Trypanosoma cruzi: desferrioxamine decreases mortality and parasitemia in infected mice through a trypanostatic effect

Desferrioxamine (DFO) is a potent iron chelator that is also known to modulate inflammation and act as an efficient antioxidant under normal conditions and under oxidative stress. Many in vitro and in vivo studies have shown the efficacy of DFO in the treatment of viral, bacterial and protozoan infections. DFO is known to reduce the intensity of Trypanosoma cruzi infections in mice even during a course of therapy that is not effective in maintaining anaemia or low iron levels. To further clarify these findings, we investigated the action of DFO on mouse T. cruzi infection outcomes and the direct impact of DFO on parasites.Infected animals treated with DFO (5 mg/animal/day) for 35 days, beginning 14 days prior to infection, presented lower parasitemia and lower cumulative mortality rate. No significant effect was observed on iron metabolism markers, erythrograms, leukograms or lymphocyte subsets.In the rapid method for testing in vivo T. cruzi susceptibility, DFO also induced lower parasitemia.In regard to its direct impact on parasites, DFO slightly inhibited the growth of amastigotes and trypomastigotes in fibroblast culture. Trypan blue staining showed no effects of DFO on parasite viability, and only minor apoptosis in trypomastigotes was observed. Nevertheless, a clear decrease in parasite mobility was detected.In conclusion, the beneficial actions of DFO on mice T. cruzi infection seem to be independent of host iron metabolism and free of significant haematological side effects. Through direct action on the parasite, DFO has more effective trypanostatic than trypanocidal properties.     

Trypanosoma cruzi: effect of benznidazole therapy combined with the iron chelator desferrioxamine in infected mice

Iron chelators have been employed in various studies aimed at evaluating the relationship between the iron status of the host and the development of infection. In the present study, the effects of benznidazole (BZ) therapy in combination with the iron chelator desferrioxamine (DFO) on the development of infection in mice inoculated with Trypanosoma cruzi Y strain have been investigated. Infected mice treated with DFO presented lower levels of parasitemia compared with infected untreated animals. Therapy with BZ for 21 days, with or without DFO, led to decreased parasitemia and reduced mortality, but BZ in combination with DFO treatment for 35 days (BZ/DFO-35) gave 0% mortality. All infected groups presented lower levels of iron in the liver, but serum iron concentrations were greater in DFO-35 and BZ/DFO-35, whereas hemoglobin levels were higher in BZ/DFO-35 and lower in DFO-35 compared with other treated groups. The percentage cure, determined from negative hemoculture and PCR results in animals that had survived for 60 days post-infection, was 18% for BZ and BZ/DFO-35, 42% for BZ combined with DFO for 21 days, and 67% for DFO-35. The results demonstrate that modification in iron stores increases BZ efficacy.     

Experimental benznidazole treatment of Trypanosoma cruzi II strains isolated from children of the Jequitinhonha Valley,Minas Gerais,Brazil, with Chagas disease

Trypanosoma cruzi strains from distinct geographic areas show differences in drug resistance and association between parasites genetic and treatment response has been observed. Considering that benznidazole (BZ) can reduce the parasite burden and tissues damage, even in not cured animals and individuals, the goal is to assess the drug response to BZ of T. cruzi II strains isolated from children of the Jequitinhonha Valley, state of Minas Gerais, Brazil, before treatment. Mice infected and treated with BZ in both phases of infection were compared with the untreated and evaluated by fresh blood examination, haemoculture, polymerase chain reaction, conventional (ELISA) and non-conventional (FC-ALTA) serologies. In mice treated in the acute phase, a significant decrease in parasitaemia was observed for all strains. Positive parasitological and/or serological tests in animals treated during the acute and chronic (95.1-100%) phases showed that most of the strains were BZ resistant. However, beneficial effect was demonstrated because significant reduction (p < 0.05%) and/or suppression of parasitaemia was observed in mice infected with all strains (acute phase), associated to reduction/elimination of inflammation and fibrosis for two/eight strains. BZ offered some benefit, even in not cured animals, what suggest that BZ use may be recommended at least for recent chronic infection of the studied region.     

DNA damage and nitric oxide synthesis in experimentally infected Balb/c mice with Trypanosoma cruzi

This study aimed to evaluate whether experimental Chagas disease in acute phase under benznidazole therapy can cause DNA damage in peripheral blood, liver, heart, and spleen cells or induce nitric oxide synthesis in spleen cells. Twenty Balb/c mice were distributed into four groups: control (non-infected animals); Trypanosoma cruzi infected; T. cruzi infected and submitted to benznidazole therapy; and only treated with benznidazole. The results obtained with the single cell gel (comet) assay showed that T. cruzi was able induce DNA damage in heart cells of both benznidazole treated or untreated infected mice. Similarly, T. cruzi infected animals showed an increase of DNA lesions in spleen cells. Regarding nitric oxide synthesis, statistically significant differences (p<0.05) were observed in all experimental groups compared to negative control, the strongest effect observed in the T. cruzi infected group. Taken together, these results indicate that T. cruzi may increase the level of DNA damage in mice heart and spleen cells. Probably, nitric oxide plays an important role in DNA damaging whereas benznidazole was able to minimize induced T. cruzi genotoxic effects in spleen cells.     

Trypanosoma cruzi: influence of predominant bacteria from indigenous digestive microbiota on experimental infection in mice

To verify the influence of some predominant components from indigenous microbiota on systemic immunological responses during experimental Chagas disease, germ-free NIH Swiss mice were mono-associated with Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp. and then infected with the Y strain of Trypanosoma cruzi. All the mono-associations predominantly induced a Th1 type of specific immune response to the infection by T. cruzi. A direct correlation was observed between a higher survival rate and increased IFN-gamma and TNF-alpha production (P<0.05) in E. faecalis-, B. vulgatus-, and Peptostreptococcus-associated mice. Moreover, higher levels of anti-T. cruzi IgG1 and anti-T. cruzi IgG2a were also found in mono-associated animals after infection. On the other hand, with the exception of E. faecalis-associated mice, mono-association induced a lower IL-10 production after infection (P<0.05) when compared with germ-free animals. Interestingly, spleen cell cultures from non-infected germ-free and mono-associated mice spontaneously produced higher levels (P<0.05) of IL-10 than cultures from infected mono-associated mice, except again for E. faecalis-associated animals. In conclusion, the presence of the components of the indigenous microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in gnotobiotic mice. However, the degree of increase in production of cytokines depends on each bacterial component.     

Trypanosoma cruzi reinfections in mice determine the severity of cardiac damage

In two murine models we studied Trypanosoma cruzi reinfection in the acute and chronic phase of experimental Chagas' disease in order to elucidate the relevance of reinfections in determining the variability of cardiac symptoms and the irreversible cardiac damage. They were followed for 120 and 600 days post infection (p.i.) for the acute and chronic model, respectively. Reinfected mice reached higher parasitaemia levels than infected mice. The survival was 33 and 21% in the chronic phase for mice reinfected in the acute phase and 13% for mice reinfected in the chronic stage at the end of the experiments. Sixty-six percent of the infected group presented electrocardiographic abnormalities (heart frequency, prolonged PQ segment or QRS complex) in the chronic stage whereas 100% of the reinfected animals exhibited electric cardiac dysfunction since 90 and 390 days p.i. for the acute and chronic reinfected model, respectively (P<0.01). Heart histopathological studies showed fibrosis and necrosis areas and mononuclear infiltrates supporting the view that parasite persistence is a major factor in continuing the tissue inflammation. This work shows that T. cruzi reinfections could be related to the variability and severity of the clinical course of Chagas' disease and that parasite persistence is involved in exacerbation of the disease.     

The increase in mannose receptor recycling favors arginase induction and Trypanosoma cruzi survival in macrophages

The macrophage mannose receptor (MR) is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. Trypanosoma cruzi antigen cruzipain (Cz) is found in the different developmental forms of the parasite. This glycoprotein has a highly mannosylated C-terminal domain that participates in the host-antigen contact. Our group previously demonstrated that Cz-macrophage (Mo) interaction could modulate the immune response against T. cruzi through the induction of a preferential metabolic pathway. In this work, we have studied in Mo the role of MR in arginase induction and in T. cruzi survival using different MR ligands. We have showed that pre-incubation of T. cruzi infected cells with mannose-Bovine Serum Albumin (Man-BSA, MR specific ligand) biased nitric oxide (NO)/urea balance towards urea production and increased intracellular amastigotes growth. The study of intracellular signals showed that pre-incubation with Man-BSA in T. cruzi J774 infected cells induced down-regulation of JNK and p44/p42 phosphorylation and increased of p38 MAPK phosphorylation. These results are coincident with previous data showing that Cz also modifies the MAPK phosphorylation profile induced by the parasite. In addition, we have showed by confocal microscopy that Cz and Man-BSA enhance MR recycling. Furthermore, we studied MR behavior during T. cruzi infection in vivo. MR was up-regulated in F4/80+ cells from T. cruzi infected mice at 13 and 15 days post infection. Besides, we investigated the effect of MR blocking antibody in T. cruzi infected peritoneal Mo. Arginase activity and parasite growth were decreased in infected cells pre-incubated with anti-MR antibody as compared with infected cells treated with control antibody. Therefore, we postulate that during T. cruzi infection, Cz may contact with MR, increasing MR recycling which leads to arginase activity up-regulation and intracellular parasite growth.     

Usefulness of the polymerase chain reaction for monitoring cure of mice infected with different Trypanosoma cruzi clonal genotypes following treatment with benznidazole

The capacity of the polymerase chain reaction (PCR) to detect the DNA of Trypanosoma cruzi was evaluated in 90 blood samples from BALB/c mice infected with T. cruzi cloned stocks of genotypes 19 and 20 (T. cruzi I) and 39 and 32 (T. cruzi II), and treated with benznidazole. The results from the fresh blood examination, hemoculture, and ELISA allowed to group the treated animals into: cured (TC), dissociated (DIS) and non-cured (NC). The PCR detected T. cruzi DNA in 50.9%, 58.3% and 100.0% of the samples from TC, DIS and NC mice, respectively. These DNA possibly derives from live T. cruzi or from recently lysed parasites, suggests that these animals are in fact not cured. The difference between the PCR results and results obtained using other techniques was statistically significant and independent of the parasite genotype. The PCR described has therefore potential to be used in cure control of treated patients.     

Trypanosoma cruzi: alteration in the lymphoid compartments following interruption of infection by early acute benznidazole therapy in mice

Disability and mortality as consequence of Chagas disease is enormous in South America. Recently, the success of the trypanocidal treatment with benznidazole, the only available drug, has been associated with the host immune response. In the current study, the impact of benznidazole administration immediately after the experimental infection with Trypanosoma cruzi was evaluated in the main lymphocyte populations in lymphoid organs. Untreated mice displayed enlargement of spleen and lymph node related to the increased frequency of T and B lymphocytes, respectively. An intense thymus involution with the depletion of CD4(+)CD8(+) double-positive thymocytes also occurred. Benznidazole treatment led to a partial reversion of the spleen and lymph node enlargement related to changes in the frequency of lymphocyte subsets due to infection. Prevention of thymus involution was achieved, with the profile of thymocyte subsets similar to that of non-infected mice. The parasitic load at the onset of T. cruzi infection seems critical to trigger immune system activation.     

Inducible cyclooxygenase released prostaglandin mediates immunosuppression in acute phase of experimental Trypanosoma cruzi infection

We investigated the possible role of prostaglandins produced by COX-2 in the immunosuppression observed during Trypanosoma cruzi infection. Con-A-stimulated splenocytes isolated from mice on days 5, 10, and 15 of infection released large amounts of PGE2 and this release was inhibited by the treatment of animals with sodium salicylate or meloxicam. The treatment of the animals with these drugs enhanced the release of IL-2 by splenocytes from T. cruzi-infected animals and significantly reduced the blood parasitemia and delayed the mortality of the infected mice. Furthermore, the release of TNF-alpha, IFN-gamma, IL-4, and IL-10 by Con-A-stimulated splenocytes obtained from infected mice on days 5, 10, and 15 of the infection was significantly inhibited by treatment of the animals with salicylate or meloxicam. In conclusion, the results suggest that the prostaglandins produced mainly by COX-2 mediate the immunosuppression observed in the acute phase of T. cruzi infection.     

A DNA vaccine encoding for TcSSP4 induces protection against acute and chronic infection in experimental Chagas disease

Immunization of mice with plasmids containing genes of Trypanosoma cruzi induces protective immunity in the murine model of Chagas disease. A cDNA clone that codes for an amastigote-specific surface protein (TcSSP4) was used as a candidate to develop a DNA vaccine. Mice were immunized with the recombinant protein rTcSSP4 and with cDNA for TcSSP4, and challenged with bloodstream trypomastigotes. Immunization with rTcSSP4 protein makes mice more susceptible to trypomastigote infection, with high mortality rates, whereas mice immunized with a eukaryotic expression plasmid containing the TcSSP4 cDNA were able to control the acute phase of infection. Heart tissue of gene-vaccinated animals did not show myocarditis and tissue damage at 365 days following infection, as compared with control animals. INF-γ was detected in sera of DNA vaccinated mice shortly after immunization, suggesting the development of a Th1 response. The TcSSP4 gene is a promising candidate for the development of an anti-T. cruzi DNA vaccine.     

Trypanosoma cruzi: the role of PGE2 in immune response during the acute phase of experimental infection

Chagas disease is characterized by cardiac lesions and a high level of PGE2. Our objective was to investigate the role of PGE2 in cardiac lesions. BALB/c mice were infected with Trypanosoma cruzi (1x10(3) trypomastigote forms) and were treated daily with PBS, meloxicam (0.5 mg/kg) or etoricoxib (0.6 mg/kg). The animals were sacrificed on the 21st day of infection and we collected the cardiac tissue and spleen cells for tissue culture. We observed that treatment with COX-2 inhibitors was able to decrease synthesis of PGE2 by spleen cells. This reduction was accompanied by reduction of the inflammatory infiltrate, parasite nets, cardiac fibrosis and fewer COX-2 positive cells in cardiac tissue obtained from these animals. In conclusion, treatment with COX-2 inhibitors, and consequent inhibition of PGE2 synthesis, was able to reduce the cardiac damage observed during the acute phase of experimental Chagas disease, thus demonstrating the involvement of this mediator in the cardiac lesion.     

Trypanosoma cruzi: Multiplex PCR to detect and classify strains according to groups I and II

A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10 fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I.     

Trypanosoma cruzi: plasma corticosterone after repetitive stress during the acute phase of infection

An increased level of plasma corticosterone is one manifestation of severe environmental or physiologic stress. The stress response mediated by the hypothalamic-pituitary-adrenal axis is already known to suppress immunoglobulin production and to impair immune function, but there are few studies relating stress and plasma corticosterone to the outcome of Trypanosoma cruzi infection. In this study, male Wistar rats were infected with the Y strain of T. cruzi and then subjected to repetitive stress by exposure to ether vapor for 1min twice a day during the acute phase of infection. Stressed animals showed decreased lytic antibody activity and lowered levels of peritoneal macrophages. Despite an increase in the weight of the spleen, histological analyses demonstrated tissue alterations, the presence of amastigote nests, and a complete absence of activated lymphoid follicles. These results suggest that stress-induced increases in plasma corticosterone can suppress the immune response and worsen tissue injury during the acute phase of T. cruzi infection.     

Morphological changes and EGF expression in the granular convoluted tubule cells of submandibular glands of Trypanosoma cruzi infected rats

We have previously demonstrated in rats that Chagas' disease affects the salivary glands, by promoting an enlargement of the submandibular gland. In order to further investigate possible functional alterations on infected submandibular glands, the objective of the present study was to analyze epidermal growth factor (EGF) expression on rat submandibular glands during Trypanosoma cruzi infection. Results demonstrated that infected rats presented lower levels of testosterone, and morphological changes in the granular convoluted tubule (GCT) cells of the submandibular glands, along with acinar enlargement and delayed ductal maturation at the developing granular ducts. Immunohistochemistry analysis additionally showed that only few cells immunolabelled with anti-EGF on infected rats during the acute phase of Chagas' disease, while after 64 and 90 days (chronic phase) of infection, EGF expression was similar to non-infected rats. The present findings suggest that at the acute phase of Chagas' disease, lower levels of testosterone may lead to a delayed maturation of GCT, which positively correlates with decreased EGF production by submandibular glands cells.     

Mouse Macrophage Galactose-type Lectin (mMGL) is Critical for Host Resistance against Trypanosoma cruzi Infection

The C-type lectin receptor mMGL is expressed exclusively by myeloid antigen presenting cells (APC) such as dendritic cells (DC) and macrophages (Mφ), and it mediates binding to glycoproteins carrying terminal galactose and α- or β-N-acetylgalactosamine (Gal/GalNAc) residues. Trypanosoma cruzi (T. cruzi) expresses large amounts of mucin (TcMUC)-like glycoproteins. Here, we show by lectin-blot that galactose moieties are also expressed on the surface of T. cruzi. Male mMGL knockout (-/-) and wild-type (WT) C57BL/6 mice were infected intraperitoneally with 10⁴ T. cruzi trypomastigotes (Queretaro strain). Following T. cruzi infection, mMGL-/- mice developed higher parasitemia and higher mortality rates compared with WT mice. Although hearts from T. cruzi-infected WT mice presented few amastigote nests, mMGL-/- mice displayed higher numbers of amastigote nests. Compared with WT, Mφ from mMGL-/- mice had low production of nitric oxide (NO), interleukin (IL)-12 and tumor necrosis factor (TNF)-α in response to soluble T. cruzi antigens (TcAg). Interestingly, upon in vitro T. cruzi infection, mMGL-/- Mφ expressed lower levels of MHC-II and TLR-4 and harbored higher numbers of parasites, even when mMGL-/- Mφ were previously primed with IFN-γ or LPS/IFN-γ. These data suggest that mMGL plays an important role during T. cruzi infection, is required for optimal Mφ activation, and may synergize with TLR-4-induced pathways to produce TNF-α, IL-1β and NO during the early phase of infection.     

Trypanosoma cruzi: sensitivity of the polymerase chain reaction for detecting the parasite in the blood of mice infected with different clonal genotypes

The polymerase chain reaction showed high sensitivity for detecting Trypanosoma cruzi in the blood of mice, independent of clonal genotype (19, 20-T. cruzi I; 32, 39-T. cruzi II) or phase of the infection (acute or chronic).     

Congenital Chagas disease: an update

Congenital infection with Trypanosoma cruzi is a global problem, occurring on average in 5% of children born from chronically infected mothers in endemic areas, with variations depending on the region. This presentation aims to focus on and update epidemiological data, research methods, involved factors, control strategy and possible prevention of congenital infection with T. cruzi. Considering that etiological treatment of the child is always effective if performed before one year of age, the diagnosis of infection in pregnant women and their newborns has to become the standard of care and integrated into the surveillance programs of syphilis and human immunodeficiency virus. In addition to the standard tests, polymerase chain reaction performed on blood of neonates of infected mothers one month after birth might improve the diagnosis of congenital infection. Recent data bring out that its transmission can be prevented through treatment of infected women before they become pregnant. The role of parasite genotypes and host genetic factors in parasite transmission and development of infection in foetuses/neonates has to be more investigated in order to better estimate the risk factors and impact on health of congenital infection with T. cruzi.     

Myenteric plexus is differentially affected by infection with distinct Trypanosoma cruzi strains in Beagle dogs

Chagasic megaoesophagus and megacolon are characterised by motor abnormalities related to enteric nervous system lesions and their development seems to be related to geographic distribution of distinct Trypanosoma cruzi subpopulations. Beagle dogs were infected with Y or Berenice-78 (Be-78) T. cruzi strains and necropsied during the acute or chronic phase of experimental disease for post mortem histopathological evaluation of the oesophagus and colon. Both strains infected the oesophagus and colon and caused an inflammatory response during the acute phase. In the chronic phase, inflammatory process was observed exclusively in the Be-78 infected animals, possibly due to a parasitism persistent only in this group. Myenteric denervation occurred during the acute phase of infection for both strains, but persisted chronically only in Be-78 infected animals. Glial cell involvement occurred earlier in animals infected with the Y strain, while animals infected with the Be-78 strain showed reduced glial fibrillary acidic protein immunoreactive area of enteric glial cells in the chronic phase. These results suggest that although both strains cause lesions in the digestive tract, the Y strain is associated with early control of the lesion, while the Be-78 strain results in progressive gut lesions in this model.     

Towards developing a diagnostic regimen for the treatment follow-up of Trypanosoma brucei gambiense

BALB/c mice infected with a high virulent strain of Trypanosoma brucei gambiense IL3707 were treated intraperitoneally (i.p.) with either Melarsoprol (Mel-B) or PSG(+) buffer as controls. The mice were subsequently monitored regularly for parasites by direct microscopic examination of their tail blood or buffy coat and by polymerase chain reaction (PCR). Mel-B was found to be an effective drug for treatment against T.b. gambiense because at the end of the first treatment schedule, all treated mice were negative for parasites even by PCR, while all the control animals were positive. Three of the five Mel-B treated mice, while parasitologically negative, were PCR positive between 53 and 80 days post infection (DPI), indicating that they still harbored an infection. All treated mice were subsequently negative for parasites even by PCR at 88 DPI. A combination of conventional microscopic examination and PCR offers a good prediction of cure following treatment of trypanosomosis.