Zigman Brener

Susceptibility and natural resistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease

The susceptibility and natural resistance to two nitroheterocyclic derivatives used clinically in Chagas disease (nifurtimox and benznidazole) were investigated in 47 Trypanosoma cruzi strains isolated from human patients, domestic vectors and sylvatic reservoirs or vectors. A large gradient of drug efficacy from 0% to 100% was detected. Drug susceptibility apparently related to geographical distribution of some T. cruzi strains was also observed. Drug resistance was identified among T. cruzi populations isolated from sylvatic vectors from an area where autochthonous human Chagas disease does not exist. Thus, natural drug-resistance of sylvatic strains might be a way of introducing this character into a T. cruzi domestic cycle. Most of the 47 studied strains were either sensitive or resistant to both compounds, an intriguing finding considering that nifurtimox and benznidazole apparently have different mechanisms of action against T. cruzi.

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.

Lytic antibody titre as a means of assessing cure after treatment of Chagas disease: a 10 years follow-up study

A complement-mediated lysis test (CoML) using living trypomastigotes was compared with conventional serological methods and with haemoculture. Over a 10 years follow-up period evidence was obtained which supported the view that chagasic patients, treated with nitroheterocyclic drugs, in whom CoML had reverted to negative, might be considered cured despite conventional serology remaining positive.

CHAGASIC PATIENTS LACK CD28 EXPRESSION ON MANY OF THEIR CIRCULATING T LYMPHOCYTES

A balanced host‐parasite interaction during Trypanosoma cruzi infection allows for the establishment of a chronic infection that can last for many years. T cells are a major element responsible for parasite specific and non‐specific immunityduring the complex immune response of the host. However, the sub‐populations of T cells involved in the response, as well as the exact mechanisms through which those cells are activated or rendered unresponsive, are not well defined. It is known thatco‐stimulatory signals, some of which are mediated via CD28, are of critical importance in the triggering of appropriate T cell responses. In this study the authors performed double‐labelling studies to determine the frequency of expression of CD28 byCD4+ and CD8+ T lymphocytes in the peripheral blood of patients with Chagas’ disease. The results show that chagasic patients throughout the spectrum of chronic clinical forms of the infection have significantly higher meanfrequencies of CD4+CD28– and CD8+CD28– T cells, as compared with non‐chagasic individuals. Considering the importance of CD28 for T‐cell activation, the observed down‐regulation or loss of CD28during infection may indicate a possible basis for observed immunoregulatory events or distinct stages of T‐cell activation in this infection. Recent evidence from patients with HIV/AIDS indicates that CD28– cell populations are morelikely to undergo apoptosis, and increased apoptosis has been observed in experimental Chagas disease.

Parasitological cure of acute and chronic experimental Chagas disease using the long-acting experimental triazole TAK-187. Activity against drug-resistant Trypanosoma cruzi strains

We investigated the activity of TAK-187, an experimental antifungal triazole with a long terminal half-life in several experimental animals, against Trypanosoma cruzi. In vitro studies showed that the minimal inhibitory concentration (MIC) against the (extracellular) epimastigote form was 0.3-1 microM, while the corresponding concentration against clinically relevant intracellular amastigotes was 1 nM. At the MIC the endogenous epimastigote C4,14-desmethyl sterols were replaced by di- and tri-methylated sterols, supporting the notion that the primary target of TAK-187 is the parasites sterol C14alpha demethylase. We investigated the in vivo activity of the compound in a murine model of acute Chagas disease, using T. cruzi strains with different susceptibilities to the drugs currently used clinically (nitrofurans and nitroimidazoles). It was found that TAK-187 given orally at 20 mg/kg induced complete protection against death and high levels (60-100%) of parasitological cures, independently of the infecting strain and even when administered every other day (e.o.d.), consistent with its long terminal half-life in mice. Other experiments, using longer treatment periods were carried out in both acute and chronic models of the disease and showed that TAK-187 given at 10-20 mg/kg e.o.d. induced 80-100% survival with 80-100% of parasitological cures of survivors in both models. No toxic side effects were observed in any of the experimental protocols. TAK-187 is a potent anti-T. cruzi compound with trypanocidal activity in vivo and should be considered for further studies as a potential specific treatment of human Chagas disease.

Trypanosoma cruzi genome project: biological characteristics and molecular typing of clone CL Brener

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. CL Brener was obtained by cloning procedures from bloodstream trypomastigotes isolated from mice infected with the CL strain. The doubling time of CL Brener epimastigotes cultured at 28 degrees C in liver infusion-tryptose (LIT) medium is 58 +/- 13 h. Differentiation to metacyclic forms is induced by incubation of epimastigotes in LIT-20% Graces medium. Metacyclics give very low parasitemia in mice, contrary to what is observed for blood forms which promote 100% mortality of the animals with inocula of 5 x 10(3) parasites. CL Brener blood forms are highly susceptible to nifurtimox, benznidazole and ketoconazole. Allopurinol is inefficient in the treatment of mice experimental infection. The clone infects mammalian cultured cells and performs the complete intracellular cycle at 33 and 37 degrees C. The molecular typing of CL Brener has been done by isoenzymatic profiles; sequencing of a 24S alpha ribosomal RNA gene domain and by schizodeme, randomly amplified polymorphic DNA and DNA fingerprinting analyses. For each typing approach the patterns obtained do not change after prolonged parasite subcultivation in LIT medium (up to 100 generations). The stability of the molecular karyotype of the clone was also confirmed.

An experimental and clinical assay with ketoconazole in the treatment of Chagas disease

Ketoconazole, an azole antifungic drug which is already in the market has also been demonstrated to be active against Trypanosoma cruzi experimental infections. In this paper we confirmed the drug effect and investigated its range of activity against different T. cruzi strains naturally resistant or susceptible to both standard drugs Nifurtimox and Benznidazole used clinically in Chagas disease. Moreover, we have shown that the association of Ketoconazole plus Lovastatin (an inhibitor of sterol synthesis), which has an antiproliferative effect against T. cruzi in vitro, failed to enhance the suppressive effect of Ketoconazole displayed when administered alone to infected mice. Finally, administration in chronic chagasic patients of Ketoconazole at doses used in the treatment of deep mycosis also failed to induce cure as demonstrated by parasitological and serological tests. The strategy of identify and test drugs which are already in the market and fortuitously are active against T. cruzi has been discussed.

Effect of specific chemotherapy on the levels of lytic antibodies in Chagas's disease.

Clinical trials with compounds active in Chagass disease have shown that after treatment parasitological diagnostic methods (xenodiagnosis) become repeatedly negative whereas conventional serology (immunofluorescence and complement fixation tests) persists steadily positive. Consequently, assessment of cure still remains controversial. This paper reports the influence of specific treatment on antibodies involved in the conventional serological diagnosis and on antibodies which bind to the living bloodstream forms and are related to host resistance. Antibodies lytic to Trypanosoma cruzi bloodstream stages were detected, through a complement-mediated lysis (CML) test, in: (a) 100% of 28 untreated patients; (b) 94% of a group of 21 treated patients in whom conventional serology remained positive, including those with persistently negative xenodiagnosis; (c) 0% of 17 normal controls. In some patients treated with a nitrofuran derivative (nifurtimox) or with a 2-nitroimidazole derivative (benznidazol), CML test became gradually negative whereas conventional serology continued to be positive. Finally, in five patients treated with benznidazol, serological tests, CML and xenodiagnosis became regularly negative, strongly suggesting parasitological cure. Those findings demonstrate a dissociation between the antibodies mediating serological diagnosis and those directed against living bloodstream parasites. Moreover, since in some patients both types of antibodies disappeared after treatment, the results suggest that cure of Chagass disease should be based not only on negative xenodiagnosis but also on the elimination of specific antibodies detectable by conventional serology and CML test.

Direct lysis of Trypanosoma cruzi: a novel effector mechanism of protection mediated by human anti-gal antibodies.

Summary Anti‐gal antibodies directed against a carbohydrate epitope present in mouse laminin (galactosyl α 1‐3 galactose) and detected in high levels in sera from patients in the acute phase of Chagas disease are responsible for the direct lysis (DL) of Trypanasoma cruzi blood forms independent of either the classic or alternative complement pathways. Furthermore, the lectins Euonymus europaeus (EE) specific for the carbohydrates gal α1‐3 gal present a similar lytic activity against T. cruzi at the same concentrations of purified anti‐gal antibodies. The DL activity was tested with several other lectins but Concanavalin A (Con A) specific for α‐D‐mannose and α‐D‐glucose was the only one also presenting lytic activity. The lectins and anti‐gal antibodies lytic activity can be inhibited by specific carbohydrates suggesting that this phenomenon is related to the capability of these lectins or anti‐gal antibodies to bind to a crucial surface component of T. cruzi. Moreover, the infectivity of T. cruzi blood forms to mice was clearly inactivated by incubation with acute chagasic sera (ACS) but not by ACS absorbed by immunoaffinity chromatography with mouse laminin, a strong evidence that high levels of anti‐gal antibodies participate in the decline of the parasitaemia from the acute to the chronic phase in Chagas disease.

Hematological changes in mice experimentally infected with Trypanosoma Cruzi

Mice inoculated with trypanosoma cruzi display an intense thrombocytopenia which is more severe in animals infected with the Y than CL strain. In animals inoculated with a T. cruzi strain which induces chronic infection (Colombiana), the number of platelets decreases as parasitemia ascends, and then reverts to normal values as soon as the acute infection merges into the chronic phase. The mechanisms involved in the thrombocytopenia are still obscure and are likely to be related to more general phenomena affecting the host rather than to direct damage of platelets or precursor cells by parasitism. Anemia and leukopenia are also present in T. cruzi infected mice.