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Background: Trypanosoma cruzi is a protozoan parasite and the etiologic agent of Chagas disease, an important public health problem in Latin America. T. cruzi is diploid, almost exclusively asexual, and displays an extraordinarily diverse population structure both genetically and phenotypically. Yet, to date the genotypic diversity of T. cruzi and its relationship, if any, to biological diversity have not been studied at the whole genome level., Results: In this study, we used whole genome oligonucleotide tiling arrays to compare gene content in biologically disparate T. cruzi strains by comparative genomic hybridization (CGH). We observed that T. cruzi strains display widespread and focal copy number variations (CNV) and a substantially greater level of diversity than can be adequately defined by the current genetic typing methods. As expected, CNV were particularly frequent in gene family-rich regions containing mucins and trans-sialidases but were also evident in core genes. Gene groups that showed little variation in copy numbers among the strains tested included those encoding protein kinases and ribosomal proteins, suggesting these loci were less permissive to CNV. Moreover, frequent variation in chromosome copy numbers were observed, and chromosome-specific CNV signatures were shared by genetically divergent T. cruzi strains., Conclusions: The large number of CNV, over 4,000, reported here uphold at a whole genome level the long held paradigm of extraordinary genome plasticity among T. cruzi strains. Moreover, the fact that these heritable markers do not parse T. cruzi strains along the same lines as traditional typing methods is strongly suggestive of genetic exchange playing a major role in T. cruzi population structure and biology.

Trypanosoma cruzi is a protozoan parasite that causes human Chagas' disease, a leading source of congestive heart failure in Central and South America. CD8+ T cells are critical for control of T. cruzi infection, and CD8+ T cells recognizing the immunodominant trans-sialidase gene-encoded peptide TSKB20 (ANYKFTLV) account for approximately 30% of the total CD8+ T-cell population at the peak of infection in C57BL/6 mice. Type I interferons (IFN-I) are pleiotropic cytokines that play a critical role in both innate and adaptive immunity against a variety of infections, but their induction and their role in infection are dictated by the infectious agent. Because type I IFNs and IFN-responsive genes are evident early after T. cruzi infection of host cells, we examined the influence of IFN-I on the development of CD8+ T-cell responses during this infection. Mice lacking the receptor for IFN-I (IFNARKO) and their wild-type counterparts both developed chronic infections and generated similar frequencies of immunodominant TSKB20- and subdominant TSKB18-specific CD8+ T cells following T. cruzi infection. In contrast, peak TSKB20-specific CD8+ T-cell responses generated during infection with vaccinia virus engineered to express TSKB20 were approximately 2.5-fold lower in IFNARKO mice than B6 mice, although after viral clearance, the frequencies of TSKB20-specific CD8+ T cells stabilized at similar levels. Together, these data suggest that IFN-I induction and biology are dependent upon the microbial context and emphasize the need to investigate various infection models for a full understanding of CD8+ T-cell development.

The synthesis of 2-(5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)hydrazone-derivatives (BTPs) and their in vitro evaluation against Trypanosoma cruzi trypomastigotes, Mycobacterium tuberculosis, Leishmania amazonensis axenic amastigotes, and six human cancer cell lines is described. The in vivo activity of the most active and least toxic compounds against T. cruzi and L. amazonensis was also studied. BTPs constitute a new family of drug leads with potential activity against infectious diseases. Due to their drug-like properties, this series of compounds can potentially serve as templates for future drug-optimization and drug-development efforts for use as therapeutic agents in developing countries., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: As many as 20 million people are living with Trypanosoma cruzi infection in Latin American, yet few receive any treatment. The major limitation in developing and evaluating potential new drugs for their efficacy is the lack of reliable tests to assess parasite burden and elimination., Methods: Adults volunteers with chronic T. cruzi infection were evaluated clinically and stratified according to the Kuschnir classification. Individuals with group 0 and group 1 clinical status were treated with benznidazole (5 mg/kg per day for 30 days). The changes in T. cruzi-specific T cell and antibody responses, as well as in clinical status, were measured periodically over the 3-5-year follow-up period and were compared with pretreatment conditions and with values in an untreated control group., Results: The frequency of peripheral interferon (IFN)-gamma-producing T cells specific for T. cruzi declined as early as 12 months after benznidazole treatment and subsequently became undetectable in a substantial proportion of treated subjects. In addition, decreases in antibody responses to a pool of recombinant T. cruzi proteins also decreased in many of these same subjects. The shift to negative IFN-gamma T cell responses was highly associated with an early increase in IFN-gamma-producing T cells with phenotypic features of effector/effector memory cells in a subset of subjects. Benznidazole treatment also resulted in an increase in naive and early differentiated memory-like CD8(+) T cells in a majority of subjects., Conclusions: Benznidazole treatment during chronic Chagas disease has a substantial impact on parasite-specific immune response that is likely indicative of treatment efficacy and cure.

Infection with the protozoan parasite Trypanosoma cruzi leads to chronic infection, with parasite persistence primarily in muscle tissue. CD8(+) T cells isolated from muscle tissue of T. cruzi-infected mice display decreased production of IFN-gamma in response to T cell receptor engagement. The expression of TGF-beta at the site of CD8(+) T cell dysfunction and parasite persistence suggested that this immunoregulatory cytokine might play a role in these processes. Mice expressing a T cell-specific dominant negative TGF-beta receptor type II (DNRII) were therefore infected with T. cruzi. Infection of DNRII mice resulted in massive CD8(+) T cell proliferation, leading to increased numbers but decreased frequencies of antigen-specific CD8(+) T cells in the spleen compared to wild-type mice. However, TGF-beta unresponsiveness failed to restore effector functions of CD8(+) T cells isolated from muscle tissue. Histological examination of skeletal muscle from T. cruzi-infected DNRII mice revealed an extensive cellular infiltrate, and DNRII mice displayed higher susceptibility to infection. Overall, while TGF-beta does not appear to be responsible for CD8(+) T cell unresponsiveness in peripheral tissue in T. cruzi-infected mice, these data suggest a role for TGF-beta in control of immunopathology in response to T. cruzi infection.

Innate and adaptive cellular immune recognition is crucial for control of the protozoan parasite Trypanosoma cruzi. T. cruzi triggers both MyD88-dependent and TRIF-dependent innate activation pathways in macrophages and dendritic cells. TLR-2 and TLR-9 recognize GPI anchors and parasite DNA, respectively; however other, as yet undefined receptors and ligands, also appear to be involved in innate recognition. CD8(+) T cells distinguish T. cruzi-infected host cells primarily via robust recognition of MHC-associated peptide epitopes from the large and highly diverse trans-sialidase family of surface proteins. To date there has been minimal investigation of linkages between innate immune recognition in vivo and the generation of adaptive cellular immune responses.

We present a cross-sectional analysis of the maturation and migratory properties of the memory CD8(+) T cell compartment, in relation to the severity of heart disease in individuals with chronic Trypanosoma cruzi infection removed from endemic areas for longer than 20 years. Subjects with none or mild heart involvement were more likely to mount T. cruzi-specific memory IFN-gamma responses than subjects with more advanced cardiac disease, and the T. cruzi-specific CD8(+) T cell population was enriched in early-differentiated (CD27(+)CD28(+)) cells in responding individuals. In contrast, the frequency of CD27(+)CD28(+)CD8(+) T cells in the total memory CD8(+) T cell population decreases, as disease becomes more severe, while the proportion of fully differentiated memory (CD27(-)CD28(-)) CD8(+) T cells increases. The analysis of CCR7 expression revealed a significant increase in total effector/memory CD8(+) T cells (CD45RA(-)CCR7(-)) in subjects with mild heart disease as compared with uninfected controls. Altogether, these results are consistent with the hypothesis of a gradual clonal exhaustion in the CD8(+) T cell population, perhaps as a result of continuous antigenic stimulation by persistent parasites.

Immune control of many intracellular pathogens, including Trypanosoma cruzi, is reported to be dependent on the production of nitric oxide. In this study, we show that mice deficient in inducible nitric oxide synthase (iNOS or NOS2) exhibit resistance to T. cruzi infection that is comparable to that of wild-type mice. This is the case for two iNOS-deficient mouse strains, Nos2(tm1Lau) and Nos2 N5, infected with the Brazil or Tulahuen strain of T. cruzi. In all cases, blood parasitemia, tissue parasite load, and survival rates are similar between wild-type and iNOS-deficient mice. In contrast, both wild-type and Nos2(tm1Lau) mice died within 32 days postinfection when treated with the nitric oxide synthase inhibitor aminoguanidine. Increased transcription of NOS1 or NOS3 is not found in iNOS-knockout (KO) mice, indicating that the absence of nitric oxide production through iNOS is not compensated for by increased production of other NOS isoforms. However, Nos2(tm1Lau) mice exhibit enhanced expression of tumor necrosis factor alpha, interleukin-1, and macrophage inflammatory protein 1alpha compared to that of wild-type mice, and these alterations may in part compensate for the lack of iNOS. These results clearly show that iNOS is not required for control of T. cruzi infection in mice.

This study sought to quantify CD8(+) T cell responses to Trypanosoma cruzi and to identify potential links between these responses and the severity of disease in humans. In the majority of patients with Chagas disease, staining with class I major histocompatibility complex tetramers and analysis of interferon (IFN)- gamma ELISPOT responses to a panel of known cytotoxic T lymphocyte target epitopes from T. cruzi failed to identify parasite-specific CD8(+) T cells. However, the frequency of individuals with positive ELISPOT responses was higher in areas of active transmission. Analysis of IFN- gamma ELISPOT responses to a parasite lysate revealed a very high frequency of responders among patients with mild clinical disease and a very low frequency of responders among those with the most severe form of the disease. These data suggest that the frequency of IFN- gamma -producing T cells in patients with chronic Chagas disease is associated with the history of recent exposure and with the clinical status of the patient.

TcruziDB (http://TcruziDB.org) is an integrated genome database for the parasitic organism Trypanosoma cruzi, the causative agent of Chagas' disease. The database currently incorporates all available sequence data (Genomic, BAC, EST) in a single user-friendly location. The database contains a variety of tools specifically designed for searching unannotated draft sequence via BLAST, keyword searches of pre-computed BLAST results, and protein motif searches. Release 1.0 of the database contains nearly 730 million bp of genome sequence from 1.1 million sequence reads generated by the TIGR-Karolinska-SBRI Trypanosoma cruzi Genome Consortium and 15 million bp of clustered EST and genomic sequence obtained from other sources. As annotation, microarray and proteomic data become available, the database will incorporate and integrate these data using the GUS (http://www.gusdb. org) relational framework.

Genetic immunization with a limited set of genes has been demonstrated to be an effective means of protecting mice from a normally lethal challenge of Trypanosoma cruzi. The goal of this study was to expand the diversity of genes assessed as genetic vaccine candidates. Screening a T. cruzi amastigote cDNA expression library with anti-amastigote monoclonal antibodies resulted in the identification of two genes, the previously identified flagellar Ca(2+) binding protein, FCaBP, and a novel homologue of the adaptin AP-3 complex beta3 subunit, Tcbeta3. A third gene, LYT1, recently identified as a secreted T. cruzi protein involved in cell lysis and infectivity, and was selected. Although peptides from all three genes were found to be targets of cytotoxic T cell responses in chronically infected mice, only immunization with LYT1 protected mice from a normally lethal challenge of T. cruzi. As an alternative to testing individual T. cruzi genes as vaccines, pools of genes from the trans-sialidase (TS) and mucin families were assessed in vaccination studies. Immunization with pools of TS but not mucin genes provided protection against a normally lethal challenge of T. cruzi. This study demonstrates that the ability of T. cruzi proteins to elicit immune responses in infected hosts does not necessarily associate with the ability to induce protection and that both the products of single genes and multi-gene families may serve as effective vaccines.