Back to the future in Chagas disease: from animal models to patient cohort studies, progress in immunopathogenesis research

Despite the wealth of information generated by trans-disciplinary research in Chagas disease, knowledge about its multifaceted pathogenesis is still fragmented. Here we review the body of experimental studies in animal models supporting the concept that persistent infection by Trypanosoma cruzi is crucial for the development of chronic myocarditis. Complementing this review, we will make an effort to reconcile seemingly contradictory results concerning the immune profiles of chronic patients from Argentina and Brazil. Finally, we will review the results of molecular studies suggesting that parasite-induced inflammation and tissue damage is, at least in part, mediated by the activities of trans-sialidase, mucin-linked lipid anchors (TLR2 ligand) and cruzipain (a kinin-releasing cysteine protease). One hundred years after the discovery of Chagas disease, it is reassuring that basic and clinical research tends to converge, raising new perspectives for the treatment of chronic Chagas disease.

A role for extracellular amastigotes in the immunopathology of Chagas disease

In spite of the growing knowledge obtained about immune control of Trypanosoma cruzi infection, the mechanisms responsible for the variable clinico-pathological expression of Chagas disease remain unknown. In a twist from previous concepts, recent studies indicated that tissue parasitism is a pre-requisite for the development of chronic myocarditis. This fundamental concept, together with the realization that T. cruzi organisms consist of genetically heterogeneous clones, offers a new framework for studies of molecular pathogenesis. In the present article, we will discuss in general terms the possible implications of genetic variability of T. cruzi antigens and proteases to immunopathology. Peptide epitopes from a highly polymorphic subfamily of trans-sialidase (TS) antigens were recently identified as targets of killer T cell (CTL) responses, both in mice and humans. While some class I MHC restricted CTL recognize epitopes derived from amastigote-specific TS-related antigens (TSRA), others are targeted to peptide epitopes originating from trypomastigote-specific TSRA. A mechanistic hypothesis is proposed to explain how the functional activity and specificity of class I MHC restricted killer T cells may control the extent to which tissue are exposed to prematurely released amastigotes. Chronic immunopathology may be exacerbated due the progressive accumulation of amastigote-derived antigens and pro-inflammatory molecules (eg. GPI-mucins and kinin-releasing proteases) in dead macrophage bodies.

Reposta proliferativa das células T contra a cruzipaina na cardiopatia chagásica crônica / T cells proliferative responses to cruzipaina in chronic chagasic cardiopathy

In this paper, we sought to determine if chronic chagasic patients with cardiopathy could be distinguished from those displaying non-chagasic cardiopathy on the basis of T cell proliferative responses to cruzipain (GP57/51), a major antigen of T. cruzi. Assays were performed with peripheral blood mononuclear cells from 24 individuals classified as follows: normal donors (n = 8), patients with non-chagasic cardiopathy (n = 8), patients with chronic chagasic cardiopathy without morbid associations (n = 8). The analysis of variance indicated that the proliferative responses stimulated by cruzipain were significantly higher in the group of chagasic patients (p = 0.0001). Turkey's multiple comparison test showed that the proliferative index medium from normal and non-chagasic cardiopathy was not significantly different from each other. We conclude that the T cell responses against T. cruzipain, as measured by proliferative indices of cells found in peripheral blood, are exclusively associated with Chagas, disease. In view of the abundance of cruzipain antigen in amastigotes it is possible that these T cell specificities contribute to the heart tissue damage observed in chronic Chagas, disease patients.

The MHC-class II restricted immune response against parasitic proteinases: clearance-receptors as an alternative pathway for their endocytic uptake into antigen presenting cells

We have recently characterized the partial structure of a relatively dominant T cell epitope from the major T. cruzi cysteinyl proteinase (GP57/51 or cruzipain), and showed that it can trigger the secretion of gamma-interferon from CD4+ lymphocytes from chagasic patients. As generally observed for soluble antigens, T cell recognition of imunogenic peptides from cruzipain requires their prior uptake by antigen presenting cells (APC). Following endocytosis by the APC, cruzipain (a lysosomal proteinase by itself) has to unsergo intracellular degradation in acidic endosomes or lysosomes. The analysis of the fine specificity of cruzipain recognition by T lymphocytes (class II restricted responses) from chronic patients has implicated epitopes mapped to the catalytic domain of cruzipain rather than to its long COOH-terminal extension. Structural differences between these two domains may render them differentially susceptible to intracellular digestion in the APC. We now suggest that the functional inactivation of extracellular cruzipain by proteinase inhibitors available in tissue fluids may increase the efficieny of T cell responses. Recent data indicate that plasma inhibitor alpha2-macroglobulin M (alpha2M) can bind covalently to cruzipain, this being followed by APC uptake by means of the alpha2 macroglobulin receptor (alpha2MR) on monocytes. Similar clearance methanisms may allow highly efficient focusing, degradation and MHC-class II presentation of T cell stimulatory peptides from proteinases originating from allergens and other pathogenic parasites.

The major cysteinyl proteinase from Trypanosoma cruzi: structural and functional properties

New opportunities to develop structure-based strategies for design of antiparasite drugs have emerged from studies of a family of structurally homologous cysteinyl proteinases, recently identified in several pathogenic parasites. Research on the major cysteinyl proteinase from T. cruzi (designated as cruzain, cruzipain or GP57/51) has rapidly progressed, due to independent studies conducted in Buenos Aires, Rio de Janeiro and San Francisco. The biochemical heterogeneity of this developmentally regulated group of glycoproteins is at least in part determined by genetic polymorphism. Distinguished from mammalian lysosomal cathepsins (L/S/B) by the presence of a long COOH-terminus, these single chained proteins are encoded by multiple copies of polymorphic genes. Nonconservative amino acid substitutions appear to concentrate in the papain-like catalytic domain, and may impart different substrate specificity properties to cruzipain variants expressed at the various stages of parasite development. Notably, the replication and differentiation of T. cruzi in infected cells is critically dependent on the activity of cathepsin L-like cruzipains, expressed by intracellular amastigotes. The goal of improving target selectivity of cruzipain inhibitors may be now pursued, based on clues derived from the recently elucidated crystal structure of recombinant cruzain. Concurrent with progress made in the study of cruzipain's role in parasite physiology, these proteinases have beem recognized as major antigens in chronically infected patients. By stimulating antigen specific T cells to release gamma-interferon to host tissues, cruzipain may further promote inflammation thereby contributing to pathology. Ongoing studies should determine if the proteolytic activity of cruzipain can also contribute to parasite-induced tissue damage or to the immunoregulatory abnormalities associated with Chagas'disease.