Chagas disease is a major public health problem in Bolivia. In the city of Cochabamba, 58% of the population lives in peripheral urban districts ("popular zones")

Here, we analysed the use of Vb-TCR regions by CD4 and CD8 T cells from acute and chronic chagasic patients using¯ow cytometry. We determined the Vb expression in cells freshly isolated from patients, as well as after in vitro stimulation with antigens derived from epimastigote (EPI) or trypomastigote (TRYPO) forms of Trypanosoma cruzi. Analysis of Vb-TCR expression of T cells freshly isolated from patients showed a decrease in Vb5 expression in the CD4 T-cell population from acutely infected individuals, whereas CD4 Vb5 T cells were found to be increased in chronic patients with the cardiac, but not indeterminate, clinical form. After culturing peripheral blood mononuclear cells (PBMC) from chronic patients with EPI or TRYPO, we found that both antigenic preparations led to a preferential expansion of CD4 Vb5 T cells. EPI stimulation also led to the expansion of CD8 Vb5 T cells, whereas TRYPO led to the expansion of this cell population only if PBMC were from cardiac and not indeterminate patients. We observed that TRYPO stimulation led to an increase in the frequency of CD4 Vb17 T cells in cultures of PBMC from indeterminate patients, whereas an increase in the frequency of CD8 Vb17 T cells was found upon TRYPO stimulation of PBMC from cardiac patients. Despite this increase in the frequency of Vb17 T-cell populations upon TRYPO stimulation, the same antigenic preparation led to a much higher expansion of Vb5 T cells. These results show a differential expression of Vb5-TCR in cells freshly isolated from chagasic patients in different stages of the disease and that parasite-speci®c antigens stimulate a portion of the T-cell repertoire with preferential usage of Vb5-TCR.

Hematophagous arthropods such as Triatoma infestans, the vector of Trypanosoma cruzi, elicit hostimmune responses during feeding. Characterization of antibody responses to salivary antigens offers the potential to develop immunologically based monitoring techniques for exposure to re-emergent triatomine bug populations in peridomestic animals. IgG-antibody responses to the salivary antigens of T. infestans have been detected in chickens as soon as 2 days after the first exposure to five adult bugs. Chickens and guinea pigs regularly exposed to this number of triatomines showed a significantly lower anti-saliva antibody titre than animals exposed to 25 adults and fifth instars of four different T. infestans strains originating from Bolivia and from Northern Chile. Highly immunogenic salivary antigens of 14 and 21 kDa were recognised by all chicken sera and of 79 kDa by all guinea pig sera. Cross-reactivity studies using saliva or salivary gland extracts from different hematophagous species, e.g. different triatomines, bed bugs, mosquitoes, sand flies and ticks, as well as chicken sera exposed to triatomines and mosquitoes, demonstrated that the 14 and 21 kDa salivary antigens were only found in triatomines. Sera from peridomestic chickens and guinea pigs in sites of known T. infestans challenge in Bolivia also recognised the 14 and 21 kDa antigens. These represent promising epidemiological markers for the detection of small numbers of feeding bugs and hence may be a new tool for vector surveillance in Chagas disease control programs.

Background Triatoma infestans is the main vector of Chagas disease in South America. As in all hematophagous arthropods, its saliva contains a complex cocktail that assists blood feeding by preventing platelet aggregation and blood clotting and promoting vasodilation. These salivary components can be immunologically recognized by their vector's hosts and targeted with antibodies that might disrupt blood feeding. These antibodies can be used to detect vector exposure using immunoassays. Antibodies may also contribute to the fast evolution of the salivary cocktail.MethodologySalivary gland cDNA libraries from nymphal and adult T. infestans of breeding colonies originating from different locations (Argentina, Chile, Peru and Bolivia), and cDNA libraries originating from F1 populations of Bolivia, were sequenced using Illumina technology. Coding sequences (CDS) were extracted from the assembled reads, the numbers of reads mapped to these CDS, sequences were functionally annotated and polymorphisms determined.Main findings/SignificanceOver five thousand CDS, mostly full length or near full length, were publicly deposited on GenBank. Transcripts that were over 10-fold overexpressed from different geographical regions, or from different developmental stages were identified. Polymorphisms were mapped to derived coding sequences, and found to vary between developmental instars and geographic origin of the biological material. This expanded sialome database from T. infestans should be of assistance in future proteomic work attempting to identify salivary proteins that might be used as epidemiological markers of vector exposure, or proteins of pharmacological interest.

We performed a cross-sectional flow cytometric analysis of peripheral blood mononuclear cells to evaluate human immunologic status during early stages of Trypanosoma cruzi infection in children. We identified major immunological features corresponding to three proposed phases of disease: early acute (EA) phase, late acute (LA) phase and recent chronic (RC) phase. EA phase was accompanied by expansion of conventional B cells, up-regulation of CD54 on monocytes and down-regulation of CD54 on T cells and not associated with monocyte-activation phenotypes or changes of natural killer (NK) population. LA phase was characterized by a selective increase in a distinct lineage of NK cells (CD16 þ CD56 -), as well as a persistent expansion of B cells and downregulation of CD54 on T cells. RC phase showed persistent low levels of CD54 molecule on T cells and an increase of B cells, mainly triggered by expansion of the B1-cell subset, as well as increased expression of human leucocyte antigen (HLA-DR) by monocytes. These findings reinforce the hypothesis that T. cruziderived antigens are able to activate NK cells before the development of T-cellmediated immunity. Moreover, our data support previous findings of increased levels of B1 lymphocytes during human Chagas' disease and show that this event is already present during initial stages of chronic infection.