Human humoral immunity to hsp70 during Trypanosoma cruzi infection

Immunologic screening of cDNA expression libraries has been widely used for the identification of DNA sequences encoding the immunologically relevant proteins of many pathogenic microorganisms. For reasons that are not entirely clear, sequences encoding 70-kDa heat shock and related proteins (hsp70), which are among the most highly conserved proteins known, have routinely been identified by this approach. Consequently, hsp70 proteins have been proposed to be involved in the autoimmune processes thought responsible for the pathogenesis of the diseases caused by some of these organisms, e.g., chronic Trypanosoma cruzi infection (Chagas' disease). Therefore, we investigated whether hsp70 might be a specific target of the human humoral immune response to T. cruzi infection, and, if so, whether humoral autoimmunity to hsp70 might play a role in pathogenesis. We found that hsp70 is indeed a major polypeptide Ag in Chagas' disease, but that the antibodies to T. cruzi hsp70 do not react with human hsp70--even though the proteins display 73% amino acid sequence identify. These results indicate that self-tolerance to hsp70 is maintained during chronic T. cruzi infection and strongly argue against a role for humoral autoimmunity to hsp70 in the pathogenesis of Chagas' disease.     

Action of Trypanosoma rangeli in infections with virulent Trypanosoma cruzi populations

In experimental murine infections with Trypanosoma rangeli it has been observed development immune response to Trypanosoma cruzi. The aim of the present work was to analyze the result of antigenic stimuli and the protective effect with T. rangeli in T. cruzi infections. Mice groups immunized with metacyclic trypomastigotes of T. rangeli (Choach -2V strain), derived from haemolymph and salivary gland and reinfected with T. cruzi virulent populations (Tulahuen strain, SA strain and Dm28c clone) from infected in vitro cells, showed decrease severity of disease outcomes, low parasitemia levels and 100% survival of all mice immunized, in comparison with groups infected only with T. cruzi populations, which demonstrated tissue affection, high parasitemia levels and the death of all animals. The above mentioned data contribute to understand the biological behaviour of T. cruzi and T. rangeli and their interaction with vertebrate host.     

Interleukin-12 but not interleukin-18 is required for immunity to Trypanosoma cruzi in mice

Protective immunity to the parasite Trypanosoma cruzi in mice depends on a pro-inflammatory T cell response involving the production of interferon-gamma (IFN-gamma). In conjunction with interleukin-12 (IL-12), IL-18 promotes the synthesis of IFN-gamma and a T helper type 1 immune response. We investigated the requirements of IL-12 and IL-18 in murine T. cruzi infection by use of C57BL/6 mice genetically deficient in either cytokine. IL-12p40(-/-) mice succumbed to infection at doses of 100 parasites, whereas IL-18(-/-) and wild-type mice resisted infectious doses up to 1000 parasites to the same extent. Levels of parasitemia were comparable between the latter groups, as were tissue parasite burdens according to quantitative real-time PCR. In contrast, IL-12p40(-/-) mice displayed vastly increased levels of parasites both in blood and in tissue. IFN-gamma concentrations in the serum of infected mice and in supernatants of splenocytes stimulated in vitro were decreased in IL-18(-/-) mice, whereas in IL-12p40(-/-) mice, IFN-gamma was undetectable in the serum and drastically reduced in cell supernatants. Levels of IL-12 production were generally comparable between wild-type and IL-18(-/-) mice, as were levels of IL-4, IL-2 and nitric oxide. Thus, the requirement for endogenous pro-inflammatory cytokines for a protective murine immune response against T. cruzi is satisfied by the expression of IL-12, while IL-18 is dispensable.     

Regulation of immunity in Trypanosoma cruzi infection

Immunity to T. cruzi is complex, involving among other components, antibody production, CD4+ helper cells, CD8+ T cells as both regulators and effectors of immunity, and possibly, double-negative T cells. In addition, several of these components have been implicated in pathogenesis in the chronic infection. Although the immunosuppression observed in the infection seems quite severe, it also appears to provide for a sufficient level of immune responsiveness to control the infection in most hosts. At the same time, immunosuppression may provide the regulatory control necessary to prevent massive chronic pathogenesis in all hosts. Continued study of the relative roles of lymphocyte populations and the products they secrete in immunity and pathogenesis may provide the understanding necessary to enhance immunity to T. cruzi without the feared consequence of increased pathogenesis.     

Cell-mediated immunity in experimental Trypanosoma cruzi infection

Infection of humans with the protozoan Trypanosoma cruzi leads to Chagas disease, or American trypanosomiasis, a disease that affects nearly 20 million people, and constitutes one of the largest socioeconomic burdens in Latin America. Much of the present knowledge on pathogenic mechanisms underlying T. cruzi infection comes from experimental murine models. Here, George A. DosReis reviews recent findings about the features of host cell-mediated immunity against the parasite and possible mechanisms leading to chronic infection.     

Interleukin-17 as an effector molecule of innate and acquired immunity against infections

Interleukin (IL)-17 is a proinflammatory cytokine which induces differentiation and migration of neutrophils through induction of cytokines and chemokines including granulocyte-colony stimulating factor and CXCL8/IL-8. IL-17-producing CD4(+) T cells (Th17) have pivotal role in pathogenesis of autoimmune diseases. IL-17 is also involved in protective immunity against various infections. IL-17 has important role in induction of neutrophil-mediated protective immune response against extracellular bacterial or fungal pathogens such as Klebsiella pneumoniae and Candida albicans. Importance of IL-17 in protection against intracellular pathogens including Mycobacterium has also been reported. Interestingly, not only CD4(+) T cells but atypical CD4(-)CD8(-) T cells expressing T cell receptor (TCR) gammadelta produce IL-17, and IL-17 producing cells participate in both innate and acquired immune response to infections. Furthermore, neutrophil induction may not be the only mechanism of IL-17-mediated protective immunity. IL-17 seems to participate in host defense through regulation of cell-mediated immunity or induction of antimicrobial peptides such as beta-defensins. In this review, we summarize recent progress on the role of IL-17 in immune response against infections, and discuss possible application of IL-17 in prevention and treatment of infectious diseases.     

Creatine phosphokinase isoenzymes and Trypanosoma cruzi infections.

1. Substantial increases in total creatine phosphokinase (CPK) and in isoenzymes from heart (CPK-MB) and skeletal muscle (CPK-MM) were observed during acute infections with the House 510 and House 11 strains of Trypanosoma cruzi. 2. In infections with the reticulotropic Tulahuen strain total CPK levels were lower and the isoenzyme pattern was essentially normal. 3. Gamma-glutamyl transpeptidase was considerably increased in the Tulahuen but not in the House 510 and House 11 infections. 4. These findings are useful in assessing tissue damage during T. cruzi infections and they also demonstrate differences between myotropic and reticulotropic strains which may aid in their taxonomic classification.     

Induction of parasite-specific helper T lymphocytes during Trypanosoma cruzi infections in mice

Development of parasite-specific T helper cells was examined in mice infected with Trypanosoma cruzi. At various times during the course of infection mice were challenged with TNP conjugated to fixed culture forms of T. cruzi (TNP-TC), and the resultant splenic plaque-forming cells (PFC) against TNP were determined. By day 10 post-infection significant responses against TNP-TC were observed but not against TNP-BSA. Infected mice that were not challenged with TNP-TC did not produce anti-TNP PFC, which demonstrated that the TNP-TC response was not the result of nonspecific B cell activation. Treatment of spleen cells from infected mice with anti-theta antiserum plus C ablated the anti-TNP-TC response when these cells were transferred to normal mice that were subsequently challenged with TNP-TC, whereas treatment of the cells with anti-Ig plus C prior to transfer had no effect on the TNP-TC response. These results demonstrate enhancement of parasite-specific Th activity of mice infected with T. cruzi and that cell-cell interaction in development of responses to neoantigens is fully functional when sensitized Th are present, even though the animals are unresponsive to heterologous antigens.     

Inositolphosphoceramide metabolism in Trypanosoma cruzi as compared with other trypanosomatids

Chagas disease is caused by Trypanosoma cruzi and is endemic to North, Central and South American countries. Current therapy against this disease is only partially effective and produces adverse side effects. Studies on the metabolic pathways of T. cruzi, in particular those with no equivalent in mammalian cells, might identify targets for the development of new drugs. Ceramide is metabolized to inositolphosphoceramide (IPC) in T. cruzi and other kinetoplastid protists whereas in mammals it is mainly incorporated into sphingomyelin. In T. cruzi, in contrast to Trypanosoma brucei and Leishmania spp., IPC functions as lipid anchor constituent of glycoproteins and free glycosylinositolphospholipids (GIPLs). Inhibition of IPC and GIPLs biosynthesis impairs differentiation of trypomastigotes into the intracellular amastigote forms. The gene encoding IPC synthase in T. cruzi has been identified and the enzyme has been expressed in a cell-free system. The enzyme involved in IPC degradation and the remodelases responsible for the incorporation of ceramide into free GIPLs or into the glycosylphosphatidylinositols anchoring glycoproteins, and in fatty acid modifications of these molecules of T. cruzi have been understudied. Inositolphosphoceramide metabolism and remodeling could be exploited as targets for Chagas disease chemotherapy.     

Cell-substrate adhesion during Trypanosoma cruzi differentiation

The transformation of Trypanosoma cruzi epimastigotes to the mammal infective metacyclic trypomastigotes (metacyclogenesis) can be performed in vitro under chemically defined conditions. Under these conditions, differentiating epimastigotes adhere to a surface before their transformation into metacyclic trypomastigotes. Scanning and transmission electron microscopy of adhered and non-adhered parasites during the metacyclogenesis process show that only epimastigotes and few transition forms are found in the first population, whereas metacyclic trypomastigotes are exclusively found in the cell culture supernatant. PAGE analysis of the [35S]methionine metabolic labeling products of adhered and non-adhered parasites shows that although most of the polypeptides are conserved, adhered parasites express specifically four polypeptides in the range of 45-50 kD with an isoelectric point of 4.8. These proteins might be involved in the adhesion process and are recognized by an antiserum against total adhered parasite proteins. This antiserum also recognized a group of 45-50 kD in the iodine-radiolabeled surface proteins of differentiating cells, providing direct evidence that these components are indeed surface antigens. The results suggest that epimastigotes must adhere to a substrate before their transformation to metacyclic trypomastigotes, being released to the medium as the metacyclogenesis process is accomplished. This could correspond to the process naturally occurring within the triatomine invertebrate host.     

Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles

The innate immune system is the first mechanism of vertebrate defense against pathogen infection. In this study, we present evidence for a novel immune evasion mechanism of Trypanosoma cruzi, mediated by host cell plasma membrane-derived vesicles. We found that T. cruzi metacyclic trypomastigotes induced microvesicle release from blood cells early in infection. Upon their release, microvesicles formed a complex on the T. cruzi surface with the complement C3 convertase, leading to its stabilization and inhibition, and ultimately resulting in increased parasite survival. Furthermore, we found that TGF-β-bearing microvesicles released from monocytes and lymphocytes promoted rapid cell invasion by T. cruzi, which also contributed to parasites escaping the complement attack. In addition, in vivo infection with T. cruzi showed a rapid increase of microvesicle levels in mouse plasma, and infection with exogenous microvesicles resulted in increased T. cruzi parasitemia. Altogether, these data support a role for microvesicles contributing to T. cruzi evasion of innate immunity.     

Differential gene expression during Trypanosoma cruzi metacyclogenesis

The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE). The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes) resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells), while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.     

Stage-specific gene expression during Trypanosoma cruzi metacyclogenesis

The process of Trypanosoma cruzi metacyclogenesis involves the transformation of noninfective epimastigotes into metacyclic trypomastigotes, which are the pathogenic form. The analysis of stage-specific genes during T. cruzi metacyclogenesis may provide insight into the mechanisms involved in the regulation of gene expression in trypanosomatids. It may also improve the understanding of the mechanisms responsible for the pathology of Chagas disease, and could lead to the identification of new targets for chemotherapy of this disease. We have demonstrated that during metacyclogenesis the expression of several genes is controlled at the translational level by an alternative regulatory mechanism. This mechanism may involve the mobilization of mRNA to the translation machinery. We have been using self-made T. cruzi microarrays to investigate the role of polysomal mobilization in modulating gene expression during metacyclogenesis.     

The karyotype of Trypanosoma cruzi Dm 28c: comparison with other T. cruzi strains and trypanosomatids

Chromosome-sized DNA molecules from Trypanosoma cruzi clone Dm 28c were analyzed and compared with other T. cruzi strains and monogenetic trypanosomatids by orthogonal field alteration gel electrophoresis. The results showed that T. cruzi Dm 28c displays at least 18 chromosomes ranging from 550 to more than 1500 kb and that in general the trypanosomatids have smaller chromosomes distributed in the size range from 300 to 1500 kb. With the exception of T. cruzi strain G49, there is no evidence of minichromosomes, suggesting they are not widely distributed among different isolates of the parasite. The hybridization of T. cruzi chromosomal Southern blots with probes for T. cruzi-specific genes showed that their location can change from one strain to another, supporting the idea of the plasticity of the parasite genome. Furthermore, the chromosome pattern is strictly conserved during the transformation of T. cruzi Dm 28c epimastigotes to metacyclic trypomastigotes, suggesting that extensive chromosomal rearrangements do not occur during at least part of the life cycle of the parasite.