Deficient regulatory T cell activity and low frequency of IL-17-producing T cells correlate with the extent of cardiomyopathy in human Chagas' disease

Background

Myocardium damage during Chagas'' disease results from the immunological imbalance between pro- and production of anti-inflammatory cytokines and has been explained based on the Th1–Th2 dichotomy and regulatory T cell activity. Recently, we demonstrated that IL-17 produced during experimental T. cruzi infection regulates Th1 cells differentiation and parasite induced myocarditis. Here, we investigated the role of IL-17 and regulatory T cell during human Chagas'' disease.

Methodology/Principal Findings

First, we observed CD4⁺IL-17⁺ T cells in culture of peripheral blood mononuclear cells (PBMC) from Chagas'' disease patients and we evaluated Th1, Th2, Th17 cytokine profile production in the PBMC cells from Chagas'' disease patients (cardiomyopathy-free, and with mild, moderate or severe cardiomyopathy) cultured with T. cruzi antigen. Cultures of PBMC from patients with moderate and severe cardiomyopathy produced high levels of TNF-α, IFN-γ and low levels of IL-10, when compared to mild cardiomyopathy or cardiomyopathy-free patients. Flow cytometry analysis showed higher CD4⁺IL-17⁺ cells in PBMC cultured from patients without or with mild cardiomyopathy, in comparison to patients with moderate or severe cardiomyopathy. We then analyzed the presence and function of regulatory T cells in all patients. All groups of Chagas'' disease patients presented the same frequency of CD4⁺CD25⁺ regulatory T cells. However, CD4⁺CD25⁺ T cells from patients with mild cardiomyopathy or cardiomyopathy-free showed higher suppressive activity than those with moderate and severe cardiomyopathy. IFN-γ levels during chronic Chagas'' disease are inversely correlated to the LVEF (P = 0.007, r = −0.614), while regulatory T cell activity is directly correlated with LVEF (P = 0.022, r = 0.500).

Conclusion/Significance

These results indicate that reduced production of the cytokines IL-10 and IL-17 in association with high levels of IFN-γ and TNF-α is correlated with the severity of the Chagas'' disease cardiomyopathy, and the immunological imbalance observed may be causally related with deficient suppressor activity of regulatory T cells that controls myocardial inflammation.     

In Vitro and In Vivo Studies of the Trypanocidal Properties of WRR-483 against Trypanosoma cruzi

Background

Cruzain, the major cysteine protease of Trypanosoma cruzi, is an essential enzyme for the parasite life cycle and has been validated as a viable target to treat Chagas'' disease. As a proof-of-concept, K11777, a potent inhibitor of cruzain, was found to effectively eliminate T. cruzi infection and is currently a clinical candidate for treatment of Chagas'' disease.

Methodology/Principal Findings

WRR-483, an analog of K11777, was synthesized and evaluated as an inhibitor of cruzain and against T. cruzi proliferation in cell culture. This compound demonstrates good potency against cruzain with sensitivity to pH conditions and high efficacy in the cell culture assay. Furthermore, WRR-483 also eradicates parasite infection in a mouse model of acute Chagas'' disease. To determine the atomic-level details of the inhibitor interacting with cruzain, a 1.5 Å crystal structure of the protease in complex with WRR-483 was solved. The structure illustrates that WRR-483 binds covalently to the active site cysteine of the protease in a similar manner as other vinyl sulfone-based inhibitors. Details of the critical interactions within the specificity binding pocket are also reported.

Conclusions

We demonstrate that WRR-483 is an effective cysteine protease inhibitor with trypanocidal activity in cell culture and animal model with comparable efficacy to K11777. Crystallographic evidence confirms that the mode of action is by targeting the active site of cruzain. Taken together, these results suggest that WRR-483 has potential to be developed as a treatment for Chagas'' disease.     

Epidemiology of mortality related to Chagas' disease in Brazil, 1999-2007

Background

Chagas'' disease is an important neglected public health problem in many Latin American countries, but population-based epidemiological data are scarce. Here we present a nationwide analysis on Chagas-associated mortality, and risk factors for death from this disease.

Methodology/Principal Findings

We analyzed all death certificates of individuals who died between 1999 and 2007 in Brazil, based on the nationwide Mortality Information System (a total of 243 data sets with about 9 million entries). Chagas'' disease was mentioned in 53,930 (0.6%) of death certificates, with 44,537 (82.6%) as an underlying cause and 9,387 (17.4%) as an associated cause of death. Acute Chagas'' disease was responsible for 2.8% of deaths. The mean standardized mortality rate was 3.36/100.000 inhabitants/year. Nationwide standardized mortality rates reduced gradually, from 3.78 (1999) to 2.78 (2007) deaths/year per 100,000 inhabitants (−26.4%). Standardized mortality rates were highest in the Central-West region, ranging from 15.23 in 1999 to 9.46 in 2007 (−37.9%), with a significant negative linear trend (p = 0.001; R² = 82%). Proportional mortality considering multiple causes of death was 0.60%. The Central-West showed highest proportional mortality among regions (2.17%), with a significant linear negative trend, from 2.28% to 1.90% (−19.5%; p = 0.001; R² = 84%). There was a significant increase in the Northeast of 38.5% (p = 0.006; R² = 82%). Bivariable analysis on risk factors for death from Chagas'' disease showed highest relative risks (RR) in older age groups (RR: 10.03; 95% CI: 9.40–10.70; p<0.001) and those residing in the Central-West region (RR: 15.01; 95% CI: 3.90–16.22; p<0.001). In logistic regression analysis, age ≥30 years (adjusted OR: 10.81; 95% CI: 10.03–10.65; p<0.001) and residence in one of the three high risk states Minas Gerais, Goiás or the Federal District (adjusted OR: 5.12; 95% CI: 5.03–5.22, p<0.001) maintained important independent risk factors for death by Chagas'' disease.

Conclusions/Significance

This is the first nationwide population-based study on Chagas mortality in Brazil, considering multiple causes of death. Despite the decline of mortality associated with Chagas'' disease in Brazil, the disease remains a serious public health problem with marked regional differences.     

The Indian triatomine genus Linshcosteus (Reduviidae)

The Indian genus Linshcosteus is revised and a key provided to the three species L.carnifex Distant; L.confumus sp.n.; L.costalis sp.n. One or both of the new species described here is the host of the egg parasite Gryon linshcostei Masner (Scelionidae) which may be used in the biological control of the Triatoma vectors of Chagas' disease in South America.     

Efficacy of novel benznidazole solutions during the experimental infection with Trypanosoma cruzi

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. About 8 million people throughout Latin America are infected causing approximately 10,000 deaths annually. Benznidazole, available as unique 100 mg tablets in many of the endemic countries, is currently the drug of choice for the specific treatment of this condition. Despite of the large number of pediatric patients infected, there are no commercial liquid dosage forms available to treat this trypanosomiasis. This work showed that novel benznidazole–water–polyethylene glycol 400 solutions are active against T. cruzi in a murine model of Chagas' disease. Present results constitute the first demonstration on the usefulness of benznidazole solutions in infected mice.     

Trypanosoma cruzi: Biological Characterization of 19 Clones Derived from Two Chronic Chagasic Patients. I. Growth Kinetics in Liquid Medium1

Nineteen clones of Trypanosoma cruzi were obtained as single-cell isolates from Triatoma infestans. Ten of the clones were isolates from a patient with chronic Chagas' disease; nine clones were isolates from a dog infected with T. cruzi strain CA-I isolated originally from a chronic chagasic patient. The growth kinetics and peak modal Coulter volume of these clones were characterized. Significant inter- and intra-group differences between growth rates and peak modal volumes were found. These data indicate that subpopulations and, consequently, genetic heterogeneity of T. cruzi exist in chronic chagasic patients. All of the clones infected vertebrate cells in vitro.     

Enantioselectivity in the metabolism of mexiletine by conjugation in female patients with the arrhythmic form of chronic Chagas' heart disease

The phenomenon of enantioselectivity in the metabolism of mexiletine (MEX) conjugation was investigated in eight female patients with the arrhythmic form of chronic Chagas' heart disease treated with racemic mexiletine hydrochloride (two 100 mg capsules every 8 hr). Blood samples were collected up to 24 hr after the administration of the morning dose, with discontinuation of the subsequent doses during the study period. Plasma concentrations of N‐hydroxymexiletine glucuronide were calculated as the difference between the concentrations of unchanged and total (unchanged + conjugated) MEX enantiomers. Total plasma MEX concentrations were analyzed by HPLC after enzymatic hydrolysis with β‐glucuronidase, the formation of diastereomeric derivatives with the chiral reagent N‐acetyl‐l ‐cysteine/o‐phthalaldehyde, and fluorescence detection. The differences in the pharmacokinetic parameters of the enantiomers were evaluated by the paired t‐test. The plasma concentrations of the (+)‐(S)‐MEX did not differ before and after enzymatic hydrolysis. The pharmacokinetic parameters calculated for (−)‐(R)‐N‐hydroxymexiletine glucuronide are presented as means (95% confidence interval): maximum plasma concentration C_max = 194.0 ng · ml⁻¹ (154.3–233.7), time to maximum plasma concentration t_max = 1.4 hr (0.3–2.5), area under the plasma concentration versus time curve AUC^0–24 = 2099.2 ng · h · ml⁻¹ (1585.6–2612.6), elimination half‐life t_1/2β = 12.8 hr (9.9–15.6) and extent of conjugation of 31.6% (24.3–38.9%). The present data indicate stereospecific conjugation of (−)‐(R)‐N‐hydroxymexiletine in the female patients with the arrhythmic form of Chagas' heart disease. Chirality 11:29–32, 1999. © 1999 Wiley‐Liss, Inc.     

Tropical diseases encountered in Canada: 1. Chagas' disease.

Chagas'' disease, or South American trypanosomiasis, is an endemic South American disease now being seen in Canada in both acute and chronic forms. It is characterized by an initial parasitemia that elicits a brisk immune response. Evidence is mounting that the debilitating chronic form, which is characterized by cardiac and visceral organ failure, results from antigenic cross-reactivity between the parasite and the human host, which generates an aberrant, destructive, cell-mediated immune response. Diagnosis, treatment and potential areas for investigation are discussed.     

L925I Mutation in the Para-Type Sodium Channel Is Associated with Pyrethroid Resistance in Triatoma infestans from the Gran Chaco Region

Background

Chagas'' disease is an important public health concern in Latin America. Despite intensive vector control efforts using pyrethroid insecticides, the elimination of Triatoma infestans has failed in the Gran Chaco, an ecoregion that extends over Argentina, Paraguay, Bolivia and Brazil.The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in domain II region of the channel have been implicated in pyrethroid resistance of several insect species.

Methods and Findings

In the present paper, we identify L925I, a new pyrethroid resistance-conferring mutation in T. infestans. This mutation has been found only in hemipterans. In T. infestans, L925I mutation occurs in a resistant population from the Gran Chaco region and is associated with inefficiency in the control campaigns. We also describe a method to detect L925I mutation in individuals from the field.

Conclusions and Significance

The findings have important implications in the implementation of strategies for resistance management and in the rational design of campaigns for the control of Chagas'' disease transmission.     

The in vivo trypanocidal effect of the diterpene 5-epi-icetexone obtained from Salvia gilliesii

The search for new compounds with trypanocidal activity is crucial for the treatment of Chagas' disease. Previous in vitro studies have shown that the diterpene 5-epi-icetexone (ICTX) is active against Trypanosoma cruzi. The aim of this work was to evaluate the effect of ICTX on the parasites in infected mice, in an experimental model that mimics the acute phase of the disease. Swiss albino mice were infected with T. cruzi and treated daily with 10 mg/kg/day ICTX (i.p.). Infected mice and mice injected with either saline or the vehicle DMSO were used as controls. Animals' survival and parasitemia were monitored once a week and histological studies were made at necropsy by the 5th week after infection. It was observed that the administration of ICTX increased the survival of mice infected, and induced a significant decrease in the parasitemia, as compared to controls. A similar protective effect was observed when animals were treated orally with benznidazole (BZN, used as a control of antiparasitic effect). By the 5th week post-infection, the presence of amastigote nests was observed within the fibers of the cardiac and skeletal muscle in controls, but not in animals treated with either ICTX or BZN. In addition, inflammatory infiltrates were observed in the tissues of controls, but not in animals treated with the drugs. We conclude that ICTX has an antiparasitic effect against T. cruzi, thus constituting an interesting option for the treatment of Chagas' disease, alone or combined with other drugs.     

Coronary Microvascular Disease in Chronic Chagas Cardiomyopathy Including an Overview on History,Pathology, and Other Proposed Pathogenic Mechanisms

This review focuses on the short and bewildered history of Brazilian scientist Carlos Chagas''s discovery and subsequent developments, the anatomopathological features of chronic Chagas cardiomyopathy (CCC), an overview on the controversies surrounding theories concerning its pathogenesis, and studies that support the microvascular hypothesis to further explain the pathological features and clinical course of CCC. It is our belief that knowledge of this particular and remarkable cardiomyopathy will shed light not only on the microvascular involvement of its pathogenesis, but also on the pathogenetic processes of other cardiomyopathies, which will hopefully provide a better understanding of the various changes that may lead to an end-stage heart disease with similar features. This review is written to celebrate the 100th anniversary of the discovery of Chagas disease.     

ISOZYME VARIABILITY IN TRYPANOSOMA CRUZI,THE AGENT OF CHAGAS' DISEASE: GENETICAL,TAXONOMICAL, AND EPIDEMIOLOGICAL SIGNIFICANCE

A genetic interpretation of the zymograms of 524 Trypanosoma cruzi stocks from various hosts and representing a broad geographical range (United States to Southern Brazil) reveals high genetic variability (only one monomorphic locus out of 15) and suggests that this parasite has a diploid structure. The data do not give any indication of Mendelian sexuality, although many opportunities are present for genetic exchange between extremely different genotypes. The population structure of T. cruzi appears to be multiclonal and complex. The natural clones evidenced by isozyme analysis are numerous (43 different ones are recorded among 121 stocks assayed at 15 gene loci) and exhibit a large range of genotypes, in a nonhierarchical structure; it is not possible to cluster them into a few strictly delimited groups which could represent natural taxa. The available data suggest that the genetic variability of T. cruzi reflects the long separate evolution of multiple clones. It is suggested that long clonal evolution may explain the present biological and medical variability of the causative agent of Chagas' disease.     

Pentoxifylline Reverses Chronic Experimental Chagasic Cardiomyopathy in Association with Repositioning of Abnormal CD8+ T-Cell Response

BackgroundChronic chagasic cardiomyopathy (CCC), the main clinical sign of Chagas disease, is associated with systemic CD8⁺ T-cell abnormalities and CD8-enriched myocarditis occurring in an inflammatory milieu. Pentoxifylline (PTX), a phosphodiesterase inhibitor, has immunoregulatory and cardioprotective properties. Here, we tested PTX effects on CD8⁺ T-cell abnormalities and cardiac alterations using a model of experimental Chagas’ heart disease.Conclusions/SignificancePTX therapy ameliorates critical aspects of CCC and repositioned CD8⁺ T-cell response towards homeostasis, reinforcing that immunological abnormalities are crucially linked, as cause or effect, to CCC. Therefore, PTX emerges as a candidate to treat the non-beneficial immune deregulation associated with chronic Chagas'' heart disease and to improve prognosis.     

The Uptake of GABA in Trypanosoma cruzi

Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na⁺, K⁺, and H⁺ on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na⁺ dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na⁺/GABA symporter energized by Na⁺‐exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.     

Control of experimental Triatoma infestans populations: effect of pour‐on cypermethrin applied to chickens under natural conditions in the Argentinean Chaco region

Among peridomestic structures, chicken coops are sites of major importance for the domestic ecology of Triatoma infestans (Hemiptera: Reduviidae). The aim of this study was to evaluate in an experimental context the effects of a cypermethrin pour‐on formulation applied to chickens on blood intake, moulting and mortality in T. infestans, under the natural climatic conditions of a region endemic for Chagas' disease. Experimental chicken huts were made of bricks and covered with plastic mosquito nets. Ninety fourth‐instar nymphs were maintained in each hut. The study used a completely random design in which chickens in the experimental group were treated with a cypermethrin pour‐on formulation. Five replicates (= huts) of the experimental and control groups were conducted. The number of live T. infestans, blood intake and moults to fifth‐instar stage were recorded at 1, 5, 20, 35 and 45 days after the application of cypermethrin. Cumulative mortality was higher in nymphs exposed to treated chickens (> 71%) than in control nymphs (< 50%) (P < 0.01). Blood intake and moulting rate were lower in nymphs fed on treated chickens than in control nymphs (P < 0.05). Pour‐on cypermethrin was able to cause significant mortality, although it did not eliminate the experimental population of T. infestans.     

Excretion/defecation patterns in Triatoma infestans populations that are,respectively, susceptible and resistant to deltamethrin

Pyrethroid resistance has been detected in Triatoma infestans (Klug) (Hemiptera: Reduviidae) specimens from different areas of Argentina and Bolivia. Genes conferring resistance can have a pleiotropic effect with epidemiological and evolutionary consequences. This research studied excretion/defecation patterns in deltamethrin‐resistant T. infestans in order to elucidate its biological performance, adaptive consequences and role in the transmission of Chagas' disease. One deltamethrin‐susceptible strain and two deltamethrin‐resistant strains were used. Fifth‐instar nymphs were fed ad libitum and their defecations recorded during and after the first or second feeding in the stadium. Resistant insects began to defecate later, defecated less, showed a lower proportion of defecating individuals and lower defecation indices compared with susceptible insects during the first hour after feeding. The number of bloodmeals in the stadium did not affect the main variables determining the pattern of defecation. The present study suggests that alterations in the excretion/defecation pattern in resistant insects entail an adaptive cost and, considering only this pattern, determine a lower capacity for transmission of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) compared with susceptible insects.     

Pathological effects of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bugs Triatoma sordida, T. pallidipennis and Dipetalogaster maxima after coprophagic infection

ABSTRACT. Developmental time and mortality in nymphs of the reduviid bugs Triatoma sordida (Stål), Triatoma pallidipennis (Stål) and Dipetalogaster maxima (Uhler) were studied in uninfected groups and in those infected with Blastocrithidia triatomae Cerisola et al. (Trypanosomatidae). In T. sordida and T. pallidipennis, major vectors of Chagas' disease in Brazil and Mexico respectively, infection with B. triatomae was associated with slight developmental retardations in the final instars, and increased mortality in the pre-adult instar. These effects were less evident in T. pallidipennis, but in this species a total infection rate of only 5–15% was achieved, compared with about 45% in T. sordida. In contrast, D. maxima was strongly affected by B. triatomae: nymphal development was retarded and complete mortality occurred in some groups exposed to infection, although not all bugs were infected.     

Interaction between cFLIPL and Itch,a ubiquitin ligase,is obstructed in Trypanosoma cruzi‐infected human cells

Death receptor‐mediated host cell apoptosis, a defense strategy for elimination by the immune system of parasite‐infected cells, is inhibited by Trypanosoma cruzi, the causative agent of Chagas' disease. It has previously been reported by us that, in infected cells, T. cruzi upregulates and exploits cFLIP_L, a mammalian inhibitor of death receptor signaling. Here it is shown that ubiquitination of cFLIP_L, leading to proteasomal degradation, is inhibited in parasite‐infected cells. The extent of expression of Itch, a protein thought to be an ubiquitin ligase for cFLIP_L, was found to be equivalent in T. cruzi‐infected and in uninfected cells. However, co‐immunoprecipitation analysis showed that the interaction between cFLIP_L and Itch is strongly inhibited in T. cruzi‐infected cells. This unique parasite strategy, which has not been reported in any other pathogen‐infected cells, may allow the host cell to accumulate cFLIP_L, eventually resulting in the inhibition of apoptosis of T. cruzi‐infected cells.     

QM/MM Molecular Dynamics Study of the Galactopyranose → Galactofuranose Reaction Catalysed by Trypanosoma cruzi UDP-Galactopyranose Mutase

The enzyme UDP-Galactopyranose Mutase (UGM) catalyses the conversion of galactopyranose into galactofuranose. It is known to be critical for the survival and proliferation of several pathogenic agents, both prokaryotic and eukaryotic. Among them is Trypanosoma cruzi, the parasite responsible for Chagas'' disease. Since the enzyme is not present in mammals, it appears as a promising target for the design of drugs to treat this illness. A precise knowledge of the mechanism of the catalysed reaction would be crucial to assist in such design. In this article we present a detailed study of all the putative steps of the mechanism. The study is based on QM/MM free energy calculations along properly selected reaction coordinates, and on the analysis of the main structural changes and interactions taking place at every step. The results are discussed in connection with the experimental evidence and previous theoretical studies.