptible or naturally resistant to the nitroderivatives benznidazole and nifurtimox, were analyzed using the following molecular markers: (i) isoenzyme patterns of six enzymes; (ii) genetic variability assayed by randomly amplified polymorphic DNA (RAPD) with two different primers; and (iii) gene probes for P-glycoprotein (TcPGP), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the ribosomal RNA gene (rDNA) and the mini-exon gene (MEX), RAPD and isoenzyme profiles divided the T. cruzi strains into three groups, whereas the gene probes divided the T. cruzi strains in two groups. Strains classified as group I or II by RAPD or zymodemes Z1 or Z2 by isoenzyme analysis were either susceptible or naturally resistant to the nitroderivatives. In contrast, strains classified as group III by RAPD and zymodeme ZB by isoenzyme analysis were only drug susceptible and showed polymorphisms for HGPRT and TcPGP. No correlation was observed between drug susceptibility and polymorphisms of rDNA and MEX. Eighteen T. cruzi strains isolated from different geographic regions were included in this study. Thus, from a total of 45 T. cruzi strains analyzed, all 19 of zymodeme B were susceptible to the experimental treatment independent of their geographic origin.

In vivo selection of a population of Trypanosoma cruzi and clones resistant to benznidazole.

A benznidazole-resistant population of Trypanosoma cruzi, Y strain, was selected after 25 successive passages (8 months) in mice treated with a single high drug dose. Initially, the resistant parasites produced a low parasitaemia level and low mortality rate in infected mice. Thereafter, the parasitaemia level and mortality rate increased to the same value obtained for mice infected with the wild-type strain. Long-term treatment with benznidazole (100 mg/kg/day) cured 71-80% of mice infected with the wild-type strain. No cure was observed in mice infected with the selected resistant parasite population. Treatment with 500 mg/kg of benznidazole at peak parasitaemia cleared all blood parasites from mice infected with wild-type parasites. No effect on parasitaemia level was observed in mice infected with the selected parasites. Benznidazole-resistant parasites showed cross-resistance to different drugs. Contrary to wild type, all clones analysed from the resistant T. cruzi population were resistant to benznidazole. Without drug pressure the resistance phenotype of clones was far more stable than that presented by the resistant population. This work demonstrates, for the first time, the in vivo selection of a population and clones of T. cruzi resistant to benznidazole, and makes available an experimental model for the study of mechanisms of drug resistance in T. cruzi.

Characterization of Trypanosoma cruzi strains isolated from chronic chagasic patients, triatomines and opossums naturally infected from the State of Rio Grande do Sul, Brazil.

Thirty-five Trypanosoma cruzi strains were isolated from chronic chagasic patients, triatomines and opossums from different municipalities of the State of Rio Grande do Sul. Parasites were characterized by means of mice infectivity, enzyme electrophoresis and randomly amplified polymorphic DNA (RAPD) analysis. Twenty-nine strains were isolated from chagasic patients, 4 from triatomines (2 from Triatoma infestans and 2 from Panstrongylus megistus) and 2 from opossums Didelphis albiventris. Thirty-three T. cruzi strains were of low and 2 strains of high virulence in mice. Both virulent strains were isolated from P. megistus. Isoenzyme analysis of the strains showed 3 different zymodemes. Eleven strains isolated from chagasic patients and 2 from D. albiventris were Z2. Eighteen strains from patients and 2 from T. infestans were ZB and 2 T. cruzi strains isolated from P. megistus were Z1. RAPD profiles obtained with 4 random primers showed a high genetic heterogeneity of the T. cruzi strains. Zymodeme 2 and ZB strains were the more polymorphic. A band sharing analysis of the RAPD profiles of Z2 and ZB strains using 3 primers, showed a very low percentage of shared bands, 20% among 13 ZB strains and 14% among 13 Z2 strains. According to the isoenzyme results, 3 T. cruzi populations were present in State of Rio Grande do Sul. Zymodeme 2 and ZB strains were found infecting man (domiciliar transmission cycle) whereas Z1 strains were found infecting the sylvatic vector P. megistus.

Anti-plasmodial and anti-trypanosomal activity of synthetic naphtho[2,3-b]thiopen-4,9-quinones.

Naphtho[2,3-b]thiophen-4,9-quinone and five derivatives were prepared using the Friedel-Crafts reaction and tandem-lithiation of aromatic diethylamides. These quinones were evaluated for their trypanocidal and anti-plasmodial activities by their effects on: (1) growth of epimastigote forms of Trypanosoma cruzi in vitro, (2) lysis of trypomastigote forms of T. cruzi in murine blood, (3) growth of Plasmodium falciparum in vitro, and (4) inhibition of the recombinant enzyme trypanothione reducatase. The parent compound, naphtho[2,3-b]thiophen-4,9-quinone (3a), was among the most active quinone tested in vitro against P. falciparum at 0.2 microM. However, it was inactive against P. berghei-infected mice treated with 2.3 mmol/kg daily for 5 days. Most of the quinones prepared were active against T. cruzi epimastigotes in culture but exhibited weak activity at 4 degrees C against trypomastigotes in murine blood as well against the enzyme trypanothione reducatase. Further structural modifications will be necessary to improve the in vivo activity of the naphthothiophenquinones.

Biological and isoenzymatic characterization of Trypanosoma cruzi strains isolated from sylvatic reservoirs and vectors from the state of Santa Catarina, Southern Brazil.

Sixty eight Trypanosoma cruzi strains were isolated in the state of Santa Catarina, Southern Brazil, from sylvatic reservoirs or naturally infected vectors and characterized by their biological behaviour in mice, morphology of bloodstream forms and isoenzyme profiles. Twenty eight strains were isolated from the triatomine bug (Panstrongylus megistus), 2 from rodents (Echimys dasythrix and Akodon sp) and 38 from opossums (Didelphis marsupialis). The infectivity in mice of 48 T. cruzi strains showed that 2 (4.2%) were of high virulence, 19 (39.6%) of medium virulence, 15 (31.2%) of low virulence and 12 (25.0%) produced subpatent parasitemia in mice. A morphological study of bloodstream trypomastigotes from 8 T. cruzi strains showed a predominance of stout forms. The isoenzyme pattern of 59 T. cruzi strains showed that 54 (91.5%) belonged to zymodeme Z1, 3 (5.1%) to zymodeme Z2 and 2 (3.4%) to mixed zymodemes, Z1 and Z2. All 34 T. cruzi strains analyzed from opossums were Z1. Three out of 5 strains isolated from adults of P. megistus collected inside houses, belonged to zymodeme Z2 and two strains exhibited mixed zymodemes, Z1 and Z2, in 5 out of 6 enzymes studied. Although the State of Santa Catarina is a non endemic region for human Chagas'disease, the presence of zymodeme Z2 parasites in the sylvatic vector, P. megistus, captured in domiciliary environments suggests the possibility of human and/or domestic mammal infection by T. cruzi.

A diterpene from Mikania obtusata active on Trypanosoma cruzi.

The diterpene ent-kaur-16-en-19-oic acid (1) was identified as the trypanocidal component of the ethanolic extract from Mikania obtusata D. C. (Asteraceae). This compound presents an IC50 of 0.5 mg/ml (1.66 mM) against the trypomastigote blood form of the Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis).

Randomly amplified polymorphic DNA (RAPD) and isoenzyme analysis of Trypanosoma rangeli strains.

Sixteen Trypanosoma rangeli strains were compared by isoenzyme and randomly amplified polymorphic DNA (RAPD) analysis. Eight strains were isolated from either Rhodnius prolixus or Homo sapiens from Honduras, Colombia and Venezuela. Another eight strains were isolated from either Panstrongylus megistus or the rodent Echimys dasythrix from the State of Santa Catarina, southern Brazil. All six T. rangeli strains isolated from P. megistus were co-infections with Trypanosoma cruzi, demonstrating an overlap of the sylvatic cycles of these parasites and that the accurate identification of species is of utmost importance. Both isoenzyme and RAPD analysis revealed two distinct groups of T. rangeli strains, one formed by the strains from Santa Catarina and the other, by the strains from Honduras, Colombia and Venezuela. With the five enzymes used, all the strains from Santa Catarina had identical profiles which overlapped with those of the other regions only in the pattern obtained with malic enzyme. Analysis of 138 RAPD bands by means of an unweighted pair group method analysis (UPGMA) phenogram using the Dice similarity coefficient allowed the separation of the two groups based on their divergence at a lower level of similarity than the phenon line. We show that the identification of T. cruzi and T. rangeli in naturally mixed infections is readily achieved by either RAPD or isoenzyme analysis.