Marta de Lana

Chemotherapy with benznidazole and itraconazole for mice infected with different Trypanosoma cruzi clonal genotypes.

ABSTRACT The benznidazole (BZ) and itraconazole (ITC) susceptibilities of a standard set of Trypanosoma cruzi natural stocks were evaluated during the acute phase and the chronic phase of experimental chagasic infection in BALB/c mice. Twenty laboratory-cloned stocks representative of the total phylogenetic diversity of T. cruzi, including genotypes 20 and 19 (T. cruzi I) and genotypes 39 and 32 (T. cruzi II), were analyzed. Our results demonstrate important differences among stocks that could be pointed out as markers of biological behavior. Members of the T. cruzi I group were highly resistant to both BZ and ITC, whereas members of the T. cruzi II group were partially resistant to both drugs, despite their susceptibilities to ITC during the chronic phase of infection. The resistance to BZ observed for T. cruzi I was mainly triggered by genotype 20 isolates, whereas resistance to ITC was due to both genotype 20 and 19 isolates. Two polar patterns of response to BZ observed for genotype 39 isolates had a major impact on the partial resistance pattern observed for members of the T. cruzi II group. Genotype 32 isolates showed a typical profile of susceptibility. The correlation between the response to treatment and phylogenetic classification of T. cruzi stocks was clearer for ITC than for BZ. In conclusion, the data presented show a correlation between phylogenetic divergence among T. cruzi stocks and their susceptibilities to chemotherapeutic agents in vivo. Our results warn of the necessity to take into account the lesser genetic subdivisions of T. cruzi stocks since the upper subdivisions (T. cruzi I and II) show a great deal of heterogeneity for in vivo drug susceptibility.

Benznidazole therapy during acute phase of Chagas disease reduces parasite load but does not prevent chronic cardiac lesions

The goals of this study were to evaluate the efficacy of benznidazole (Bz) treatment in decreasing of the parasitic load during the acute phase of experimental Chagas disease and to analyze its influence in the development of cardiac chronic alterations in mice inoculated with drug-resistant Trypanosoma cruzi strains. Our results showed that the early Bz treatment (started at 4th day of infection) was efficient in reducing the parasite load in animals from both acute and chronic phase of the infection. Moreover, this reduction in the parasite load could not be associated with the intensity of the cardiac chronic lesions. The histopathological evaluation of cardiac tissue of Bz-treated mice showed three different patterns of response: (1) presence of a small number of inflammatory cells and fibrotic area similar to noninfected mice; (2) similar intensity of inflammatory infiltrate and smaller fibrotic area in relation to nontreated animals; (3) similar intensity of inflammatory infiltrated and fibrosis area among the Bz-treated and nontreated animals. Each specific pattern was obtained with different T. cruzi strain, suggesting that the pattern of the heart lesions in chronic phase of Bz-treated animals was T. cruzi strain dependent but not related with drug resistance levels.

Are increased frequency of macrophage-like and natural killer (NK) cells, together with high levels of NKT and CD4+CD25high T cells balancing activated CD8+ T cells, the key to control Chagas’ disease morbidity?

The immunological response during early human Trypanosoma cruzi infection is not completely understood, despite its role in driving the development of distinct clinical manifestations of chronic infection. Herein we report the results of a descriptive flow cytometric immunophenotyping investigation of major and minor peripheral blood leucocyte subpopulations in T. cruzi‐infected children, characterizing the early stages of the indeterminate clinical form of Chagas’ disease. Our results indicated significant alterations by comparison with uninfected children, including increased values of pre‐natural killer (NK)‐cells (CD3– CD16+ CD56–), and higher values of proinflammatory monocytes (CD14+ CD16+ HLA‐DR++). The higher values of activated B lymphocytes (CD19+ CD23+) contrasted with impaired T cell activation, indicated by lower values of CD4+ CD38+ and CD4+ HLA‐DR+ lymphocytes, a lower frequency of CD8+ CD38+ and CD8+ HLA‐DR+ cells; a decreased frequency of CD4+ CD25HIGH regulatory T cells was also observed. These findings reinforce the hypothesis that simultaneous activation of innate and adaptive immunity mechanisms in addition to suppression of adaptive cellular immune response occur during early events of Chagas’ disease. Comparative cross‐sectional analysis of these immunophenotypes with those exhibited by patients with late chronic indeterminate and cardiac forms of disease suggested that a shift toward high values of macrophage‐like cells extended to basal levels of proinflammatory monocytes as well as high values of mature NK cells, NKT and regulatory T cells, may account for limited tissue damage during chronic infection favouring the establishment/maintenance of a lifelong indeterminate clinical form of the disease. On the other hand, development of an adaptive cell‐mediated inflammatory immunoprofile characterized by high levels of activated CD8+ cells and basal levels of mature NK cells, NKT and CD4+ CD25HIGH cells might lead to late chronic pathologies associated with chagasic heart disease.

Immunogenicity of a killed Leishmania vaccine with saponin adjuvant in dogs

Abstract Cellular and humoral immune responses of dogs to a candidate vaccine, composed of Leishmania braziliensis promastigote protein plus saponin as adjuvant, have been investigated as a pre-requisite to understanding the mechanisms of immunogenicity against canine visceral leishmaniasis (CVL). The candidate vaccine elicited strong antigenicity related to the increases of anti-Leishmania IgG isotypes, together with higher levels of lymphocytes, particularly of circulating CD8+ T-lymphocytes and Leishmania chagasi antigen-specific CD8+ T-lymphocytes. As indicated by the intense cell proliferation and increased nitric oxide production during in vitro stimulation by L. chagasi soluble antigens, the candidate vaccine elicited an immune activation status potentially compatible with effective control of the etiological agent of CVL.

Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-γ, TNF-α, and low IL-10 production during the acute infection phase.

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions.

Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains

In this work, the susceptibility to benznidazole of two parental Trypanosoma cruzi strains, Colombian and Berenice-78, was compared to isolates obtained from dogs infected with these strains for several years. In order to evaluate the susceptibility to benznidazole two groups of mice were infected with one of five distinct populations isolated from dogs as well as the two parental strains of T. cruzi. The first group was treated with benznidazole during the acute phase and the second remained untreated controls. The animals were considered cured when parasitological and serological tests remained persistently negative. Mice infected with the Colombian strain and its isolates Colombian (A and B) did not cure after treatment. On the other hand, all animals infected with Berenice-78 were cured by benznidazole treatment. However, 100%, 50% and 70% of cure rates were observed in animals infected with the isolates Berenice-78 B, C and D, respectively. No significant differences were observed in serological profile of infected control groups, with all animals presenting high antibody levels. However, the ELISA test showed differences in serological patterns between mice inoculated with the different T. cruzi isolates and treated with benznidazole. This variability was dependent on the T. cruzi population used and seemed to be associated with the level of resistance to benznidazole.

Caracterização biológica comparativa das cepas Berenice e Berenice-78 de Trypanosoma cruzi isoladas da mesma paciente em diferentes períodos

The Berenice-78 strain of T. cruzi is very different from the Berenice strain isolated 16 years earlier from the same pacient. The authors verified is high infectivity and low virulence for C3H inbred mice that survived the acute phase of infection. In these animals, it was verified that the tropism of parasites was more accentuated for cardiac and skeletal musculature and the parasitaemic level progressively increased with sucessive blood passages with posterior stability.

A killed Leishmania vaccine with sand fly saliva extract and saponin adjuvant displays immunogenicity in dogs

Summary A vaccine against canine visceral leishmaniasis (CVL), comprising Leishmania braziliensis promastigote protein, sand fly gland extract (SGE) and saponin adjuvant, was evaluated in dog model, in order to analyse the immunogenicity of the candidate vaccine. The vaccine candidate elicited strong antigenicity in dogs in respect of specific SGE and Leishmania humoral immune response. The major saliva proteins recognized by serum from immunized dogs exhibited molecular weights of 35 and 45kDa, and were related to the resistance pattern against Leishmania infection. Immunophenotypic analysis revealed increased circulating CD21+ B-cells and CD5+ T-cells, reflected by higher counts of CD4+ and CD8+ T-cells. The observed interaction between potential antigen-presenting cells (evaluated as CD14+ monocytes) and lymphocyte activation status indicated a relationship between innate and adaptive immune responses. The higher frequency in L. chagasi antigen-specific CD8+ T-lymphocytes, and their positive association with intense cell proliferation, in addition to the progressively higher production of serum nitric oxide levels, showed a profile compatible with anti-CVL vaccine potential. Further studies on immunological response after challenge with L. chagasi may provide important information that will lead to a better understanding on vaccine trial and efficacy.

Etiological treatment during early chronic indeterminate Chagas disease incites an activated status on innate and adaptive immunity associated with a type 1-modulated cytokine pattern.

Pro-inflammatory immune response is usually associated with Chagas disease pathogenesis, but is also relevant to treatment effectiveness. Cross-sectional studies have suggested that this activated state may persist for years after therapeutic intervention. However, short-term longitudinal investigation has suggested that the Benznidazole treatment (Bz-treatment) leads to decreased immunological activation. In order to elucidate this issue, we performed a longitudinal study to evaluate the immunological status following Bz-treatment during early indeterminate Chagas disease. Our results demonstrated that Bz-treatment led to higher activation status of circulating monocytes but was negatively associated with the number of IL-12(+)CD14(+) cells. Moreover, Bz-treatment triggered a high frequency of circulating CD3(-)CD16(+)CD56(-) NK cells, in addition to elevated activation status associated with a type 1-modulated cytokine pattern. Bz-treatment induced substantial T and B-cell activation status associated with an overall IL-10 modulated type 1 cytokine profile. In summary, these findings provide new information regarding immune activation status following the etiological treatment of Chagas disease. These results suggest that in addition to the increased number of activated leukocytes in the peripheral blood, Bz-treatment may also involve a qualitative change in their functional capacity that drives their activation state toward a modulated cytokine profile. These changes may account for the benefits of etiological treatment of Chagas disease.

Trypanosoma cruzi high infectivity in vitro is related to cardiac lesions during long-term infection in Beagledogs

Trypanosoma cruzi is a hemoflagelate parasite associated with heart dysfunctions causing serious problems in Central and South America. Beagle dogs develop the symptoms of Chagas disease in humans, and could be an important experimental model for better understanding the immunopathogenic mechanisms involved in the chagasic infection. In the present study we investigated the relation among biological factors inherent to the parasite (trypomastigote polymorphism and in vitro infectivity) and immunoglobulin production, inflammation, and fibrosis in the heart of Beagle dogs infected with either T. cruzi Y or Berenice-78 strains. In vitro infectivity of Vero cells as well as the extension of cardiac lesions in infected Beagle was higher for Y strain when compared to Berenice-78 strain. These data suggested that in vitro infectivity assays may correlate with pathogenicity in vivo. In fact, animals infected with Y strain, which shows prevalence of slender forms and high infectivity in vitro, presented cardiomegaly, inflammation, and fibrosis in heart area. Concerning the immunoglobulin production, no statistically significant difference was observed for IgA, IgM or IgG levels among T. cruzi infected animals. However, IgA together IgM levels have shown to be a good marker for the acute phase of Chagas disease.