Chagas Disease Diagnostic Applications: Present Knowledge and Future Steps.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a lifelong and debilitating illness of major significance throughout Latin America and an emergent threat to global public health. Being a neglected disease, the vast majority of Chagasic patients have limited access to proper diagnosis and treatment, and there is only a marginal investment into R&D for drug and vaccine development. In this context, identification of novel biomarkers able to transcend the current limits of diagnostic methods surfaces as a main priority in Chagas disease applied research. The expectation is that these novel biomarkers will provide reliable, reproducible and accurate results irrespective of the genetic background, infecting parasite strain, stage of disease, and clinical-associated features of Chagasic populations. In addition, they should be able to address other still unmet diagnostic needs, including early detection of congenital T. cruzi transmission, rapid assessment of treatment efficiency or failure, indication/prediction of disease progression and direct parasite typification in clinical samples. The lack of access of poor and neglected populations to essential diagnostics also stresses the necessity of developing new methods operational in point-of-care settings. In summary, emergent diagnostic tests integrating these novel and tailored tools should provide a significant impact on the effectiveness of current intervention schemes and on the clinical management of Chagasic patients. In this chapter, we discuss the present knowledge and possible future steps in Chagas disease diagnostic applications, as well as the opportunity provided by recent advances in high-throughput methods for biomarker discovery.

Multiple overlapping epitopes in the repetitive unit of the shed acute-phase antigen from Trypanosoma cruzi enhance its immunogenic properties.

The repetitive shed acute-phase antigen (SAPA) from Trypanosoma cruzi was thoroughly mapped by SPOT peptides and phage display strategies, showing that a single SAPA repeat is composed of multiple overlapping B-cell epitopes. We propose that this intricate antigenic structure constitutes an alternative device to repetitiveness in order to improve its immunogenicity.

Tandem amino acid repeats from Trypanosoma cruzi shed antigens increase the half-life of proteins in blood.

Proteins containing amino acid repeats are widespread among protozoan parasites. It has been suggested that these repetitive structures act as immunomodulators, but other functional aspects may be of primary importance. We have recently suggested that tandem repeats present in Trypanosoma cruzi trans-sialidase stabilize the catalytic activity in blood. Because the parasite releases trans-sialidase, this delayed clearance of the enzyme might have implications in vivo. In the present work, the ability of repetitive units from different T. cruzi molecules in stabilizing trans-sialidase activity in blood was evaluated. It is shown that repeats present on T. cruzi shed proteins (antigens 13 and Shed-Acute-Phase-Antigen [SAPA]) increase trans-sialidase half-life in blood from 7 to almost 35 hours. Conversely, those repeats present in intracellular T. cruzi proteins only increase the enzyme half-life in blood up to 15 hours. Despite these results, comparative analysis of structural and catalytic properties of both groups of chimeric enzymes show no substantial differences. Interestingly, antigens 13 and SAPA also increase the persistence in blood of chimeric glutathione S-transferases, thus suggesting that this effect is inherent to these repeats and independent of the carrier protein. Although the molecular basis of this phenomenon is still uncertain, its biotechnological potential can be envisaged.

The repetitive domain of Trypanosoma cruzi trans-sialidase enhances the immune response against the catalytic domain.

Trypanosoma cruzi trans-sialidase consists of a C-terminal domain composed essentially of immunodominant amino acid repeat units (SAPA-repeats) and an amino region responsible for the enzymatic activity (catalytic domain). To investigate the possible function(s) of SAPA-repeats, recombinant trans-sialidases either containing or lacking the C-terminal region were tested in mice. The presence of SAPA-repeats in the intravenously injected protein has two consequences. First, they enhance the persistence of the trans-sialidase activity in blood. Second, SAPA-repeats promoted the production of antibodies directed to the catalytic domain that inhibit trans-sialidase activity. These results suggest that SAPA-repeats modulate the trans-sialidase activity in blood.

Regulation of ovarian follicle differentiation in gonadotrophin-stimulated rats.

The aim of the present study was to investigate the regulation of the in vitro DNA synthesis of ovarian cells recovered from prepubertal rats 48 h after administration of pregnant mare's serum gonadotrophin alone (granulosa cells) or followed by human chorionic gonadotrophin (luteal cells). Isolated granulosa cells were cultured in serum-free medium, different stimuli added for periods of 48 h, and 3H-thymidine incorporation was measured. Both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) inhibited 3H-thymidine incorporation by cultured granulosa cells in a dose-dependent manner (FSH: 10, 100, 200 ng/mL = 26, 41, 49% inhibition, respectively; LH: 0.1, 1, 10 ng/mL = 11, 37, 75% inhibition, respectively). On the other hand, estradiol was found to stimulate 3H-thymidine incorporation in granulosa cells (Estradiol: 5, 50, 500 ng/mL = 17, 37, 76% stimulation, respectively). In luteal cells, the rate of basal 3H-thymidine incorporation was very low (granulosa cells: 2560 +/- 310; luteal cells: 661 +/- 92 cpm/100,000 cells) and not modified by any stimulus. To determine the possible production of an inhibitory growth factor by the early corpus luteum, 3H-thymidine incorporation by granulosa cells was assessed in the presence of 10% conditioned media (CM) recovered from luteal cell cultures. A marked inhibition both in basal and estradiol-stimulated 3H-thymidine incorporation was observed (74 and 76% of inhibition, respectively). Results suggest that an inhibitory growth factor produced by luteal cells after luteinizing gonadotrophin stimulus could be involved in the differentiation of growing follicles to corpus luteum.

A putative pyruvate dehydrogenase alpha subunit gene from Trypanosoma cruzi.

A full-length DNA clone encoding a putative pyruvate dehydrogenase alpha subunit (E1 alpha) gene was isolated from a Trypanosoma cruzi (RA strain) DNA library. Sequencing of this clone revealed it to encode a 378 amino acid protein (M(r) 42774) with high sequence similarity to E1 alpha obtained from different sources. The highest score is obtained with human E1 alpha: 43,3% similarity. Southern blot analysis is consistent with the existence of a single copy of this putative T. cruzi E1 alpha gene per haploid genome in different parasite strains. Expression of this gene was demonstrated by Northern blot analysis and its trans-splicing acceptor site was identified by Polymerase Chain Reaction-mediated amplification of its cDNA.